Handbook of Plant and Crop Stress

THIRD EDITION

HANDBOOK OF PLANT AND

CROP STRESS

BOOKS IN SOILS, PLANTS, AND THE ENVIRONMENT

Editorial Board Agricultural Engineering

Robert M. Peart, University of Florida, Gainesville

Crops

Mohammad Pessarakli, University of Arizona, Tucson

Environment

Kenneth G. Cassman, University of Nebraska, Lincoln

Irrigation and Hydrology

Donald R. Nielsen, University of California, Davis

Microbiology Wageningen,

Jan Dirk van Elsas, Research Institute for Plant Protection, The Netherlands

Plants Maryland

L. David Kuykendall, U.S. Department of Agriculture, Beltsville, Kenneth B. Marcum, Arizona State University, Tempe

Soils

Jean-Marc Bollag, Pennsylvania State University, University Park Tsuyoshi Miyazaki, University of Tokyo, Japan

Soil Biochemistry, Volume 1, edited by A. D. McLaren and G. H. Peterson Soil Biochemistry, Volume 2, edited by A. D. McLaren and J. Skujins Soil Biochemistry, Volume 3, edited by E. A. Paul and A. D. McLaren Soil Biochemistry, Volume 4, edited by E. A. Paul and A. D. McLaren Soil Biochemistry, Volume 5, edited by E. A. Paul and J. N. Ladd Soil Biochemistry, Volume 6, edited by Jean-Marc Bollag and G. Stotzky Soil Biochemistry, Volume 7, edited by G. Stotzky and Jean-Marc Bollag Soil Biochemistry, Volume 8, edited by Jean-Marc Bollag and G. Stotzky Soil Biochemistry, Volume 9, edited by G. Stotzky and Jean-Marc Bollag Organic Chemicals in the Soil Environment, Volumes 1 and 2, edited by C. A. I. Goring and J. W. Hamaker Humic Substances in the Environment, M. Schnitzer and S. U. Khan Microbial Life in the Soil: An Introduction, T. Hattori Principles of Soil Chemistry, Kim H. Tan Soil Analysis: Instrumental Techniques and Related Procedures, edited by Keith A. Smith Soil Reclamation Processes: Microbiological Analyses and Applications, edited by Robert L. Tate III and Donald A. Klein Symbiotic Nitrogen Fixation Technology, edited by Gerald H. Elkan

Soil–Water Interactions: Mechanisms and Applications, Shingo Iwata and Toshio Tabuchi with Benno P. Warkentin Soil Analysis: Modern Instrumental Techniques, Second Edition, edited by Keith A. Smith Soil Analysis: Physical Methods, edited by Keith A. Smith and Chris E. Mullins Growth and Mineral Nutrition of Field Crops, N. K. Fageria, V. C. Baligar, and Charles Allan Jones Semiarid Lands and Deserts: Soil Resource and Reclamation, edited by J. Skujins Plant Roots: The Hidden Half, edited by Yoav Waisel, Amram Eshel, and Uzi Kafkafi Plant Biochemical Regulators, edited by Harold W. Gausman Maximizing Crop Yields, N. K. Fageria Transgenic Plants: Fundamentals and Applications, edited by Andrew Hiatt Soil Microbial Ecology: Applications in Agricultural and Environmental Management, edited by F. Blaine Metting, Jr. Principles of Soil Chemistry, Second Edition, Kim H. Tan Water Flow in Soils, edited by Tsuyoshi Miyazaki Handbook of Plant and Crop Stress, edited by Mohammad Pessarakli Genetic Improvement of Field Crops, edited by Gustavo A. Slafer Agricultural Field Experiments: Design and Analysis, Roger G. Petersen Mechanisms of Plant Growth and Improved Productivity: Modern Approaches, edited by Amarjit S. Basra Selenium in the Environment, edited by W. T. Frankenberger, Jr. and Sally Benson Plant–Environment Interactions, edited by Robert E. Wilkinson Handbook of Plant and Crop Physiology, edited by Mohammad Pessarakli Handbook of Phytoalexin Metabolism and Action, edited by M. Daniel and R. P. Purkayastha Soil–Water Interactions: Mechanisms and Applications, Second Edition, Revised and Expanded, Shingo Iwata, Toshio Tabuchi, and Benno P. Warkentin Stored-Grain Ecosystems, edited by Digvir S. Jayas, Noel D. G. White, and William E. Muir Agrochemicals from Natural Products, edited by C. R. A. Godfrey Seed Development and Germination, edited by Jaime Kigel and Gad Galili Nitrogen Fertilization in the Environment, edited by Peter Edward Bacon Phytohormones in Soils: Microbial Production and Function, William T. Frankenberger, Jr. and Muhammad Arshad Handbook of Weed Management Systems, edited by Albert E. Smith Soil Sampling, Preparation, and Analysis, Kim H. Tan Soil Erosion, Conservation, and Rehabilitation, edited by Menachem Agassi Plant Roots: The Hidden Half, Second Edition, Revised and Expanded, edited by Yoav Waisel, Amram Eshel, and Uzi Kafkafi Photoassimilate Distribution in Plants and Crops: Source–Sink Relationships, edited by Eli Zamski and Arthur A. Schaffer Mass Spectrometry of Soils, edited by Thomas W. Boutton and Shinichi Yamasaki Handbook of Photosynthesis, edited by Mohammad Pessarakli Chemical and Isotopic Groundwater Hydrology: The Applied Approach, Second Edition, Revised and Expanded, Emanuel Mazor Fauna in Soil Ecosystems: Recycling Processes, Nutrient Fluxes, and Agricultural Production, edited by Gero Benckiser

Soil and Plant Analysis in Sustainable Agriculture and Environment, edited by Teresa Hood and J. Benton Jones, Jr. Seeds Handbook: Biology, Production, Processing, and Storage, B. B. Desai, P. M. Kotecha, and D. K. Salunkhe Modern Soil Microbiology, edited by J. D. van Elsas, J. T. Trevors, and E. M. H. Wellington Growth and Mineral Nutrition of Field Crops, Second Edition, N. K. Fageria, V. C. Baligar, and Charles Allan Jones Fungal Pathogenesis in Plants and Crops: Molecular Biology and Host Defense Mechanisms, P. Vidhyasekaran Plant Pathogen Detection and Disease Diagnosis, P. Narayanasamy Agricultural Systems Modeling and Simulation, edited by Robert M. Peart and R. Bruce Curry Agricultural Biotechnology, edited by Arie Altman Plant–Microbe Interactions and Biological Control, edited by Greg J. Boland and L. David Kuykendall Handbook of Soil Conditioners: Substances That Enhance the Physical Properties of Soil, edited by Arthur Wallace and Richard E. Terry Environmental Chemistry of Selenium, edited by William T. Frankenberger, Jr., and Richard A. Engberg Principles of Soil Chemistry, Third Edition, Revised and Expanded, Kim H. Tan Sulfur in the Environment, edited by Douglas G. Maynard Soil–Machine Interactions: A Finite Element Perspective, edited by Jie Shen and Radhey Lal Kushwaha Mycotoxins in Agriculture and Food Safety, edited by Kaushal K. Sinha and Deepak Bhatnagar Plant Amino Acids: Biochemistry and Biotechnology, edited by Bijay K. Singh Handbook of Functional Plant Ecology, edited by Francisco I. Pugnaire and Fernando Valladares Handbook of Plant and Crop Stress, Second Edition, Revised and Expanded, edited by Mohammad Pessarakli Plant Responses to Environmental Stresses: From Phytohormones to Genome Reorganization, edited by H. R. Lerner Handbook of Pest Management, edited by John R. Ruberson Microbial Endophytes, edited by Charles W. Bacon and James F. White, Jr. Plant–Environment Interactions, Second Edition, edited by Robert E. Wilkinson Microbial Pest Control, Sushil K. Khetan Soil and Environmental Analysis: Physical Methods, Second Edition, Revised and Expanded, edited by Keith A. Smith and Chris E. Mullins The Rhizosphere: Biochemistry and Organic Substances at the Soil–Plant Interface, Roberto Pinton, Zeno Varanini, and Paolo Nannipieri Woody Plants and Woody Plant Management: Ecology, Safety, and Environmental Impact, Rodney W. Bovey Metals in the Environment, M. N. V. Prasad Plant Pathogen Detection and Disease Diagnosis, Second Edition, Revised and Expanded, P. Narayanasamy Handbook of Plant and Crop Physiology, Second Edition, Revised and Expanded, edited by Mohammad Pessarakli

Environmental Chemistry of Arsenic, edited by William T. Frankenberger, Jr. Enzymes in the Environment: Activity, Ecology, and Applications, edited by Richard G. Burns and Richard P. Dick Plant Roots: The Hidden Half, Third Edition, Revised and Expanded, edited by Yoav Waisel, Amram Eshel, and Uzi Kafkafi Handbook of Plant Growth: pH as the Master Variable, edited by Zdenko Rengel Biological Control of Major Crop Plant Diseases edited by Samuel S. Gnanamanickam Pesticides in Agriculture and the Environment, edited by Willis B. Wheeler Mathematical Models of Crop Growth and Yield, Allen R. Overman and Richard Scholtz Plant Biotechnology and Transgenic Plants, edited by Kirsi-Marja Oksman Caldentey and Wolfgang Barz Handbook of Postharvest Technology: Cereals, Fruits, Vegetables,Tea, and Spices, edited by Amalendu Chakraverty, Arun S. Mujumdar, G. S. Vijaya Raghavan, and Hosahalli S. Ramaswamy Handbook of Soil Acidity, edited by Zdenko Rengel Humic Matter in Soil and the Environment: Principles and Controversies, edited by Kim H. Tan Molecular Host Plant Resistance to Pests, edited by S. Sadasivam and B. Thayumanayan Soil and Environmental Analysis: Modern Instrumental Techniques, Third Edition, edited by Keith A. Smith and Malcolm S. Cresser Chemical and Isotopic Groundwater Hydrology, Third Edition, edited by Emanuel Mazor Agricultural Systems Management: Optimizing Efficiency and Performance, edited by Robert M. Peart and W. David Shoup Physiology and Biotechnology Integration for Plant Breeding, edited by Henry T. Nguyen and Abraham Blum Global Water Dynamics: Shallow and Deep Groundwater, Petroleum Hydrology, Hydrothermal Fluids, and Landscaping, , edited by Emanuel Mazor Principles of Soil Physics, edited by Rattan Lal Seeds Handbook: Biology, Production, Processing, and Storage, Second Edition, Babasaheb B. Desai Field Sampling: Principles and Practices in Environmental Analysis, edited by Alfred R. Conklin Sustainable Agriculture and the International Rice–Wheat System, edited by Rattan Lal, Peter R. Hobbs, Norman Uphoff, and David O. Hansen Plant Toxicology, Fourth Edition, edited by Bertold Hock and Erich F. Elstner Drought and Water Crises: Science,Technology, and Management Issues, edited by Donald A. Wilhite Soil Sampling, Preparation, and Analysis, Second Edition, Kim H. Tan Climate Change and Global Food Security, edited by Rattan Lal, Norman Uphoff, B. A. Stewart, and David O. Hansen Handbook of Photosynthesis, Second Edition, edited by Mohammad Pessarakli Environmental Soil-Landscape Modeling: Geographic Information Technologies and Pedometrics, edited by Sabine Grunwald Water Flow in Soils, Second Edition, Tsuyoshi Miyazaki Biological Approaches to Sustainable Soil Systems, edited by Norman Uphoff, Andrew S. Ball, Erick Fernandes, Hans Herren, Olivier Husson, Mark Laing, Cheryl Palm, Jules Pretty, Pedro Sanchez, Nteranya Sanginga, and Janice Thies

Plant–Environment Interactions, Third Edition, edited by Bingru Huang Biodiversity in Agricultural Production Systems, edited by Gero Benckiser and Sylvia Schnell Organic Production and Use of Alternative Crops, Franc Bavec and Martina Bavec Handbook of Plant Nutrition, edited by Allen V. Barker and David J. Pilbeam Modern Soil Microbiology, Second Edition, edited by Jan Dirk van Elsas, Janet K. Jansson, and Jack T. Trevors Functional Plant Ecology, Second Edition, edited by Francisco I. Pugnaire and Fernando Valladares Fungal Pathogenesis in Plants and Crops: Molecular Biology and Host Defense Mechanisms,Second Edition, P. Vidhyasekaran Handbook of Turfgrass Management and Physiology, edited by Mohammad Pessarakli Soils in the Humid Tropics and Monsoon Region of Indonesia, Kim H. Tan Handbook of Agricultural Geophysics, edited by Barry J. Allred, Jeffrey J. Daniels, and M. Reza Ehsani Environmental Soil Science, Third Edition, Kim H. Tan Principles of Soil Chemistry, Fourth Edition, Kim H. Tan Handbook of Plant and Crop Stress, Second Edition, edited by Mohammad Pessarakli

THIRD EDITION

HANDBOOK OF PLANT AND

CROP STRESS Edited by

Mohammad Pessarakli

Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2011 by Taylor and Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number: 978-1-4398-1396-6 (Hardback) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http:// www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

In the memory of my beloved parents, Fatemeh and Vahab, who, regretfully, did not live to see this work and my other works, which, in no small part, resulted from their gift of many years of unconditional love.

Contents Preface............................................................................................................................................xvii Acknowledgments............................................................................................................................xxi Editor............................................................................................................................................ xxiii Contributors....................................................................................................................................xxv

Part I  Soil Salinity and Sodicity Problems Chapter 1 Soil Salinity and Sodicity as Particular Plant/Crop Stress Factors...............................3 Mohammad Pessarakli and I. Szabolcs Chapter 2 Soil Salinity Development, Classification, Assessment, and Management in Irrigated Agriculture............................................................................................... 23 Shabbir A. Shahid and Khalil ur Rahman Chapter 3 Soil Salinization and Management Options for Sustainable Crop Production........... 41 Donald L. Suarez Chapter 4 Influence of Sodium on Soils in Humid Regions........................................................ 55 Rafif K. Srour, Louis M. McDonald, V.P. (Bill) Evangelou

Part II  Plant/Crop Tolerance and Stressful Conditions Chapter 5 Oxidative Stress and Antioxidative Defense Systems in Plants Growing under Abiotic Stresses................................................................................................. 89 Pallavi Sharma, Ambuj Bhushan Jha, and Rama Shanker Dubey Chapter 6 Antioxidant Protection during Abiotic Stresses........................................................ 139 Dagmar Procházková and Nad’a Wilhelmová Chapter 7 Biochemical Mechanisms for the Maintenance of Oxidative Stress under Control in Plants.............................................................................................. 157 Diego G. Arias, Claudia V. Piattoni, Sergio A. Guerrero, and Alberto A. Iglesias

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Chapter 8 Plant Hormone Functions in Abiotic and Biotic Stress Responses........................... 191 Radomíra Vanková Chapter 9 Role of Proline in Plant Response to Drought and Salinity...................................... 213 Bruria Heuer Chapter 10 Role of Dehydrins in Plant Stress Response............................................................. 239 Klára Kosová, Ilja Tom Prášil, and Pavel Vítámvás Chapter 11 Behavior of Water in Plants at Low and Ultralow Temperatures............................. 287 Jiří Zámečník and Miloš Faltus

Part III Plants and Crops Responses: Physiology, Cellular, and Molecular Biology, and Microbiological Aspects under Salt, Drought, Heat, Cold, Light, and Other Stressful Conditions Chapter 12 Germination of Seeds and Propagules under Salt Stress.......................................... 321 Abdul Wahid, Muhammad Farooq, Shahzad M.A. Basra, Ejaz Rasul, and Kadambot H.M. Siddique Chapter 13 Response of Crop Plants to Nitrogen Stress: Opportunities to Increase Nitrogen Use Efficiency............................................................................................ 339 Jagadish Rane, Manabu Ishitani, and Idupulapati M. Rao Chapter 14 Photosynthesis and Light Stress in a Model Plant: Role of Chloroplast Transporters.......................................................................................................... 361 Cornelia Spetea and Benoît Schoefs Chapter 15 Photosynthetic Pigment Apparatus in Northern Plants............................................ 391 Tamara Golovko, Olga Dymova, Yakov Yatsco, and Galina Tabalenkova Chapter 16 Modifications of the Carotenoid Metabolism in Plastids: A Response to Stress Conditions...................................................................................................407 Pascale Moulin, Yves Lemoine, and Benoît Schoefs Chapter 17 Thermoluminescence Study of Photosystem II Activity in Resurrection Plant Haberlea rhodopensis during Desiccation................................................................ 435 Liliana T. Maslenkova, Violeta N. Peeva, Yuliana K. Markovska, and Yuzeir Zeinalov

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Chapter 18 Carbon Metabolism and Plant Stress........................................................................ 447 Carlos M. Figueroa, Alberto A. Iglesias, and Florencio E. Podestá Chapter 19 Protein Synthesis by Plants under Stressful Conditions........................................... 465 Pallavi Sharma and Rama Shanker Dubey Chapter 20 Heat Shock Proteins and Acquisition of Thermotolerance in Plants........................ 519 Saaimatul Huq and Hitoshi Nakamoto Chapter 21 Effect of Low Temperatures on the Structure of Plant Cells: Structural, Biochemical, and Molecular Aspects........................................................................ 535 L’udmila Slováková, Ildikó Matušíková, Ján Salaj, and Ján Hudák Chapter 22 Effects of UV-B Radiation on Plants: Molecular Mechanisms Involved in UV-B Responses................................................................................................... 565 Brian R. Jordan Chapter 23 Effect of High Temperature and UV-A Radiation on Photosystem II...................... 577 E.L. Apostolova and A.G. Dobrikova

Part IV  Plant and Crop Responses to Pollution Stress Chapter 24 Plant Responses to Toxic Metal Stress...................................................................... 595 Elena Masarovičová, Katarína Král’ová, and František Šeršeň Chapter 25 Heavy Metal Pollution: Damage and Defense Strategies in Plants.......................... 635 Flavia Navari-Izzo and Nicoletta Rascio Chapter 26 Heavy Metals and Plastid Metabolism...................................................................... 675 Katalin Solymosi and Martine Bertrand Chapter 27 Plant Responses to Cadmium and Mercury Stress................................................... 713 Elena Garmash, Svetlana Skugoreva, and Tamara Golovko

Part V Plant and Crop Responses to Weeds, Pests, Pathogens, and Agrichemical Stress Conditions Chapter 28 Stress in Plants and Crops Induced by Parasitic Weeds........................................... 735 Andrea Cavalieri and Asghar Heydari

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Chapter 29 Involvement of Insect Pests in Plant and Crop Stress............................................... 747 Stefano Speranza, Angelo Mazzaglia, Antoine Harfouche, and Asghar Heydari Chapter 30 Stress in Plants and Crops Induced by Herbicide-Mediated Alteration in the Population and Activity of Root-Associated Microorganisms....................... 773 Asghar Heydari and Iraj J. Misaghi Chapter 31 Stress in Plants and Crops Induced by Fungal Pathogens......................................... 787 Asghar Heydari and Giorgio M. Balestra

Part VI Genetic Factors and Plant/ Crop Genomics under Stress Chapter 32 Genetic Factors Affecting Abiotic Stress Tolerance in Crops.................................. 803 Arun Kumar Joshi Chapter 33 Genetic Improvement of Cold Hardiness in Bermudagrass...................................... 851 Yanqi Wu and Jeffrey A. Anderson Chapter 34 Candidate Gene Expression Involved in Plant Drought Resistance.......................... 867 Yiwei Jiang and Ying Wang

Part VII Examples of Empirical Investigations of Specific Plants and Crops Grown in Salt, Drought, and Other Environmental Stress Conditions Chapter 35 Responses of Green Beans (Phaseolus vulgaris L.) in Terms of Dry Matter Production, Nitrogen Uptake, and Water Absorption under Salt-Stress Conditions............................................................................................................. 879 Mohammad Pessarakli Chapter 36 Physiology and Molecular Biology of the Effects of Salinity on Rice..................... 899 R.K. Singh and T.J. Flowers Chapter 37 Landscape under Water-Stress Conditions................................................................ 941 Atif Riaz

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Chapter 38 Turfgrass Nutrient Management under Stresses: A Part of Integrated Stress Management.............................................................................................................. 963 Haibo Liu, Nick Menchyk, Frank Bethea, and Christian Baldwin Chapter 39 Nutrient Management of Golf Course Putting Greens under Stresses..................... 987 Haibo Liu, Nick Menchyk, Frank Bethea, and Christian Baldwin

Part VIII Climatic Changes, Elevated Carbon Dioxide, and Plant/Crop Responses Chapter 40 Plant Biomass and Stem Juice of the C4 Sugarcane at Elevated Growth CO2 and Temperature...................................................................................................... 1019 Joseph C.V. Vu and Leon H. Allen Jr.

Part IX Future Promises: Improving Plant and Crop Adaptation/Tolerance and Cultivation under Stressful Conditions Chapter 41 Improving Crop Resistance to Abiotic Stresses through Seed Invigoration........... 1031 Muhammad Farooq, Abdul Wahid, Shahzad M.A. Basra, and Kadambot H.M. Siddique Chapter 42 Plant Stress Physiology: Physiological and Biochemical Strategies Allowing Plants/Crops to Thrive under Ionic Stress............................................................... 1051 Hans-Werner Koyro, N. Geissler, R. Seenivasan, and Bernhard Huchzermeyer Chapter 43 Role of Acacia ampliceps in Managing Salt-Affected Lands................................. 1095 Nico Marcar, Shoaib Ismail, Arunee Yuvaniyama, and Raziuddin Ansari Chapter 44 Adaptive Strategies of Tropical Forage Grasses to Low Phosphorus Stress: The Case of Brachiaria Grasses..............................................................................1111 Annabé E. Louw-Gaume, Idupulapati M. Rao, Emmanuel Frossard, and Alain J. Gaume Chapter 45 Forgotten Link in Improving Crop Salt Tolerance under Brackish Irrigation: Lateral Soil Salinity Gradients around Roots..........................................................1145 Uwe Schleiff

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Chapter 46 Improving Crop Production on Saline Soils in Arid Regions: Do We Need a Different Approach to Develop and Select Plants for These Regions?................ 1153 Shafqat Farooq and F. Azam

Part X  Beneficial Aspects of Stress Chapter 47 Salinity-Induced Enhancement of Horticultural Crop Quality................................1173 Catherine M. Grieve Index............................................................................................................................................. 1195

Preface The dynamic and ever-expanding knowledge of environmental stresses, and their effects on plants and crops, has resulted in the compilation of a large volume of information since the second edition of the Handbook of Plant and Crop Stress was presented to scientists and professionals. This fact necessitated that this unique, comprehensive source of information be revised and all the new findings in this field be included in this updated edition. Like the first and second editions, the new edition is a unique, comprehensive, and complete collection of the issues on stress imposed on plants/crops. More than 80% of the materials in this edition are entirely new and have been included under new titles. The remaining 20% have been updated. Therefore, almost 90% of the materials are new, thus creating what could be considered a new version. Out of the 47 chapters, only 11 have been taken from the second edition, but have been substantially revised and updated. The other 36 chapters are entirely new. Since the early 1900s, soil/plant scientists have observed that plant growth and crop yields decreased under salinity, drought, and/or other environmental stress conditions. Reduction in plant growth was reported as a result of a modification in the physiological process and environmental conditions that control growth. Stresses imposed on plants due to pollution or applications of agrichemicals have recently attracted the attention of scientists, investigators, and environmentalists in agriculture and related areas. The mechanisms by which salinity, drought, high/low temperatures or heat, high/low pH, high/low light, nutrient deficiency, pollution, agrichemicals, climatic changes, or any other stresses affect plant metabolism, thereby reducing plant growth and development, are still not completely understood. Among plant physiological processes, the change in nutrient uptake and metabolism induced by salt, drought, and/or other stress factors is commonly accepted by scientists as one of the most important factors responsible for abnormal plant metabolism, reduced growth, and decreased crop yield. The need for minimizing the effects of salt, drought, extreme temperatures, extreme pH, extreme light, pollution, agrichemicals, climatic changes, or any other environmental stresses on plant growth and crop yield is vital. Thus, a greater awareness of these stress factors, and problems associated with them, is essential to scientists, producers, and all involved in the field of agriculture. This handbook is a comprehensive, up-to-date reference book that addresses issues and concerns related to plant and crop stress effectively. While there are many reference books on soil salinity, sodicity, specific plant/crop salt and water stress, pollution, and other environmental stresses, all of these exist in relative isolation, covering only one specific topic. To solve plant and crop stress problems efficiently and effectively requires the accountability and coordination of all the factors and their interrelationship with plant/crop stress. While several authors have competently covered the many areas separately, the areas are, nonetheless, interrelated and should be covered comprehensively in a single text. Thus, the purpose of this book is to fill this niche. The new and updated third edition of the Handbook of Plant and Crop Stress has been written by more than 100 contributors from 27 countries, who are among the most competent and knowledgeable scientists, specialists, and researchers in the field of agriculture. It is intended to serve as a resource for preparing for lectures as well as for conducting research. Scientists, agricultural researchers, agricultural practitioners, and students will benefit from this unique comprehensive guide, which covers plant stress problems from the soil to the atmosphere. As with other fields, accessibility of knowledge is among the most critical factors involved with crop stress problems. Without due consideration of all the elements contributing to a specific crop stress problem, it is unlikely that a permanent solution will be achieved. Therefore, this book covers as many of the factors as possible. To further facilitate the accessibility of the desired information xvii

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Preface

in the areas of stress covered in this collection, the book has been divided into 10 parts. These include Soil Salinity and Sodicity Problems; Plant/Crop Tolerance and Stressful Conditions; Plants and Crops Responses, Physiology, Cellular and Molecular Biology, and Microbiological Aspects under Salt, Drought, Heat, Cold, Light, and Other Stressful Conditions; Plant and Crop Responses to Pollution Stress; Plant and Crop Responses to Weeds, Pests, Pathogens, and Agrichemical Stress Conditions; Genetic Factors and Plant/Crop Genomics under Stress; Examples of Empirical Investigations of Specific Plants and Crops Grown in Salt, Drought, and Other Environmental Stress Conditions; Climatic Changes, Elevated Carbon Dioxide, and Plant/Crop Responses; Future Promises: Improving Plant and Crop Adaptation/Tolerance and Cultivation under Stressful Conditions; and Beneficial Aspects of Stress. Each of these parts consists of one or more chapters so as to discuss, independently, as many aspects of the stresses as possible. Part I consists of four chapters, including “Soil Salinity and Sodicity as Particular Plant/ Crop Stress Factors”; “Soil Salinity Development, Classification, Assessment, and Management in Irrigated Agriculture”; “Soil Salinization and Management Options for Sustainable Crop Production”; and “Influence of Sodium on Soils of Humid Regions”. These chapters explain soil as a medium of crop growth, soil salinity, and sodicity problems, as well as the effects of soil salinity and sodicity on plant/crop growth. Part II consists of seven chapters as follows: “Oxidative Stress and Antioxidative Defense Systems in Plants Growing under Abiotic Stresses”; “Antioxidant Protection during Abiotic Stresses”; “Biochemical Mechanisms for the Maintenance of Oxidative Stress under Control in Plants”; “Plant Hormone Functions in Abiotic and Biotic Stress Responses”; “Role of Proline in Plant Response to Drought and Salinity”; “Role of Dehydrins in Plant Stress Response; and Behavior of Water in Plants at Low and Ultralow Temperatures”. These chapters address plants and crop mechanisms of stress tolerance. Part III consists of the following 12 chapters: “Germination of Seeds and Propagules under Salt Stress”; “Response of Crop Plants to Nitrogen Stress: Opportunities to Increase Nitrogen Use Efficiency”; “Photosynthesis and Light Stress in a Model Plant: Role of Chloroplast Transporters”; “Photosynthetic Pigments Apparatus in the Northern Plants”; “Modifications of the Carotenoid Metabolisms in Plastids: A Response to Stress Conditions”; “Thermoluminescence Study of Photosystem II Activity in Resurrection Plant Haberlea rhodopensis during Desiccation”; “Carbon Metabolism and Plant Stress”; “Protein Synthesis by Plants under Stressful Conditions”; “Heat Shock Proteins and Acquisition of Thermotolerance in Plants”; “Effect of Low Temperatures on the Structure of Plant Cells: Structural, Biochemical, and Molecular Aspects”; “Effects of UV-B Radiation on Plants: Molecular Mechanisms Involved in UV-B Responses”; and “Effect of High Temperature and UV-A Radiation on Photosystem II”. Each of these chapters provides in-depth information on each of these topics. Part IV consists of four chapters, including “Plant Responses to Toxic Metal Stress”; “Heavy Metal Pollution: Damage and Defense Strategies in Plants”; “Heavy Metals and Plastid Metabolism”; and “Plant Responses to Cadmium and Mercury Stress”. These chapters provide detailed information on plants/crops influenced by pollution generated from either the soil, water, or the atmosphere. Part V also consists of four chapters as follows: “Stress in Plants and Crops Induced by Parasitic Weeds”; “Involvement of Insect Pests in Plant and Crop Stress”; “Stress in Plants and Crops Induced by Herbicide-Mediated Alteration in the Population and Activity of Root-Associated Microorganisms”; and “Stress in Plants and Crops Induced by Fungal Pathogens”. These chapters discuss the interactions between weeds, pests, pathogens, and agrichemicals, and plants/crops and the potential problems caused by the application of agrichemicals to plants/crops. Part VI consists of three chapters, including “Genetic Factors Affecting Abiotic Stress Tolerance in Crops”; “Genetic Improvement of Cold Hardiness in Bermudagrass”; and “Candidate Gene Expression Involved in Plant Drought Resistance”. These chapters present detailed and comprehensive information of all available materials on these subjects.

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Several examples of empirical investigations of specific plants and crops grown under salt, drought, and/or other stress conditions are covered in Part VII, which consists of five chapters, presenting various plants and crops with different degrees of tolerance. These chapters include “Responses of Green Beans (Phaseolus vulgaris L.) in Terms of Dry Matter Production, Nitrogen Uptake, and Water Absorption under Salt Stress Conditions”; “Physiology and Molecular Biology of the Effects of Salinity on Rice”; “Landscaping under Water Stress Conditions”; “Turfgrass Nutrient Management under Stresses: A Part of Integrated Stress Management”; and “Nutrient Management of Golf Course Putting Greens under Stresses”. Due to recent climatic changes and increase in CO2 levels, plant resistance to these changes must be considered for cultivation under these conditions. Therefore, Part VIII, which consists of a single chapter entitled “Plant Biomass and Stem Juice of the C4 Sugarcane at Elevated Growth CO2 and Temperature” presents the latest information on this subject. Part IX provides evidence and guidance on plants and crops that can be used under stressful conditions. This part consists of the following six chapters: “Improving Crop Resistance to Abiotic Stresses through Seed Invigoration”; “Physiological and Biochemical Strategies Allowing Plants/Crops to Thrive under Ionic Stress”; “Role of Acacia ampliceps in Managing Salt-Affected Lands”; “Adaptive Strategies of Tropical Forage Grasses to Low Phosphorus Stress: The Case of Brachiariagrasses”; “Forgotten Link in Improving Crop Salt Tolerance Research under Brackish Irrigation: Lateral Soil Salinity Gradients around Roots”; and “Improving Crop Production on Saline Soils in Arid Regions: Do We Need a Different Approach to Develop and Select Plants for These Regions?” The important subject of beneficial aspects of stress, which has received very little attention, is covered in Part X. This part consists of a unique chapter entitled “Salinity-Induced Enhancement of Horticultural Crop Quality,” which presents available information on this subject. Numerous figures and tables have been provided in this book to facilitate the comprehension of the materials that have been presented. An extensive index has also been generated to further increase accessibility to the desired information. Mohammad Pessarakli

Acknowledgments I would like to express my appreciation for the assistance that I received from the secretarial and administrative staff of the School of Plant Sciences, College of Agriculture and Life Sciences, the University of Arizona, Tucson, Arizona. The continuous encouragement and support of the school Director, Dr. Kenneth Feldmann, and the school Associate Director, Dr. Dennis T. Ray, for my editorial work, especially the books, is always greatly appreciated. Dr. Feldmann and Dr. Ray, your encouraging words have certainly been a driving force for the successful completion of this project. In addition, I would like to express my sincere gratitude to John Sulzycki (senior editor, Taylor & Francis Group, CRC Press) who supported this project and my previous book projects from their initiation to their completion. My sincere acknowledgments also to Randy Brehm (editor, Taylor & Francis Group, CRC Press), whose professionalism, patience, proactiveness, and hard work were instrumental in the completion of this project. This job would certainly not have been completed as smoothly and rapidly without Randy’s valuable support and sincere efforts. I am also indebted to Jill Jurgensen, senior project coordinator, for the professional and careful handling of the book. Jill, many thanks to you for your extraordinary patience and thoroughness in handling this huge book as well as my previous book projects. Also, the sincere efforts and the hard work of the copy editor and the acquisition editor can never be forgotten. The collective sincere efforts and invaluable contributions of several competent scientists, specialists, and experts in the field of plant/crop stress made it possible to produce this unique source for those seeking information on this subject. Each and every one of these contributors and their contributions are greatly appreciated. Last but not least, I thank my wife, Vinca, a high school science teacher, and my son, Mahdi, a fourth-year medical college student, who supported me during the course of this project.

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Editor Dr. Mohammad Pessarakli is a research associate professor and a teaching faculty member in the School of Plant Sciences, College of Agriculture and Life Sciences, the University of Arizona, Tucson, Arizona and senior lecturer in the Department of Plant Sciences, College of Agriculture and Life Sciences, the University of Arizona. He received his BS (1977) in environmental resources in agriculture and his MS (1978) in soil management and crop production from Arizona State University, Tempe, Arizona, and his PhD (1981) in soil and water science from the University of Arizona, Tucson, Arizona. Tucson, Arizona. His work at the University of Arizona includes research and extension services as well as teaching courses in turfgrass science, management, and stress physiology. He is the editor of the Handbook of Plant and Crop Stress and the Handbook of Plant and Crop Physiology (both titles published by the Taylor & Francis Group, CRC Press), and the Handbook of Photosynthesis and the Handbook of Turfgrass Management and Physiology also published by Taylor & Francis Group, CRC Press. He has written 13 book chapters, is an editorial board member of the Journal of Plant Nutrition and Communications in Soil Science and Plant Analysis and the Journal of Agricultural Technology, a member of the Book Review Committee of the Crop Science Society of America, and a reviewer for Crop Science, Agronomy, Soil Science Society of America, and HortScience. He is also the author or coauthor of nearly 90 journal articles. Dr. Pessarakli is an active member of the Agronomy Society of America, Crop Science Society of America, and Soil Science Society of America, among others. He is an executive board member of the American Association of the University Professors (AAUP), Arizona Chapter. He is a well-known, internationally recognized scientist and scholar and an esteemed member (invited) of Sterling Who’s Who, Marques Who’s Who, Strathmore Who’s Who, Madison Who’s Who, and Continental Who’s Who, as well as numerous honor societies (i.e., Phi Kappa Phi, Gamma Sigma Delta, Pi Lambda Theta, Alpha Alpha Chapter). He is a certified professional agronomist and a certified professional soil scientist (CPAg/SS) designated by the American Registry of the Certified Professionals in Agronomy, Crop Science, and Soil Science. He is also a United Nations consultant on agriculture for underdeveloped countries. His research work on environmental stress and his expertise on plants and crops is internationally recognized. For more information on Dr. Pessarakli, please visit http://ag.arizona.edu/pls/faculty/pessarakli.htm

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Contributors Leon H. Allen Jr. Chemistry Research Unit Center for Medical, Agricultural, and Veterinary Entomology U.S. Department of Agriculture Agricultural Research Service Gainesville, Florida Jeffrey A. Anderson Department of Horticulture and Landscape Architecture Oklahoma State University Stillwater, Oklahoma Raziuddin Ansari Institute of Sustainable Halophyte Utilisation University of Karachi Karachi, Pakistan E.L. Apostolova Institute of Biophysics Bulgarian Academy of Sciences Sofia, Bulgaria Diego G. Arias Facultad de Bioquímica y Ciencias Biológicas Laboratorio de Enzimología Molecular y Laboratorio de Bioquímica Microbiana Instituto de Agrobiotecnología del Litoral Consejo Nacional de Investigaciones Científicas y Técnicas Universidad Nacional del Litoral Ciudad Universitaria Santa Fe, Argentina F. Azam Nuclear Institute for Food and Agriculture (NIFA) Peshawar, Pakistan Christian Baldwin Jacklin Seed by Simplot Post Falls, Idaho

Giorgio M. Balestra Department of Plant Protection University of Tuscia Viterbo, Italy Shahzad M.A. Basra Department of Crop Physiology University of Agriculture Faisalabad, Pakistan Martine Bertrand National Institute for Marine Sciences and Techniques Conservatoire National des Arts et Metiers Cherbourg-Octeville, France Frank Bethea Department of Environmental Horticulture Clemson University Clemson, South Carolina Andrea Cavalieri Department of Crop Production University of Tuscia Viterbo, Italy A.G. Dobrikova Institute of Biophysics Bulgarian Academy of Sciences Sofia, Bulgaria Rama Shanker Dubey Faculty of Science Department of Biochemistry Banaras Hindu University Varanasi, India Olga Dymova Institute of Biology Russian Academy Sciences Syktyvkar, Komi Republic, Russia

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V.P. (Bill) Evangelou (Deceased) Department of Agronomy Iowa State University Ames, Iowa Miloš Faltus Department of Molecular Biology Crop Research Institute Prague, Czech Republic Muhammad Farooq Department of Agronomy University of Agriculture Faisalabad, Pakistan Shafqat Farooq Pakistan Atomic Energy Commission Islamabad, Pakistan Carlos M. Figueroa Facultad de Bioquímica y Ciencias Biológicas Laboratorio de Enzimología Molecular Instituto de Agrobiotecnología del Litoral Consejo Nacional de Investigaciones Científicas y Técnicas Universidad Nacional del Litoral Ciudad Universitaria Santa Fe, Argentina T.J. Flowers School of Life Sciences University of Sussex Brighton, United Kingdom Emmanuel Frossard Institute of Plant Sciences Eidgenössische Technische Hochschule Zürich Lindau, Switzerland Elena Garmash Institute of Biology Russian Academy of Sciences Syktyvkar, Komi Republic, Russia

Contributors

Tamara Golovko Institute of Biology Russian Academy Sciences Syktyvkar, Komi Republic, Russia Catherine M. Grieve U.S. Department of Agriculture Agricultural Research Service U.S. Salinity Laboratory Riverside, California Sergio A. Guerrero Facultad de Bioquímica y Ciencias Biológicas Laboratorio de Enzimología Molecular y Laboratorio de Bioquímica Microbiana Consejo Nacional de Investigaciones Científicas y Técnicas Universidad Nacional del Litoral Ciudad Universitaria Santa Fe, Argentina Antoine Harfouche Department of Forest Environment and Resources University of Tuscia Viterbo, Italy Bruria Heuer Institute of Soil, Water and Environmental Sciences Agricultural Research Organization Volcani Center Bet Dagan, Israel Asghar Heydari Plant Diseases Research Department Iranian Research Institute of Plant Protection Tehran, Iran

Alain J. Gaume Syngenta Crop Protection Stein, Switzerland

Bernhard Huchzermeyer Institute of Botany Leibniz Universitaet Hannover, Germany

N. Geissler Institute of Ecophysiology Justus-Liebig University Giessen, Germany

Ján Hudák Faculty of Natural Sciences Comenius University Bratislava, Slovak Republic

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Contributors

Saaimatul Huq Department of Biochemistry and Molecular Biology Saitama University Saitama, Japan Alberto A. Iglesias Facultad de Bioquímica y Ciencias Biológicas Laboratorio de Enzimología Molecular y Laboratorio de Bioquímica Microbiana Instituto de Agrobiotecnología del Litoral Consejo Nacional de Investigaciones Científicas y Técnicas Universidad Nacional del Litoral Ciudad Universitaria Santa Fe, Argentina Manabu Ishitani Agrobiodiversity Research Area International Center for Tropical Agriculture Cali, Colombia Shoaib Ismail International Centre for Biosaline Agriculture Dubai, United Arab Emirates Ambuj Bhushan Jha Faculty of Science Department of Biochemistry Banaras Hindu University Varanasi, India and Crop Development Centre Department of Plant Sciences College of Agriculture and Bioresources University of Saskatchewan Saskatoon, Saskatchewan, canada Yiwei Jiang Department of Agronomy Purdue University West Lafayette, Indiana Brian R. Jordan Faculty of Agriculture and Life Sciences Department of Wine, Food and Molecular Biosciences Centre for Viticulture and Oenology Lincoln University Christchurch, New Zealand

Arun Kumar Joshi Department of Genetics and Plant Breeding Institute of Agricultural Sciences Banaras Hindu University Varanasi, India and Centro Internacional de Mejoramiento de Maíz y Trigo Kathmandu, Nepal Klára Kosová Department of Genetics and Plant Breeding Crop Research Institute Prague, Czech Republic Hans-Werner Koyro Institute of Ecophysiology Justus-Liebig University Giessen, Germany Katarína Král’ová Faculty of Natural Sciences Institute of Chemistry Comenius University Bratislava, Slovak Republic Yves Lemoine Université Lille Nord de France Lille, France and Centre National de la Recherche Scientifique Laboratoire d’Océanologie et de Géosciences Université des Sciences et Technologies de Lille Villeneuve d’Ascq, France Haibo Liu Department of Environmental Horticulture Clemson University Clemson, South Carolina Annabé E. Louw-Gaume Institute of Plant Sciences Eidgenössische Technische Hochschule Zürich Lindau, Switzerland

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Nico Marcar Sustainable Ecosystems Division Commonwealth Scientific and Industrial Research Organisation Canberra, Australian Capital Territory Australia Yuliana K. Markovska Faculty of Biology University of Sofia Sofia, Bulgaria Elena Masarovicˇová Faculty of Natural Sciences Department of Soil Science Comenius University Bratislava, Slovak Republic Liliana T. Maslenkova Institute of Plant Physiology Bulgarian Academy of Sciences Sofia, Bulgaria Ildikó Matušíková Institute of Plant Genetics and Biotechnology Nitra, Slovak Republic Angelo Mazzaglia Department of Plant Protection University of Tuscia Viterbo, Italy Louis M. McDonald Division of Plant and Soil Sciences West Virginia University Morgantown, West Virginia Nick Menchyk Department of Environmental Horticulture Clemson University Clemson, South Carolina Iraj J. Misaghi Department of Plant Pathology University of Riverside Riverside, California

Contributors

Pascale Moulin Unité Mixte de Recherche Laboratoire de Biologie et Biotechnologies Marines Institut Français de Recherche pour l’exploitation de la Mer Université de Caen—Physiologie et Ecophysiologie des Mollusques Marins Esplanade de la Paix Caen, France Hitoshi Nakamoto Department of Biochemistry and Molecular Biology and Institute for Environmental Science and Technology Saitama University Saitama, Japan Flavia Navari-Izzo Dipartimento di Chimica e Biotecnologie Agrarie Università di Pisa Pisa, Italia Violeta N. Peeva Institute of Plant Physiology Bulgarian Academy of Sciences Sofia, Bulgaria Mohammad Pessarakli School of Plant Sciences The University of Arizona Tucson, Arizona Claudia V. Piattoni Facultad de Bioquímica y Ciencias Biológicas Laboratorio de Enzimología Molecular y Laboratorio de Bioquímica Microbiana Instituto de Agrobiotecnología del Litoral Consejo Nacional de Investigaciones Científicas y Técnicas Universidad Nacional del Litoral Ciudad Universitaria Santa Fe, Argentina

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Contributors

Florencio E. Podestá Facultad de Ciencias Bioquímicas y Farmacéuticas Centro de Estudios Fotosintéticos y Bioquímicos Consejo Nacional de Investigaciones Científicas y Técnicas Universidad Nacional de Rosario Rosario, Argentina Ilja Tom Prášil Department of Genetics and Plant Breeding Crop Research Institute Prague, Czech Republic Dagmar Procházková Institute of Experimental Botany Academy of Sciences of the Czech Republic Prague, Czech Republic Khalil ur Rahman Research Associate, Halophytes International Center for Biosaline Agriculture Dubai, United Arab Emirates Jagadish Rane Agrodiversity Research Area International Center for Tropical Agriculture Cali, Colombia Idupulapati M. Rao Agrodiversity Research Area International Center for Tropical Agriculture Cali, Colombia Nicoletta Rascio Dipartimento di Biologia Università di Padova Padova, Italia Ejaz Rasul Department of Biology Foreman Christian College Lahore, Pakistan Atif Riaz Institute of Horticultural Sciences University of Agriculture Faisalabad, Pakistan Ján Salaj Institute of Plant Genetics and Biotechnology Nitra, Slovak Republic

Uwe Schleiff Wolfenbuettel, Germany Benoît Schoefs Unité Mixte de Recherche Centre National de la Recherche Scientifique Le Centre de Microbiologie du Sol et de L’Environnement Institut National de la Recherche Agronomique Université de Bourgogne Plante Microbe Environnement Dijon, France R. Seenivasan School of Bio Sciences and Technology Vellore Institute of Technology University Vellore, Tamil Nadu, India František Šeršenˇ Faculty of Natural Sciences Institute of Chemistry Comenius University Bratislava, Slovak Republic Shabbir A. Shahid Salinity Management Scientist International Center for Biosaline Agriculture Dubai, United Arab Emirates Pallavi Sharma Faculty of Science Department of Biochemistry Banaras Hindu University Varanasi, India and Department of Plant Sciences College of Agriculture and Bioresources University of Saskatchewan Saskatoon, Saskatchewan, Canada Kadambot H.M. Siddique The UWA Institute of Agriculture The University of Western Australia Crawley, Western Australia, Australia R.K. Singh Plant Breeding Genetics & Biotechnology Division International Rice Research Institute (IRRI) Eastern and Southern Africa Regional Office Dar-Es-Salaam, Tanzania

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Contributors

Svetlana Skugoreva Institute of Biology Russian Academy of Sciences Syktyvkar, Komi Republic, Russia

Pavel Vítámvás Department of Genetics and Plant Breeding Crop Research Institute Prague, Czech Republic

L’udmila Slováková Faculty of Natural Sciences Comenius University Bratislava, Slovak Republic

Joseph C.V. Vu Chemistry Research Unit U.S. Department of Agriculture Agricultural Research Service Center for Medical, Agricultural, and Veterinary Entomology Gainesville, Florida

Katalin Solymosi Department of Plant Anatomy Institute of Biology Eötvös University Budapest, Hungary Stefano Speranza Department of Plant Protection University of Tuscia Viterbo, Italy Cornelia Spetea Division of Molecular Genetics Department of Physics, Chemistry and Biology Linköping University Linköping, Sweden Rafif K. Srour Division of Plant and Soil Sciences West Virginia University Morgantown, West Virginia Donald L. Suarez U.S. Department of Agriculture Agriculture Research Service U.S. Salinity Laboratory Riverside, California

Abdul Wahid Department of Botany University of Agriculture Faisalabad, Pakistan Ying Wang Department of Forestry and Natural Resources Purdue University West Lafayette, Indiana Nad’a Wilhelmová Institute of Experimental Botany Academy of Sciences of the Czech Republic Prague, Czech Republic Yanqi Wu Department of Plant and Soil Sciences Oklahoma State University Stillwater, Oklahoma Yakov Yatsco Institute of Biology Russian Academy Sciences Syktyvkar, Komi Republic, Russia

I. Szabolcs (Deceased) Research Institute for Soil Science and Agricultural Chemistry Hungarian Academy of Science Budapest, Hungary

Arunee Yuvaniyama Salinity Research and Development Section and Development Department Bangkok, Thailand

Galina Tabalenkova Institute of Biology Russian Academy Sciences Syktyvkar, Komi Republic, Russia

Jirˇ í Zámecˇník Department of Molecular Biology Crop Research Institute Prague, Czech Republic

Radomíra Vanková Laboratory of Hormonal Regulations in Plants Institute of Experimental Botany AS CR Prague, Czech Republic

Yuzeir Zeinalov Institute of Biophysics Bulgarian Academy of Sciences Sofia, Bulgaria

Part I Soil Salinity and Sodicity Problems

Salinity and Sodicity 1 Soil as Particular Plant/Crop Stress Factors Mohammad Pessarakli and I. Szabolcs† Contents 1.1 Introduction............................................................................................................................... 3 1.2 Significance of Soils in Respect of Crop Stress........................................................................ 4 1.3 Place and Role of the Soil in Nature.......................................................................................... 4 1.4 Extension and Global Distribution of Salt-Affected Soils.........................................................6 1.5 Development and Grouping of Salt-Affected Soils, Particular Plant/Crop Growth Stress Factors.............................................................................................................................7 1.6 Reclamation of Salt-Affected Soils, Relieve or Elimination of Particular Plant/Crop Stress Factors........................................................................................................................... 11 1.7 Concluding Remarks............................................................................................................... 15 References......................................................................................................................................... 15

1.1  Introduction Soil salinity and sodicity are among the major agricultural problems limiting plant growth and development throughout the world [2,6–8,10,12–18,22,23,26,29,30,32–35,40,42,48–52,54,59–63, 66,67,71–74,80,83–86,100,105–107,109,110,112,114,115,121–124,132–134,136–144,149–152]. Salinity and sodicity problems in agriculture have an ancient history, and presently have become a very cumbersome problem in agricultural and farming activities [153]. These problems are especially of great concern for countries that their economies rely to a great extent on agriculture. Salinity and sodicity problems are common in arid and semiarid regions, where rainfall is insufficient to leach salts and excess sodium ions out of the rhizosphere. In addition, these areas often have high evaporation rates, which can encourage increase in salt concentration at the soil surface. The arid and semiarid regions include almost one-third of the world’s land [80,108,114]. According to the Food and Agricultural Organization (FAO) [46] of the United Nations, total salt-affected area of the world has been estimated to be over 800 million ha. The presence of a cliche horizon and/or a cemented hardpan layer at varying depths plus insufficient precipitation for leaching often adds to the salt accumulation in these soils. Newly established irrigation projects, with improper planning and management practices, may also add salts to soils [88]. Soil salinity and sodicity problems are present in nearly every irrigated area of the world and also occur on nonirrigated croplands and rangelands. Thus, virtually no land is immune to salinization. Therefore, for sustaining life on earth, control of these problems and finding new ways to utilize these extensive saline and sodic soils and water resources, at least for agricultural purposes, are vital and urgent. Reclamation, or at least minimizing the effect of salinity and/or sodicity, is important †

Deceased.

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Handbook of Plant and Crop Stress

and necessary. In this respect, proper utilization of water for both plant growth and soil salinity and sodicity control is probably of the greatest importance. The main focus of this introductory chapter is to summarize general information on salt-affected (saline and sodic) soils, factors influencing their formation and reclamation, and discussing salinity and sodicity as plant/crop stress factors.

1.2  Significance of Soils in Respect of Crop Stress As far as all the crops are grown on soils, soil properties have substantial influence on the life conditions of plants and crops. In nature, usually particular plant species grow on specific soils. Thus, specific relationships exist between a particular soil and the vegetation cover of that specific soil. For example, Kreeb et al. [68] investigated soil and vegetation relationships associated with sodic–saline soil surfaces. Plant development and successful crop production require proper soil conditions, including adequate water and nutrient supply. Unfavorable soil conditions, environmental stress [2,31,130,135], salinity and/or sodicity [6–8,13,14,18,22,25,26,29,34,40,51,54,60,62,66,71,83,85,91–94, 96,97,115,124,132,133,136,137,139–142,148,149,151], and inadequate nutrient supply [98,145] have an adverse effect on the life of the plants, sometimes seriously hindering their effective production. Based on the above facts, we can speak of stress factors originating in the soil, i.e., such unfavorable soil conditions which cause, or contribute to, the stress factors plants and crops are exposed to. It is impossible to list all or most of such factors in a short introductory chapter. Therefore, the authors limit the range of this chapter to a general description of soil behavior and its function in nature and production as well as to an outline of one of the most serious factors originating salt-affected soils. For more in-depth information regarding salt-affected soils, the readers are referred to more comprehensive available sources [9,21,24,27,37,41,43,69,77–79,82,87,97,106,113,119,125,128,129,146,147].

1.3  Place and Role of the Soil in Nature It is generally accepted that the soil is a substantial part of the environment, comprising different substances and forming a special kind of ecosystem inside the given ecosystem, with various properties and attributes. It is also accepted that the soil of the continents is of high diversity, which is dealt with by several branches of soil science, e.g., taxonomy, classification, survey, mapping, etc. The soil, or the pedosphere, which is an environmental synonym of the soils of a given territory, has a specific place in nature. It is a natural body, similar to rocks, waters, or biota, in the sense that they too have their own materials, mass and energy fluxes, development, and regularities. This fact should be mentioned because, not only in newspapers but also in technical literature, soils are frequently treated either as living substances or as nonbiological substances. Neither of these approaches is correct, because one of the characteristics of the soil is its complexity, the fact that it contains both living and nonliving substances, forming due to both biotic and abiotic processes. The soil as a natural body is inseparable from the rocks and the crust of weathering on the surface of the continents from which it has developed, on the one hand, and from the biological processes on the other hand. The main characteristics that distinguish the soil from the rocks is the result of biological processes: the production of organic matters by the activities of microorganisms, plants, invertebrates, and other animals, and, finally, human beings, which transforms the rocks into soils, capable of supplying plants and crops with nutrients and water, and being an anchor for their establishment and stands on the land. The processes of soil formation started concurrently with the appearance of life on the continents and continued during the billions of years of interactions between living substances and rocks under the influence of climatic conditions, with particular regard to the action of water, vegetation cover, organism (both macro and micro), geomorphological patterns, and the time factor. As a result of their interactions, specific mass and energy fluxes formed the different soil types in various environmental conditions.

5

Soil Salinity and Sodicity as Particular Plant/Crop Stress Factors

Bio

h sp

sph e

re

Atm o

ere

Ecosystem

s ho L it

ph

er e

dr os ph e

re

Soil

y H

FIGURE 1.1  Schematic diagram of the interaction of lithosphere, atmosphere, biosphere, hydrosphere, ecosystems, and soils. (From Szabolcs, I., Salt-Affected Soils, CRC Press, Boca Raton, FL, 1989.)

With the appearance of the human race on the face of the earth, even changes in the environment became different. Due to human activities, the natural processes affected by biotic and abiotic factors accelerated, and several others that were unknown or minimal before developed. The role of soils in nature is complex and multisided, including biospheric, hydrospheric, and lithospheric functions. Their interaction is illustrated in Figure 1.1 [128]. Figure 1.1 clearly shows that the soil is a specific body related to the ecosystem. Even the word “soil” is very often used as a synonym of ecosystem when characterizing the given ecological conditions in a certain place. If we want to be precise, we must agree that the ecosystem includes the pedon, in other words, the soils. However, the soil includes different phases (solid, liquid, gaseous), living and nonliving substances, plants, animals, microbes, and has its own energy and material fluxes. Therefore, it can be considered an ecosystem in itself. In this respect, when speaking of soils versus their plant cover, we can consider the soils of a given location as the basis, ladder, and foothold, for instance, that in savannas or in the tropical belt, a well-defined plant cover develops and very often the soil properties promote or limit the living conditions of certain plant species or associations. Based upon the above considerations, it can be accepted that certain soil types, when discussed as the habitat for certain plant associations, are often named as the ecosystem of the plant association concerned, as the pedon includes, apart from the plants, most of the components of the ecosystem. Evidently, the soil, as a specific natural entity, is far from being identical with the vegetation, and, in spite of their close correlation, direct conversion between soil types and vegetation is hardly possible. Still there are soil types that, more or less, determine the ecological function for certain types of vegetation either by providing beneficial conditions for their development or by limiting the ecological conditions for other types of vegetation. This is perhaps best demonstrated in the case of salt-affected soils where high electrolyte contents of extreme pH conditions limit the development of the majority of plants and serve as a habitat only for such species that can survive or tolerate the unfavorable conditions caused by the salinity and sodicity of the soil. For example, the grass Leptochloa fusca that grows vigorously on the salt-affected soils can tolerate extremely saline and sodic (alkaline) conditions [69]. This species is also well adapted to the waterlogging encountered on saline and sodic (alkaline) soils. Saltgrass (Distichlis spicata) is another example of a highly salt-tolerant plant species that grows vigorously on saline and sodic soils [76,89–92,94–96]. In fact, the intensive investigations of the senior author

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of this chapter and his coworkers on this plant species have found that this grass performed better than control when some salt was added to it during its establishment period, and so far it has been the most salt- and drought-tolerant species compared to the other highly salt-tolerant halophytes that have been tested by this investigator [89,90,94]. Other investigators [39,68,97,111] have also reported on the soil and vegetation relationships that specific plant types are adapted and growing on specific habitats. In such respects, salt-affected soils can be considered as habitat or ecosystems for halophytes, and, if we agree on this, correlations can be found between the different types of salt-affected soils and their flora and fauna as components of the ecosystem. In order to cast light on both the theoretical and practical aspects of such considerations, it is necessary to describe briefly the properties and grouping of salt-affected soils with regard to the possibilities of the occurrence and distribution of halophytes and xerophytes developing on them.

1.4  Extension and Global Distribution of Salt-Affected Soils Nearly 10% of the total land surface is covered with different types of salt-affected soils. Table 1.1 demonstrates the distribution of salt-affected soils in the world [65]. Table 1.1 shows that no continent on our globe is free from salt-affected soils. They are distributed not only in deserts and semidesert TABLE 1.1 Salt-Affected Soils on the Continents and Subcontinents Continent North America Mexico and Central America South America Africa South Asia North and Central Asia South-East Asia Australasia Europe Total

FIGURE 1.2  Global distribution of the salt-affected soils.

Area (Millions ha) 15.7 2.0 129.2 80.5 87.6 211.7 20.0 357.3 50.8 954.8

Soil Salinity and Sodicity as Particular Plant/Crop Stress Factors

7

regions, but also frequently occur in fertile alluvial plains, river valleys, and coastal areas, close to densely populated areas and irrigation systems [27,37,41,45,46,78,87,108,112,128,129]. Figure 1.2 shows the distribution of salt-affected soils throughout the world [129].

1.5  Development and Grouping of Salt-Affected Soils, Particular Plant/Crop Growth Stress Factors In spite of the fact that the properties and attributes of salt-affected soils have been well known for a long time, it is appropriate to give a brief definition of this group of soils right at the start, because the salinity and sodicity (alkalinity) as well as the acidity of soils are substantial stress factors, seriously affecting the productivity of the land [2,7,8,10,12–18,22,23,25,26, 28–33,35,36,40,42, 48 –52,54,56,59 – 63,67,70 –74,80,83 –87,98 –10 0,105 –107,109,110,112,114,115,121–124, 128–130,132–133,143–145,148–150,153]. Salt-affected (i.e., saline, saline–sodic, and sodic) soils usually have low biological activity both because of osmotic and ionic effects of salts and due to limitation of carbonaceous substrates. Rao and Pathak [103] reported that microbial growth was depressed in sodic (alkali) soils due to, at least in part, limitation in carbon substrate (carbon stress), and in saline soils due to salt stress. For detailed information on the formation of salt-affected soils, the readers are referred to Szabolcs [128,129] and Pessarakli [87]. Salt-affected soils can be characterized as soils formed under the dominant influence of different salts in their solid or liquid phases, which will then have a decisive influence on the development, characteristics, physical, chemical, and biological properties, and eventually the fertility of the soil. Whenever and wherever this phenomenon occurs, it produces specific formations of soils where the high electrolyte concentration and its consequences overshadow the former soilforming processes or former soil properties and environmental conditions, often radically changing them. High electrolyte concentration is the only common feature of all salt-affected soils. Their chemistry, morphology, pH, and many other properties may be different, depending on the character of salinization and/or alkalization. Salt-affected soils, in the broader sense, can be divided into the following groups:

1. Saline soils that develop under the influence of electrolytes of sodium salts with nearly neutral reaction [dominantly sodium sulfate (Na2SO4), sodium chloride (NaCl), and seldom sodium nitrate (NaNO3)]. These soils occur mainly in arid and semiarid regions and form a major part of all the salt-affected soils of the world. High contents of soluble salts accumulated in these soils can significantly decrease their value and productivity. 2. Sodic (alkali) soils that develop under the influence of electrolytes capable of alkaline hydrolysis [mainly sodium carbonate (Na2CO3) and sodium bicarbonate (NaHCO3) and seldom sodium silicate (Na2SiO3) and sodium bisilicate (NaHSiO3)]. This group is well extended in practically all the climatic regions from the humid tropics to beyond the polar circles, and their total salt content is usually lower than that of saline soils, sometimes even strongly sodic (alkaline). Virgin sodic (alkali) soils have a high pH and high exchangeable sodium (Na) percentage (ESP) and are often barren. Sodic soils exhibit poor physical conditions that adversely influence water and air movement in the soils. Sodicity causes soil erodibility and impairs plant growth [82,87]. 3. Salt-affected soils that mostly develop owing to the presence of calcium sulfate (CaSO4) [gypsiferous soils] or, rarely, in the presence of calcium chloride (CaCl2). Gypsiferous soils can mainly be found in the arid and semiarid regions of North America, North Africa, the Near East, Middle East, and Far East, and also in Australia.

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4. Salt-affected soils that develop under the influence of magnesium salts. This group occurs in arid, semiarid, and even semi-humid regions, and has a particular significance, especially those soils that have a heavy texture. 5. Acid-sulfate soils whose salt content is composed mainly of Al2(SO4)3 and Fe2(SO4)3. This type of salt-affected soils is broadly extended in the tidal marsh areas along the seashores of all the continents. These soils are particularly common in North Europe, the western and eastern coastlines of Africa, along the coastline of South-East India, and develop on sulfurous marine sediments.

Inland acid-sulfate soils can also be found in different areas of the world, such as the western territories of the United States, Asia Minor, and China. Such soils develop as a result of fluvial glacial processes and have had no connection with seashores in recent geological times. Evidently, the different groups of salt-affected soils have diverse physicochemical and biological properties besides the one they have in common, i.e., a comparatively high electrolyte content. The grouping of the salt-affected soils and their properties causing plant and crop stress are presented in Table 1.2. The five groups in Table 1.2 represent the formations of different salt-affected soils described above, indicating their chemical types, the environmental conditions where they dominate or occur, the pattern of their main adverse effect on production, and the basic methods of their reclamation. For detailed information on formation and reclamation of salt-affected soils, see Szabolcs [128,129] and Pessarakli [87]. In Table 1.2, the adverse properties of different salt-affected soils causing crop stress are also included. From these, it is clear that, in various groups, different properties are responsible for hindering the development of plants and crops by causing stress. In saline soils, it is the high salt concentration in the solid and liquid phases that results in high osmotic pressure, hindering the normal development of plants, the stress factor is the salinity with TABLE 1.2 Grouping of Salt-Affected Soils and Their Properties Causing Plant and Crop Stress Types of Salt-Affected Soils Saline

Electrolyte(s) Causing Salinity and/or Sodicity

Environment

Sodium chloride and sulfate (in extreme cases nitrate) Sodium ions capable of alkaline hydrolysis

Arid and semiarid

Magnesium

Magnesium ions

Semiarid and semi-humid

Gypsiferous

Calcium ions (mainly CaSO4) Ferric and aluminum ions (mainly sulfates)

Semiarid and arid

Sodic

Acid sulfate

Semiarid, semi-humid, and humid

Seashores and lagoons with heavy, sulfate-containing sediments, diluvial inland slopes and depressions

Properties Causing Plant and Crop Stress

Methods for Reclamation

High osmotic pressure of soil solution, toxic effect of chlorides High (alkali) pH, poor water physical conditions

Removal of excess salt (leaching) Lowering of neutralizing the high pH by chemical amendments Chemical amendments, leaching Alkaline amendments Liming

Toxic effect, high osmotic pressure, Ca deficiency Low (acidic) pH toxic effect High acidity and the toxic effect of aluminum

Soil Salinity and Sodicity as Particular Plant/Crop Stress Factors

9

all its disadvantageous consequences of plant life. Apart from this, some compounds of the salt content of these soils, e.g., chlorides as toxic elements, also act as one of the stress factors. In sodic (alkali) soils, as a rule, not the high salt concentration but the sodic (alkaline) pH value is the stress factor, particularly in cases where there is a high concentration of sodium carbonate in the solid and liquid phases of the soil. The high pH hinders the life function of crops and limits their development. In another group of sodic (alkali) soils, which sometimes does not have very alkaline pH value (solonetz type), the comparatively low concentration of sodium salts capable of sodic (alkaline) hydrolysis constitutes a stress factor through its action, resulting in poor water physical properties in the soil. As a consequence of this phenomenon, the wilting point in the soil increases and the plants suffer from water deficiency, even in wet soils, due to the swelling of clay saturated with sodium ions (Na+). In magnesium soils, which have not been adequately studied, the combination of toxic effect, calcium deficiency, and poor soil physical properties are the stress factors. In gypsiferous soils, the acidic pH and sometimes the toxic effect of the high gypsum content, contribute to the appearance of stress factors for plant and crop life in areas with large extensions of intensively gypsiferous soils. In acid-sulfate soils, the very high acidity, with a pH sometimes below 2, poses stress with all the adverse effects of extreme acidity. Furthermore, the high aluminum content of the soil solution has an intensive toxic effect. Apart from this, the temporary or permanent waterlogging in such soils acts as a stress factor, hindering the normal air and nutrient regime, necessary for plant life, in these soils. Besides the salt-affected soils developing due to natural soil-forming processes, the so-called secondary salt-affected soils have an increasing importance that is both scientific and practical. Secondary salt-affected soils are those that have been salinized due to man-made factors, mainly as a consequence of improper methods of irrigation. The extension of secondary salt-affected soils is rather sizeable, and this adverse process is as old as irrigated agriculture itself. Old civilization in Mesopotamia, China, and Pre-Columbian America fell in consequence of the salinization of irrigated land. The process is also advancing vigorously at present and more than half of all the irrigated lands in the world are under the influence of secondary salinization and/or alkalization. When speaking of the man-made factors of salinization, we also have to mention potential saltaffected soils that are not salt-affected at present, but in case of the extension of irrigation, deforestation, overgrazing, and other man-made measures, can and will be salinized unless the necessary preventive procedures are undertaken in due time. No global records are available of the size of potential salt-affected soils; however, the area that they cover is larger than that of existing salt-affected soils. Secondary salt-affected soils can be divided into the following two categories: 1. Secondary formation of salt-affected soils caused by irrigation. In spite of the negative experiences, the salinization of irrigated and surrounding areas has not diminished. On the contrary, it is still on the increase. According to the estimates of the FAO and UNESCO (the United Nations’ Educational, Scientific and Cultural Organization), as much as half of all the existing irrigation systems of the world are, more or less, under the influence of secondary salinization, alkalization, and waterlogging. This phenomenon is very common not only in old irrigation systems but also in areas where irrigation has only recently begun. According to the estimates of the above mentioned agencies, 10 million ha of irrigated land are abandoned yearly because of the adverse effects of salinity due to irrigation, mainly secondary salinization and alkalinization. The mentioned losses and damages are not evenly distributed among the irrigating countries. In some of them, the damage may be relatively small, while in others it actually constitutes the major problem in agriculture or even in the national economy of the country in question. In this respect, unfortunately, there are countless sad examples.

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In Pakistan, Ahmad [4] carried out statistical analyses in respect of secondary salinized land. According to his data, out of 35 million ac (approximately 16 million ha) of total irrigated territory, salinized areas account for 5.3 million ac (approximately 2.4 million ha) after a few years of irrigation. He indicated among the causes of secondary salinization in Pakistan the joint effect of irrigation and ground water. According to Zavaleta [147], practically all irrigated alluvial soils in Peru show the features of salinity and sodicity (alkalinity). It is known from FAO reports [45,46] and the papers of Kovda [64] that more than 40% of irrigated soils in Iraq and Iran are affected by secondary salinization. A country report by FAO [45] on salinity in Syria estimated the adverse effects of salinity as follows: a. In more than 20,000 ha, salinity developed to a level where these soils had to be taken out of cultivation, and the loss is estimated at a total of 30,000 ton of cotton per year. b. In about 30,000 ha, the yield decreased by 50% and the total loss is estimated at 20,000 ton of cotton per year. c. In about 60,000 ha, the yield decreased by 20%, and the total loss is estimated at about 18,000 ton of cotton per year. At present, no continent is free from the occurrence of this very serious phenomenon. In Argentina, 50% of the 40,000 ha of land irrigated in the nineteenth century are now salinized. In Australia, secondary salinization and alkalization take place in the valley of the river Murray, and in Northern Victoria 80,000 ha have been affected. The same phenomena can be observed in Alberta, Canada, and similar processes have been recorded in the northern states of the United States, where irrigation was introduced much later than in the dry west. It is noteworthy that these last examples, and many other irrigated regions, are far from being arid areas and the majority of salt accumulations are associated with the sodium salts capable of sodic (alkaline) hydrolysis, and not with the neutral sodium salts that we are familiar with in desert and semidesert areas. The more recent reports of the FAO [46] of the United Nations estimated the saltaffected areas due to irrigation in the developing countries, including the above mentioned ones (Iran, Iraq, Pakistan, and Syria) much higher than the previous reports. 2. Secondary formation of salt-affected soils caused by human activities other than irrigation. When speaking of secondary salinization, most people think of irrigation and drainage. However, there are also other anthropogenic factors causing this adverse phenomenon. It is true that the majority of secondary salt-affected soils develop as a result of improper methods of irrigation, but there are other human effects that more and more often trigger this process in many places, both in arid and humid areas. Some of these anthropogenic processes are, including but not limited to, the following: a. Overgrazing This process occurs mainly in arid and semiarid regions, where the natural soil cover is poor and scarcely satisfies the fodder requirement of rather extensive animal husbandry. If, due to overgrazing, the natural vegetation is sparse or annihilated, progressive salinization develops and, step by step, the scarcity of the plant cover becomes increasingly pronounced. Sometimes, the process ends in desertification because even the poor pasture diminishes and no other fodder resources are available. According to Theunissen [131], the gradual decline in the ecological condition of natural pastures as a result of overgrazing and the application of insufficient management decisions, coupled with the detrimental effects of long-term drought, has left extensive areas of high potential grazing land in southern Africa in urgent need of restoration. However, due to the limited number of grasses currently available for rehabilitating and restoring the vast number of different habitats encountered, selecting indigenous grasses suitable for restoration of denuded areas in the arid and semiarid grasslands of Southern Africa was initiated.

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b. Deforestation in semi-humid and semiarid areas Particularly, in the last few decades, it has became evident that deforestation results in many tropical and subtropical countries in the salinization and alkalization of soils due to the effects of soil migration both in the upper and the lower layers. In South East India, e.g., vast territories of former forest land became intensely saline and sodic (alkaline) in a few years after the annihilation of the woods. Similar phenomena occurred in the forest steppe areas in Russia, Iran, East-Central Europe, and Latin America. c. Salinization caused by contamination with chemicals In spite of the fact that the amount of chemicals applied in agriculture is practically negligible, in comparison to the salt content of several soils, we have considered the fact that this kind of salinization more and more often occurs in modern intensive agricultural production, particularly in greenhouses and intensive farming systems. When production takes place in semi-closed systems (e.g., greenhouses), where the chemicals applied will not be removed regularly, the accumulation of salts or their components becomes possible in the upper layer of the soil, resulting in salinity and sodicity (alkalinity). In Japan, the Netherlands, and other countries with intensive agriculture, and particularly horticulture, such type of salinization more and more frequently appears, causing serious losses of crop yields. d. The accumulation of airborne or waterborne salts Due to the concentration of industrial plants, the emission of chemical compounds may accumulate in the soil and, if their concentration is high enough, they result in salt accumulation in the upper layer of the soil.    A similar phenomenon appears when, due to water system regulations, sludge water disposal, and other hydrotechnical measures, water with considerable salt concentration contaminates the upper soil layer, causing salinization and/or alkalization.

1.6  Reclamation of Salt-Affected Soils, Relieve or Elimination of Particular Plant/Crop Stress Factors The major environmental stresses caused by soil salinity and sodicity have existed long before the agricultural practices have started. Soil salinity and sodicity have a substantial effect in reducing agricultural production worldwide [2,6–8,10,12–20,22,23,26,29,30,32–35,40,42,48–52,54,59–63, 66,67,71–73,80,83–86,100,105,107,109,110,112,114,115,121–124,132–134,136–144,148–150,152]. This has a major impact on increased food and feed insecurity globally, particularly in developing countries that are more prone and vulnerable to salinization and desertification due to lack of advanced technology, adequate education, and other socioeconomical and technological problems. Population growth and increasing demand for food and agricultural products necessitate using the salt-affected soils and marginal lands for food production. These soils are needed for the agricultural extension and, hence, reclamation is required. Reclamation is needed on the millions of hectares of slowly permeable saltaffected (i.e., saline–sodic and sodic) soils throughout the world [2,46,55,63,87,112,124,150]. Different techniques of reclamation and preventive measures or management practices are used for reclamation of salt-affected soils and reducing the salt contents of the growth medium or to find more stress-tolerant plant/crop species and cultivars via genetic engineering to combat salinity stress. These management practices were aimed to enable plants to grow in saline and sodic conditions to utilize salt-affected soils for agricultural practices and food production [2,3,5,10,12, 15–20,23,28,30,32,33,35,38,42,44,47–49,52,56,59,61,63,69,72,80,81,84,86,87,99,100,105,107–110, 114,117–119,121–124,126,131,134,138,143,144,149,150,152]. Saline soils are usually reclaimed by leaching the salts out of the soil through irrigation and drainage systems, whereas reclamation of sodic (alkaline) soils requires application of chemical amendments followed by the leaching process.

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Present recommendations for reclamation of the salt-affected soils are usually based only on relatively simple and often empirical relations. Various amendments and management strategies have been used for reclamation of the salt-affected soils. To evaluate particular reclamation strategies, some specific considerations should be noted as follows:

1. The quantity of water needed 2. The quality of water needed 3. The quantity of amendments to be used 4. The type(s) of amendment(s) to be used 5. The time required for reclamation to be completed

Chemical reactions such as cation exchange, precipitation, and dissolution of solid phases (reclamation amendments) and the soil hydraulic properties and corresponding changes in the water flow and solute transport rates must be considered [119]. Among the various reclamation practices, usually, a combination of added gypsum amendment and crop rotation has been proven the best. Reclamations of salt-affected (saline–sodic and sodic) soils by chemical amendments has become cost-intensive and requires high capital investment, and are not always a practical solution to the problem of soil salinity and sodicity. Therefore, biotic approach such as cultivation of salinity- and ­sodicity-tolerant plants and crops on salt-affected soils, i.e., “saline agriculture,” may be another alternative. Cultivation of different salinity- and sodicity-tolerant plant types and species have been used by several investigators, i.e., grasses [44,62,69,75,76,89–91,93–97,99,131,140,148], agronomic crops [5,15,16,19,20,23,42,47,48,61,63,72,73,81,86,108,114,121–123,149,150], forest species, and trees [10,28,54,117,118,126] for reclamation purposes. These plants can mobilize the native lime (calcium carbonate, CaCO3) in these soils through root action, a substitute for the chemical approach. Qadir et al. [97], studying the combination of chemical amendments and biological (using plants) reclamation technique, reported that the soil treated with gypsum at a high rate (100% GR, grade reagent) removed the greatest amount of Na+ from the soil columns and resulted in a marked decrease in soil salinity (EC, electrical conductivity) and sodicity, sodium absorption ratio (SAR), and ESP (exchange sodium percentage). The performance of grass treatment in enhancing the leaching of Na+ was between the gypsum treatments. According to Kumar [69] and Qadir [97], the grass, Leptochloa fusca, was very useful and effective in the reclamation of salt-affected soils. This plant can tolerate extremely saline and sodic (alkaline) conditions. Since its growth is not affected by gypsum application, planting with Leptochloa is an alternative biological rather than a chemical method for the reclamation of sodic (alkaline) soils. This plant is also well adapted to the waterlogging encountered on saline and sodic (alkaline) soils. The plant improves the soil’s physical, chemical, and biological properties so that within 2 or 3 years many commercial and forage crops can be grown on the soil [69]. Leptochloa excretes salts through specialized glands and is, therefore, reasonably palatable to farm animals. It must be noted that because of its vigorous growth on sodic (alkaline) soils, Leptochloa does not allow satisfactory growth of companion plant species, especially in the initial years of soil reclamation. Subramaniam and Babu [126] also used a forest shrub species for reclamation of sodic soils. According to these investigators [126], Sophora mollis, which is a shrub to medium-sized tree and is used for both fodder and firewood, can be used in the reclamation of sodic (alkaline) soils. Kilic et al. [63] investigated the salt-removing capacity of purslane (Portulaca oleracea L.) by studying different stress criteria and by tracking its salt removal from germination to harvest. The results of their study showed that purslane could cumulatively remove considerable amounts of salt from the soil if practical to cultivate as an intercrop all year round. Saltgrass (Distichlis spicata) that has been found the only vegetation cover on a highly sodic (alkaline) soil in Wilcox Playa, Arizona [92] can also be very effective in reclamation of saline

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and sodic soils. As mentioned earlier, the senior author of this chapter and his coworkers found this grass to be very high salt-tolerant plant species that grows vigorously on saline and sodic soils [76,89–92,94–96]. Compared to the other highly salt-tolerant halophytes that have been tested by this investigator [89,90,94], so far, this grass has proven the most salt and drought tolerant of all the tested species. Although slow, definite improvement is achieved in the physicochemical properties of the saltaffected soils by encouraging the vegetation growth on such lands. The tree species in general are effective in improving the soil properties as reflected by the changes in physicochemical characteristics of the soil such as bulk density (BD), water holding capacity (WHC), hydraulic conductivity (HC) and pH, EC, OC (organic carbon), N (nitrogen) and exchangeable cations (Na+ and Ca++) [117]. Due to the low biological activity and depressed microbial growth of salt-affected (i.e., saline, saline–sodic, and sodic) soils, there is a need for applying organic amendments (i.e., plant residue or manure) during sodic (alkali) soil reclamation. In reclamation of saline soils, organic amendments must be applied following the leaching process. Kumar et al. [70] conducted a combination of biological and chemical reclamation study on a highly sodic (alkaline) soil. These investigators [70] found that rice produced satisfactory yields in the first year of gypsum application, but sorghum and Sesbania yields were very poor. The yield of Leptochloa was not affected by gypsum application. In their crop rotation practice, Kumar et al. [70] reported that the green forage yield of sorghum was greatest when sorghum followed Leptochloa grown for 2 years and the harvested grass was left to be decomposed on the site. In a biological reclamation study of saline soils, Helalia et al. [53] reported that amshot grass significantly reduced the soil salinity compared to either ponding or gypsum application, and this grass produced a higher fresh yield than clover cultivated in such soils. The above findings indicate that biological reclamation with the salinity- or sodicity-tolerant plants (i.e., Leptochloa, grasses, shrubs, or trees) is a proper substitute for chemical reclamation with gypsum, and the former has an economic advantage over the latter. Yildirim et al. [144] evaluated the effects of selected biological treatment on direct seeded and transplanted squash plant growth and mineral contents under salinity stress. These investigators reported that salinity negatively affected growth of squash; however, biological treatments significantly increased fresh weight compared to nontreated plants that were under salt stress. They also found biological treatments increased the uptake of potassium compared to the nontreated control in both direct seeded and transplanted squash. Based on their results, these investigators concluded that alteration of mineral uptake may be one mechanism for the alleviation of salt stress, and the use of biological treatments may provide a means of facilitating plant growth under salt stress conditions. Compost or any other organic materials is recommended to be used during the reclamation process of the salt-affected soils. The results of a field experiment conducted by Avnimelech et al. [21] verified that compost application improved both physical and chemical conditions of saline and sodic (alkaline) soils. Compost application to such soils is expected to release acids, which would ultimately lead to the replacement of exchangeable sodium by calcium. In addition, compost application would stabilize soil structure and enhance plant growth. These investigators [21] found that the municipal solid waste compost application was equivalent or even superior to the addition of gypsum, the most common amendment used to reclaim sodic (alkaline) soils. This was evident from the substantial increase in crop yields. The combined application of compost and gypsum raised yields to the levels equal to that of the commercial fields. In a field experiment, Batra et al. [24] compared the microbiological and chemical amelioration of a highly deteriorated sodic (alkaline) soil using two reclamation technologies:

1. Growing Karnal grass (Leptochloa fusca) as a first crop with no chemical amendment (biological reclamation) 2. Gypsum application as a chemical amendment for different crop rotations

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Handbook of Plant and Crop Stress

These investigators [24] reported that the microbiological properties changed more than the chemical properties of sodic (alkali) soil as the time period advanced. In a biological reclamation study carried out on saline soils, Apte and Thomas [11] found that a brackish water, nitrogen-fixing cyanobacterium, Anabaena torulosa, could successfully grow and fix nitrogen on moderately saline soils (EC of 5–8.5 dS m−1). These investigators [11] reported that cyanobacterium exhibited high rates of nitrogen fixation and substantially enriched the nitrogen status of saline soils. However, permanent removal of Na+ from saline soils using cyanobacteria or any other microorganisms may not be possible, since Na+ is released back into the soil subsequent to the death and decay of cyanobacteria or other microorganisms. Amelioration of soil salinity by simultaneous application of Anabaena torulosa during crop growth seems to be an attractive possibility for reclamation, especially since it can also supplement the nitrogen requirement of the crops growing on these soils. Blue-green algae that tolerate excess Na and grow extensively on the soil surface in wet seasons were found effective in sodic soils reclamation [102]. However, a permanent reclamation of such soils by using only blue-green algae as a biological amendment to achieve sodic (alkali) soil reclamation is neither possible nor comparable with an effective chemical amendment such as gypsum. In the reclamation process of the saline soils, de Villiers et al. [39] compared different annual and perennial species. Of the six species tested, the perennials seemed to be more effective and better suited for rehabilitation purposes under saline soil conditions. The type of chemical compound being used also influences the reclamation process of saltaffected soils. Sharma and Upadhyay [116] reported that, among the up-to-date known chemical compounds, cyclohexathiazenium chloride (S-6N-4)-2+Cl-2 is the best and the most suitable chemical to reclaim the sodic (alkaline) soil at any pH of the soil. When good quality water is not available for leaching the salts out of the soil, low-quality water can be used for the initial stages of reclamation. In this regard, Singh and Bajwa [120] studied the effects of gypsum and sodic irrigation on the precipitations of Ca++ and removal of Na+ from a sodic soil reclaimed with different levels of gypsum and growth of rice in a greenhouse experiment. Dubey and Mondal [43] also used low-quality saline water in conjunction with organic and inorganic amendments for initial stages of reclamation of sodic soils. Using low-quality water, Joshi and Dhir [58] evaluated the rehabilitation of degraded sodic soils using residual sodium carbonate water (low-quality water) combined with gypsum treatment and found that the combination treatment was effective in lowering the soil SAR (sodium absorption ratio) and improved water infiltration rate. In the first year of gypsum treatment, it was possible to establish the crop. In the second year, moderate productions of wheat (2610 kg ha−1) and raga (Brassica sp.) (2000 kg ha−1) were obtained [58]. Using the most common technique, irrigation water and drainage system, for reclamation of the salt-affected soils, the results of an investigation carried out by Millette et al. [77] demonstrated the ability of fall irrigation to leach salts from the surface soil during a period of low consumptive use, which could lead to reclamation. Long-term monitoring would be required to determine whether a further and permanent decline in salinity could be achieved. Concerning other reclamation materials and techniques, results of Jones et al. [57] indicate that acid whey is effective in reclaiming sodic soil by lowering ESP, SAR, and pH and by improving infiltration rate. Rao and Leeds Harrison [104] used simulation models for desalinization of a drained two-layered saline soil using surface irrigation for different water management practices to increase leaching efficiency. Based on image elements and their correlation with the ground features, Rao et al. [101] suggested categorizing sodic soils in moderately and strongly sodic groups. The delineation thus made would help the execution of a reclamation program for sodic soils at the study sites. Abdel-Hamid et al. [1] monitored soil salinity in the northern Nile delta Egypt by using data collected via landsat and geographic information system (GIS). The collected data were used in making recommendations for reclamation of the saline soils of the Nile delta area. The vast area of salt-affected soils still remains a burden for the societies, particularly the undeveloped countries, in need of adequate resources to reclaim them with the available technology

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involving initial heavy investments. The process of degradation, which has been due to reckless destruction of vegetation, can be reversed by reestablishment of vegetative cover, which results in slow but definite improvement in such soils. This phenomenon has been demonstrated a great deal by various parameters influencing the soil welfare in several investigations which show a positive sign of improvement both in terms of physical and chemical properties of the salt-affected soils. Such soils should, therefore, be brought under any type of vegetation (i.e., sod, shrub, tree) cover, if not found economical for regular farming and growing agronomic crops, and taken care by the community for posterity [117]. Even by the execution of the reclamation processes, nutrient status and their behavior in saltaffected soils (i.e., saline–sodic and sodic soils) during reclamation by crop rotation and chemical amendments requires a comprehensive assessment. This is because, usually, during the leaching process of the soluble salts and the exchangeable sodium, some soil nutrients are also lost and leached out of the soil. In this regard, several investigators [28,36,98,127,145] have studied nutrient status and behavior during the reclamation processes. Swarup et al. [127] reported the effect of gypsum on the behavior of soil phosphorus during the reclamation of a sodic soil. According to Bhojvaid et al. [28], soil nutrient status under the tree plantation was higher than that of the nonsodic farm soil. This finding confirms that successful tree plantation may restore the productivity and fertility of highly degraded sodic soils. Regardless, the techniques used in reclamation of salt-affected soils, post-reclamation management practices, i.e., proper choice of crops, crop rotation, method of irrigation, quality and quantity of water used for irrigation and reclamation, fertilization, and the economics of reclamation must be taken into consideration and followed to achieve successful results.

1.7  Concluding Remarks In this chapter, information has been given on the important functions of the soil in relation to soiloriginated stress factors for plant and crop growth and development as well as a little more detailed information of particular problems related to salt-affected soils, their formation and reclamation. The properties of the stress factors for plant and crop growth originating in soil are diverse and multisided. We know comparatively little about the up-to-date orientation and, particularly, for finding methods to improve the situation and ensure better plant and crop growth and development. Therefore, target-oriented studies of the different kinds of soil-originated stress factors for plant and crop growth and development are necessary so that the complex correlations and actions in the soil–plant–water system can be disclosed with the purpose of a better characterization of stress factors on the one hand, and improving the environmental and production conditions on the other hand.

References

1. Abdel-Hamid, M.A., D. Shrestha, and C. Valenzuela. 1992. Delineating mapping and monitoring of soil salinity in the northern Nile delta Egypt using landsat data and a geographic information system. Egyptian Journal of Soil Science, 32(3):463–481. 2. Adcock, D., A.M. McNeill, G.K. McDonald, and R.D. Armstrong. 2007. Subsoil constraints to crop production on neutral and alkaline soils in south-eastern Australia: A review of current knowledge and management strategies. Australian Journal of Experimental Agriculture, 47(11):1245–1261. 3. Afzal, I., S.M.A. Basra, A. Hameed, and M. Farooq. 2006. Physiological enhancements for alleviation of salt stress in wheat. Pakistan Journal of Botany, 38(5):1649–1659. 4. Ahmad, N. 1965. A review of salinity-alkalinity status of irrigated soils of West Pakistan. Agrokemia es Talajtan, 14(Suppl.):117–154. 5. Ahmad, S., J.D.H. Keatinge, A. Ali, and B.R. Khan. 1992. Selection of barley lines suitable for spring sowing in the arid highlands of Baluchistan. Sarhad Journal of Agriculture, 8(1):49–56. 6. Ahmadi, A., Y. Emam, and M. Pessarakli. 2009. Response of various cultivars of wheat and maize to salinity stress. Journal of Agriculture, Food, and Environment (JAFE), 7(1):123–128.

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138. Wahid, A., M. Perveen, S. Gelani, and S.M.A. Basra. 2007. Pretreatment of seed with H2O2 improves salt tolerance of wheat seedlings by alleviation of oxidative damage and expression of stress proteins. Journal of Plant Physiology, 164(3):283–294. 139. Wang, D. and M.C. Shannon. 1999. Emergence and seedling growth of soybean cultivars and maturity groups under salinity. Plant and Soil, 214(1–2):117–124. 140. Wang, D., J.A. Poss, T.J. Donovan, M.C. Shannon, and S.M. Lesch. 2002. Biophysical properties and biomass production of elephant grass under saline conditions. Journal of Arid Environments, 52(4):447–456. 141. Wilson, C. and J.J. Read. 2006. Effect of mixed-salt salinity on growth and ion relations of a barnyardgrass species. Journal of Plant Nutrition, 29(10):1741–1753. 142. Wilson, C., X. Liu, S.M. Lesch, and D.L. Suarez. 2006. Growth response of major U.S. cowpea cultivars I. Biomass accumulation and salt tolerance. HortScience, 41(1):225–230. 143. Winicov, I. and D.R. Bastola. 1999. Transgenic overexpression of the transcription factor Alfin1 enhances expression of the endogenous MsPRP2 gene in alfalfa and improves salinity tolerance of the plants. Plant Physiology, 120(2):473–480. 144. Yildirim, E., A.G. Taylor, and T.D. Spittler. 2006. Ameliorative effects of biological treatments on growth of squash plants under salt stress. Scientia Horticulturae, 111(1):1–6. 145. Yobterik, A.C. and V.R. Timmer. 1994. Nitrogen mineralization of agroforestry tree mulches under saline soil conditions. In: Advances in Geoecology, 27, Soil Erosion, Land Degradation and Social Transition (R.B. Bryan, ed.), pp. 181–194, Catena Verlag, Destedt, Germany. 146. Zahow, M.F. and C. Amrhein. 1992. Reclamation of a saline sodic soil using synthetic polymers and gypsum. Soil Science Society of America Journal, 56(4):1257–1260. 147. Zavaleta, G.G. 1965. The nature of saline and alkaline soils of the Peruvian coastal zone. Agrokemia es Talajtan, 14(Suppl.):415–425. 148. Zehra, A. and M.A. Khan. 2007. Comparative effect of NaCl and sea salt on germination of halophytic grass Phragmites karka at different temperature regimes. Pakistan Journal of Botany, 39(5):1681–1694. 149. Zhang, H.X., J.N. Hodson, J.P. Williams, and E. Blumwald. 2001. Engineering salt-tolerant Brassica plants: Characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation. Proceedings of the National Academy of Sciences of the United States of America, 98(22):12832–12836. 150. Zhang, Q.T., M. Inoue, K. Inosako, M. Irshad, K. Kondo, G.Y. Qiu, and S.P. Wang. 2008. Ameliorative effect of mulching on water use efficiency of Swiss chard and salt accumulation under saline irrigation. Journal of Food Agriculture and Environment, 6(3–4):480–485. 151. Zhao, G.Q., B.L. Ma, and C.Z. Ren. 2007. Growth, gas exchange, chlorophyll fluorescence, and ion content of naked oat in response to salinity. Crop Science, 47(1):123–131. 152. Zhu, H., G.H. Ding, K. Fang, F.G. Zhao, and P. Qin. 2006. New perspective on the mechanism of alleviating salt stress by spermidine in barley seedlings. Plant Growth Regulation, 49(2–3):147–156. 153. Zhu, J.K. 2001. Plant salt tolerance. Trends in Plant Sciences, 6:66–71.

Salinity Development, 2 Soil Classification, Assessment, and Management in Irrigated Agriculture Shabbir A. Shahid and Khalil ur Rahman Contents 2.1 Introduction.............................................................................................................................24 2.1.1 Salinity and Sodicity: A Global Scale Problem..........................................................24 2.1.2 Salinity and Sodicity...................................................................................................24 2.1.3 Causes of Salinity Development..................................................................................25 2.1.3.1 Salinity Development: Hypothetical Cycle...................................................26 2.1.3.2 Dryland Salinity Development.....................................................................26 2.1.4 Damage due to Salinity...............................................................................................26 2.1.5 Quick Facts about Salinity and Plant Growth............................................................. 27 2.1.6 Indicators of Soil Salinization..................................................................................... 27 2.1.7 Classes of Soil Salinity and Plant Growth.................................................................. 27 2.2 Classification of Salt-Affected Soils........................................................................................ 27 2.2.1 U.S. Salinity Laboratory Staff (Richards, 1954) Classification.................................. 27 2.2.2 FAO-UNESCO Classification (1974)..........................................................................28 2.2.3 USDA: Soil Survey Division Staff Classification (1993).............................................28 2.2.4 USDA-NRCS (Keys to Soil Taxonomy, 2010) Classification...................................... 28 2.2.5 Russian System of Salinity Classification...................................................................28 2.3 Salinity Assessment................................................................................................................. 29 2.3.1 Remote Sensing and Soil Salinity............................................................................... 29 2.3.2 Conventional Methods................................................................................................. 29 2.3.3 Modern Methods......................................................................................................... 30 2.4 Soil Salinity in Irrigated Fields and Relative Yield Prediction............................................... 31 2.5 Salinity Management and Reclamation................................................................................... 31 2.5.1 Physical Method.......................................................................................................... 31 2.5.2 Hydrological Method................................................................................................... 33 2.5.2.1 Modern Irrigation Methods.......................................................................... 34 2.5.3 Chemical Method........................................................................................................ 35 2.5.4 Biological Method....................................................................................................... 35 2.5.4.1 Serial Biological Concentration of Salts....................................................... 35 2.5.4.2 Biosaline Agriculture (Practicing Salt-Tolerant Crops)................................ 36 2.5.5 Alternatives for Using Marginal Saline Lands............................................................ 36

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2.6 Economic, Environmental, and Social Losses due to Soil Salinity........................................ 37 2.7 Research–Extension–Farmer Link.......................................................................................... 37 2.8 Summary................................................................................................................................. 37 References......................................................................................................................................... 38

2.1  Introduction Arid zones receive inadequate and irregular precipitation to accomplish leaching of salts originally present in the soil profile. Normally when the precipitation is more than 1000 mm per annum, salinity should not develop. This is not the case in arid zones; therefore, salts accumulate in soils. Salt buildup in concentrations detrimental to plant growth is a constant threat in irrigated crop production. In arid and semiarid regions, evapotranspiration is higher than the total annual rainfall. Therefore, rainfall contributes insignificantly to groundwater recharge, and hence there is a general shortage of fresh quality water to offset the total agriculture water demand in these countries. The shortage of fresh water necessitates the use of marginal quality ground water, such as brackish and saline, for irrigated agriculture. This is highly demanded in water-scarce regions. The improper use of saline/brackish water in irrigated agriculture often introduces salinity and sodicity problems and the soil if not properly managed can reach a condition where it cannot be exploited to its full production capacity. Under such conditions, irrigated agriculture has faced the challenge of sustaining its productivity for centuries, particularly soil and water salinity, poor irrigation, and drainage management continue to plague agriculture especially in arid and semiarid regions (Tanji, 1996). If soil becomes saline and sodic, it creates plant- and soil-related problems that restrict plant growth through undermining soil quality, and hence many plants either fail to grow in saline soils or their growth is retarded significantly; however, few plants grow well on saline soils (Maas, 1990). Therefore, soil salinity often restricts options for cropping in a given area. Because of this reason, understanding salinity in irrigated agriculture fields is essential for their precise management. Salinity management is highly site specific and depends on factors such as site characteristics, nature of soils, and local hydrological conditions. Soil salinity and sodicity are global issues and not restricted to one country or region. Once the soil salinity and sodicity are diagnosed and site characteristics are established, ­integrated management and reclamation strategies specific to the site can be formulated for better results and long-time sustainability of irrigated agriculture.

2.1.1  Salinity and Sodicity: A Global Scale Problem Planet Earth consists of land surface of about 13.2 × 109 ha, out of which only 7 × 109 ha are arable and only 1.5 × 109 ha are cultivated (Massoud, 1981). Of the cultivated lands, about 0.34 × 109 ha (23%) are saline and another 0.56 × 109 (37%) are sodic. Older estimates (Szabolcs, 1989) suggest 10% of the total arable land to be affected by salinity and sodicity, and extends over more than 100 countries and almost all continents.

2.1.2  Salinity and Sodicity Some people get confused between salinity and sodicity. Salinity is a measure of the concentration of all the soluble salts in soil or water. It is expressed as decisiemens per meter (dS m−1) or millisiemens per centimeter (mS cm−1). If we want to keep our soils productive, we need to identify potential salinity problems and be ready with remedies or actions to help reduce the effects or avoid them in the first place. Sodicity is a measure of sodium ions in soil or water relative to calcium and magnesium ions (Richards, 1954). It is expressed either as the sodium adsorption ratio (SAR) or as

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the exchangeable sodium percentage (ESP). If the SAR of the soil equals or is greater than 13 or ESP equals or is greater than 15, the soil is termed sodic (Richards, 1954).

2.1.3  Causes of Salinity Development There may be a number of causes (Plate 2.1) of soil salinity development: (1) inherent soil salinity (parent material); (2) seawater intrusion to coastal areas; (3) uses of brackish/saline water in farming and urban landscapes area; (4) restricted drainage developed into a high water table; (5) low rainfall; and (6) high rate of evapotranspiration. The rainfall contributes 10–200 kg salts per year per ha, depending on the vicinity of the area to the sea or inland. In farming areas, the continuous pumping of ground water and subsequent use for irrigation purposes (recycling) usually lowers the water table; however, this practice resulted in an increase in water salinity and covert normal soils to become saline with low productivity. These soils need attention for their management and reclamation. Pumping groundwater to alleviate surface salinity and to lower water table is an effective way, with the condition that ground brackish water is not used directly for irrigation, but with some management, e.g., conjunctive or cyclic use. In areas where water table is high and persistent, the imbalance in the natural water, the clearing of vegetation and the general absence of deep rooted trees, and the absence of adequate drainage cause soil salinity. As the groundwater rises, it brings salt to the surface through capillary rise and subsequent evaporation, which can be harmful to plants by reducing yields. The quality of the groundwater used for irrigation and its rates of recharge are critical too. These considerations make land-water management in irrigated arid lands a delicate task. Subject to these limitations, irrigation helps reduce risks linked to soil moisture stress and enhances yield.

(a)

(b)

(c)

(d)

Plate 2.1  Soil salinization, waterlogging, and plant growth. (a) Patchy salinity in wheat field. (b) Salt accumulation through sea water intrusion—salt flat. (c) Affect of salinity on date palm trees. (d) Waterlogging in forestry field.

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The groundwater usually rises 0.6–1.5 m or more in the soil above the water table by capillarity, depending upon texture, structure, and other factors. The water reaching the surface evaporates, leaving a salt-deposit typical of saline soils. Generally, water table below 2 m is considered safe for irrigated agriculture. 2.1.3.1  Salinity Development: Hypothetical Cycle Recently, Shahid et al. (2010) have published a hypothetical salinization development cycle in irrigated agriculture fields (secondary salinization). Figure 2.1 depicts poor irrigation and drainage management and high temperature as the main causes of secondary salinization. 2.1.3.2  Dryland Salinity Development In Australia, dryland salinity is very common and is developed due to clearing of trees to convert the area to arable agriculture. In the former, the rainfall is compensated through high evapotranspotration with no or insignificant leaching; in the latter case, low evapotranspiration relative to former lead the extra rain to leach down and with poor drainage condition, water table developed and subsequent evaporation caused dryland soil salinization.

2.1.4  Damage due to Salinity An exact estimate of losses caused due to salinity in an area is difficult to know; however, it is clear that losses may be quite considerable, and high cost of work to be done to control salinity must also be added. Different types of salinity damage are; saline water tables can cause productive land to become barren; soil salinity also enhances “erosion” and loss of farm income; salinity can deteriorate the quality of drinking water; in salt-affected areas, roads and building foundations are weakened by high salty water tables and high water table also affects biological activity in the soil.

Salt water irrigation

Ionic imbalance

Soil impermeability due to high Na

Salt accumulation

Leaching Seepage from the system

Needs more water

Water table rise

Ionic imbalance

Restriction to leaching

Capillary rise and evaporation

Figure 2.1  Hypothetical salinization cycle. (From Shahid, S.A. et al., Mapping and monitoring of soil salinization, remote sensing, GIS, modeling, electromagnetic induction and conventional methods—case studies, in Proceedings of International Conference on Management of Soil and Groundwater Salinization in Arid Regions, Sultan Qaboos University, Sultanate of Oman, January 11–14, 2010. Volume 1: Keynote papers and Abstracts, pp. 59–97.)

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2.1.5  Quick Facts about Salinity and Plant Growth Proper plant selection is one way to moderate yield reductions caused by excessive soil salinity. The stage of plant growth has a direct bearing on salt tolerance. Generally, the more mature the plant, the more tolerant it is to salt. Most fruit trees are more sensitive to salt than are vegetable, field, and forage crops and generally, vegetable crops are more sensitive to salt than are field and forage crops.

2.1.6  Indicators of Soil Salinization Once soil salinity is developed in irrigated agriculture fields, it starts showing its effects on soil properties and plant growth. The white salt crust, reduced plant vigor, salt stain on dry soil surface, affected area worsen after rainfall, marked changes in leaf color and shape, and presence of naturally growing halophytes and trees are either dead or dying are the indicators of soil salinity, which can be observed in the field without laboratory analyses.

2.1.7  Classes of Soil Salinity and Plant Growth Electrical conductivity of the soil saturation extract (ECe) is the standard measure of salinity. Richards (1954) has described general relationship of ECe and plant growth. Class 0 Nonsaline 1 Very slightly saline 2 Slightly saline 3 Moderately saline 4 Strongly saline

ECe (dS m−1)

Plant Growth

0–2 2–4 4–8 8–16 >16

Salinity effects mostly negligible Yields of very sensitive crops may be restricted Yields of many crops restricted Only tolerant crops yield satisfactory Only a few very salt-tolerant crops yield satisfactory

2.2  Classification of Salt-Affected Soils A soil, which contains sufficient soluble salts in the root-zone to impair the growth of crop plants, is defined as “saline.” However, because salt injury depends on species, variety, growth stage, environmental factors, and nature of the salts, it is very difficult to define a saline soil precisely. The definitions are based on salt content either alone or in conjunction with texture, morphology, or hydrology (Richards, 1954; Northcote and Skene, 1972; FAO-UNESCO, 1974; Soil Science Society of America, 1978). The most widely accepted definition of a saline soil is one that gives an electrical conductivity of extract from saturated soil paste (ECe exceeding 4 dS m−1 at 25°C), while FAOUNESCO (1974) mapped soils with ECe exceeding 15 dS m−1 as strongly saline soils or solonchaks.

2.2.1  U.S. Salinity Laboratory Staff (Richards, 1954) Classification The term “salt-affected” soil is being used more commonly to include saline, saline-sodic, and sodic soils, which are clearly differentiated by Richards (1954). The term “alkali” to describe soils with excess exchangeable sodium (ES) is being discouraged due to its ambiguity (Overstreet et al., 1951). Saline soils are those which have pHs usually less than 8.5, ECe > 4 dS m−1 and ESP < 15. The high ECe with low ESP tends to flocculate soil particles into aggregates. The soils are usually recognized by the presence of white salt-crust during some part of the year. Permeability is either greater or equal to those of similar normal soils. Saline-sodic soils contain sufficient soluble salts (ECe > 4 dS m−1) to interfere with the growth of most crop plants and sufficient ESP (>15) to affect the soil properties and plant growth adversely by the degradation of soil structure. The pHs may be less or more than 8.5.

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Sodic soils contain ESP >15 and ECe < 4 dS m−1 and pHs generally range between 8.5 and 10 and may even be as high as 11. The low ECe and high ESP tends to deflocculate soil aggregates and hence lower their permeability.

2.2.2  FAO-UNESCO Classification (1974) Salt-affected soils (halomorphic soils) are also indicated on the soil map of the world (1:5,000,000) by FAO-UNESCO (1974) as solonchaks and solonetz. The origin of both solonchak and solonetz are Russian. Solonchaks are soils with high salinity (ECe > 15 dS m−1) within 125 cm of the soil surface. The FAO-UNESCO (1974) divided solonchaks into four mapping units: (1) orthic solonchaks—the most common solonchaks; (2) gleyic solonchaks—with groundwater influencing the upper 50 cm; (3) takyric solonchaks—solonchaks in cracking clay soils and; (4) mollic solonchaks—solonchaks with dark colored surface layer, often high in organic matter. Soils with a lower salinity than solonchaks, but higher than 4 dS m−1 are mapped as “saline phase” of other soil units. A “solonetz” is a sodium-rich soil that has an ESP > 15. The solonetz are subdivided into three mapping units: (1) orthic solonetz—the most common solonetz; (2) gleyic solonetz—those soils with groundwater influence in the upper 50 cm, and (3) mollic solonetz—the soils with a dark colored surface layer, often high in organic matter. Soils with a lower ESP than a solonetz, but higher than 6, are mapped as a “sodic phase” of other soil units.

2.2.3  USDA: Soil Survey Division Staff Classification (1993) The following classes of salinity are used if the EC has not been measured, but salinity is inferred (Soil Survey Division Staff, 1993). These are: class 0 (nonsaline, 0–2 dS m−1); class 1 (very slightly saline, 2–4 dS m−1), class 2 (slightly saline, 4–8 dS m−1), class 3 (moderately saline, 8–16 dS m−1), and class 4 (strongly saline ≥ 16 dS m−1). The class 0 shows no visible salts on the soil surface and plant growth is not affected by salinity/sodicity. In classes 1 and 2, the plant growth may be uneven or patchy. Salts are generally present in small-sized patches (Plate 2.1a), which do not cover more than 25% area collectively. In class 3, the plant growth on these soils is very patchy and the salts are fairly visible on the soil surface. The area in class 4 lies unused and may support some salt-tolerant plants.

2.2.4  USDA-NRCS (Keys to Soil Taxonomy, 2010) Classification The Keys to Soil Taxonomy (USDA-NRCS, 2010) system of classification has hierarchies of groups of soils (taxa). In this system, the true salt-affected soils belong to the order “Aridisols” and suborder salids. At the third level of classification, there are great groups named Natrargids, which are argids with a high ESP and are equivalent to solonetz on the soil map of the world (FAO-UNESCO, 1974). At this third level (great group) of classification, there are also the aquisalids (Plate 2.2a—salids with water table within 1 m from soil surface) and haplosalids (Plate 2.2b—where water table is below 1 m or even deeper) in the suborder of salids, which are equivalent to solonchaks on the soil map of the world.

2.2.5  Russian System of Salinity Classification In the Russian classification, the solonchaks may be “external solonchaks” with the soluble salts throughout the whole soil or internal solonchaks with soluble salts in the subsoil or substratum only. The solonchaks are subdivided according to the composition of salts. The following types have been recognized: nitrate, nitrate-chloride, chloride, chloride-sulfate, sulfate-chloride, sulfate-soda, soda, and borate solonchaks. The external solonchaks are of different types (flooded, puffed, sabkha),

Soil Salinity Development, Classification, Assessment, and Management

(a)

29

(b)

Plate 2.2  Soil salinity classes (USDA-NRCS): (a) aquisalids—water table at 40 cm and (b) haplosalids— water table at 140 cm.

sometimes the subdivision is made according to the origin of the salt, e.g., closed basin, marine, allochthonous air blown, and anthropic.

2.3  Salinity Assessment Accurate measurement is essential to understand soil salinity problem for better management, to improve crop yield and to maintain root zone soil health. If the salinity could be measured, it could be managed. A reliable salinity assessment method is required. The choice of the method, however, depends on purpose, size of the area, depth of soil to be assessed, number and frequency of measurement, accuracy required, and available resources. There are a number of soil salinity assessment tools, such as salinity monitor maps prepared over a period of time to assess present salinity problem and to predict future salinity risk to the area, salinity indicators on soil surface, vegetative indicators, conventional salinity tests (EC 1:1 or 1:5; ECe) and modern methods (Geophysical—EM38; salinity sensors).

2.3.1  Remote Sensing and Soil Salinity Remote sensing acquires information about the Earth’s surface without actually being in contact with it. The fundamentals of remote sensing in soil salinity assessment and examples of such studies from the Middle East, Kuwait, Abu Dhabi Emirate, and Australia have been described recently by Shahid et al. (2010). The remote sensing imagery picks surface reflection and provides general salinity information of the area; however, it lacks information about root zone salinity, which requires other conventional (EC meters) and modern methods (EMI and salinity probes) to be used. The combination of salinity maps taken over period of time and digital elevation model (DEM) help predict salinity risk in the area (Furby et al., 1995, 1998).

2.3.2  Conventional Methods Soil salinity measurement is made on georeferenced (using GPS) field sampling, and laboratory analysis of extract from saturated soil paste (Plate 2.3a) by EC meter is accepted as the standard way of soil salinity assessment, expressed as desisiemens per meter (dS m−1) or millisiemens per centimeter (mS cm−1). This is due to the amount of water that a soil holds at saturation, is related to soil texture, surface area, clay content, and cation-exchange capacity. The lower soil:water ratios (1:1, 1:2, 1:5) are also used in many laboratories; however, the results require calibration with ECe to select salt-tolerant crops.

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(a)

(b)

(c)

(d)

(e)

(f )

Plate 2.3  Salinity assessment methods: (a) saturation extract collection, (b) salinity surveys by EM38, (c)  activity meter and probe, (d) placing sensor in the root zone, (e) buried sensor and smart interface and (f) instant viewing of EC on smart datalogger.

2.3.3  Modern Methods The salinity assessment and management at farm level help farmers improve crop productivity. The conventional field sampling and laboratory analysis is a tedious, expensive, and time-consuming process. Other quicker and modern methods can be used in the field salinity mapping, such as electromagnetic induction (EMI-EM38) and activity meter with salinity probe. The EM38 (Plate 2.3b) is most commonly used in agricultural surveys and for rapid assessment of the soil’s apparent electrical conductivity (ECa) in millisiemens per meter (mS m−1). The EM38 has transmitter and receiving coils. The transmitter coil induces an electrical current into the soil and the receiving coil records the resulting electromagnetic field. The EM38 provides a maximum of 1.5 and 0.75 m depth of exploration in vertical and horizontal dipole modes, respectively. EC mapping is one of the simplest, least expensive salinity measurement tools. Integration of GIS with salinity data results in salinity maps and help farmers interpret yield variations and in understanding subtle salinity differences across agricultural fields, allowing them to develop more precise management zones and, ultimately, potentially higher yields. Activity meter with salinity probe (Plate 2.3c) is handy equipment and gives instant apparent electrical conductivity (ECa) information in mS cm−1 and g L−1. The German-made PNT3000 COMBI + model is commonly used in agriculture, horticulture, and landscape sites for rapid salinity assessment and monitoring. It provides an extended EC-measuring range from 0 to 20 mS cm−1 and from 20 to 200 mS cm−1. The unit includes stainless steel measuring electrode 250 mm long for direct soil salinity measurements; EC-plastic probe with platinum-plated ring sensors and highquality aluminum-carrying case. The operation is convenient and simple; only one button makes the full operation possible. It is essential to validate ECa values with ECe from same fields. In both cases, the ECa must be correlated to ECe for crop salt tolerance. The most modern salinity logging system (Plate 2.3d through f) is real-time dynamic automated salinity logging system (RTASLS). In this system, ceramic sensors are buried in the rootzone where salinity monitoring is required. Each salinity sensor is fitted with an external smart interface that consists of an integrated microprocessor containing all the required information to

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allow autonomous operation of the sensor, including power requirements and logging interval. The smart interface resolution is 16 bit, offering highly precise and accurate recording of the salinity sensor. The smart interface is connected to DataBus, which leads to Smart Datalogger. The Smart Datalogger searches the DataBus and automatically identifies the number of salinity sensors connected and begin logging them at the predetermined intervals. Instantaneous readings from sensors can be viewed on the logger’s display directly in the field without the need for a laptop. Data can also be accessed in the field by memory stick or remotely using a mobile phone modem. This data is then available for graphing and interpretation in Excel (Shahid et al., 2009a).

2.4  Soil Salinity in Irrigated Fields and Relative Yield Prediction Crops can tolerate salinity up to certain levels without a measurable loss in yield (this is called threshold level). As a general rule, the more the salt tolerant the crop, the higher the threshold level. At salinity levels greater than the threshold, crop yield reduces linearly as salinity increases. Using the salinity values in a salinity/yield model developed by Maas and Hoffman (1977), predictions of expected yield loss can be made. Maas and Hoffman expressed salt tolerance of crops by the following relationship:

Yr = 100 − s(ECe − t)

where Yr is the percentage of the yield of crop grown in saline conditions relative to that obtained on nonsaline conditions t is the threshold salinity level where yield decrease begins s is the percent yield loss per increase of 1 ECe (dS m−1) in excess of t Salinity mapping at the farm level and Table 2.1 may be used as a guide to predict yield losses.

2.5  Salinity Management and Reclamation It is essential to keep the plant root zone salinity below crop threshold level to get higher production and to maintain soil health. This requires careful management and reclamation of irrigated agricultural fields. The main objectives of management and reclamation should be to bring more soils under cultivation, to increase the yield per unit area, and to increase the water and fertilizer use efficiency, and to improve livelihood of the farmers. Efficient, effective, and long-term reclamation of saline soils require the lands to be well leveled before leaching is initiated, additional supply of good quality water is required and good subsurface drainage is essential. The physical, hydraulic, chemical, and biological techniques are the methods of soil reclamation.

2.5.1  Physical Method Physical method includes land leveling, salts scraping, deep ploughing and tillage, subsoiling and sanding. In order to remove salts through leaching or flushing, leveling (preferable laser leveling) is a prerequisite to allow uniform distribution of water. The objective is to leach the salts or flush from the surface if a near surface restrictive layer is present. The leveling process may compact the soil due to heavy machinery used, subsoiling or chiseling should follow this practice. In certain cases, salt crusts formed at surface can be removed by mechanical means. In small agricultural farms, salt scraping is the simplest and most economic way of reclaiming saline soils. Scraping can minimize

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Table 2.1 General Threshold (t) and Slope (s) Values to Calculate Crop Yield as a Function of Soil Salinity for Various Crops Crops Alfalfa (Medicago sativa) Barley for grain (Hordeum vulgare) Bean (Phaseolus vulgaris) Bean, dry edible (Phaseolud vulgaris) Cabbage (Brassica oleracea) Carrot (Daucus carota) Clover (Trifolium spp.) Corn for grain (Zea mays) Corn for silage (Zea mays) Cucumber (Cucumis sativus) Date (Phoenix dactylifera) Lettuce (Latuca sativa) Onion (Allium cepa) Pepper (Capsicum annum) Potato (Salanum tuberosum) Radish (Raphanus sativus) Sorghum for grain (Sorghum bicolor) Soybean (Glycine max) Spinach (Spinacia oleracea) Sugar beet (Beta vulgaris) Tomato (Lycopersicum esculentum) Wheat for grain (Triticum aestivum)

Threshold (t) ECe (dS m−1) 2.0 8.0 1.0 1.0 1.8 1.0 1.5 1.7 1.8 2.5 4.0 1.3 1.2 1.5 1.7 1.2 6.8 5.0 2.0 7.0 2.5 6.0

Slope (s) % Yield Loss per 1 ECe (dS m−1) above (t) 7.3 5.0 18.9 19.0 9.7 14.1 12.0 12.0 7.4 13.0 3.6 13.0 16.1 14.1 12.0 13.0 16.0 20.0 7.6 5.9 9.9 7.1

Source: Hoffman, G.J., Water quality criteria for irrigation. Biological System Engineering University of Nebraska, Institute of Agricultural and Natural Resources. Publication No. EC 97–782, 2001. Notes: s, % yield loss per 1 ECe (dS m−1) increase above t (ECe) value; t, salinity threshold ECe (dS m−1), where yield is optimum.

the salts temporarily; however, they can reappear with a continuous feed of ground water to the surface. Low salinity in the rootzone can be achieved through tillage practices by manipulating the soil surface condition, i.e., bed shape and irrigation management (Plate 2.4a). It is very well recognized that salts tend to accumulate on the ridges top away from the wet ridge shoulder (Plate 2.4b) when furrow irrigation is adopted. Placing the seeds on off-center slope of the single row will put the seed (Plate 2.4a) in minimum salinity and optimum moisture condition. Under high salinity, the alternate row should be left unirrigated; this will ensure maximum accumulation of salts in the unirrigated area and leave the irrigated furrows free of salts and fit for planting seeds (Plate 2.4b and c). Subsoiling is particularly important for disrupting the dense layers (Plate 2.4d) at depth to enhance permeability (Shahid et al., 2009b). In the absence of subsoiling, flushing should be preferred over leaching; the latter compounds the salinity problem in the root zone due to the dense layer. Subsoiling is important while reclaiming sodic soils after the addition of a suitable amendment such as gypsum and watering the field. If the soil surface to be reclaimed is very heavy textured, mixing of sand, “sanding,” to the surface can change the texture permanently, and the soils become more permeable and easy to reclaim. This practice also provides a favorable environment for plant growth compared to the original soil without sanding.

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(a)

(b)

(c)

(d)

33

Plate 2.4  Seed bed, salinity development, and plant growth: (a) Seed placement on furrow shoulder, (b) salt accumulation on ridge tops, (c) barley plants growing on ridge shoulder, and (d) dense layer need subsoiling.

2.5.2  Hydrological Method Hydrological method is concerned with water use and drainage. In irrigated agriculture, the objective is to free the root zone from salts through leaching to lower depths and the subsequent drainage and surface flushing of dissolved salts. The rootzone salinity may increase if the net downward movement of salts is less than the salt input from irrigation, and salt water flux to surface. Therefore, salt balance must be kept under control, and this is a function of irrigation water salinity and to the success of drainage system. Traditionally, saline soils have been reclaimed by flooding or by ponding water. In general, the depth of soil leached is roughly equal to the depth of water infiltrated during leaching. In order to leach the salts, the leaching requirement (LR) is very important. The LR is the calculated fraction (depth) or quantity of water that must pass through the rootzone to maintain the EC of the drainage water at or below some specified level. The recent trend is to minimize this LR in order to prevent raising the groundwater and minimize the load to drainage system (Mashali, 1995). Methods of LR calculation and to predict the losses in yield due to salinity are described by Rhoades (1992). Timely leaching is important to assure root zone salinity is not exceeded above crop salinity tolerance limit for extended periods of time or critical stage of plant growth. In normal conditions, leaching can be accomplished at each irrigation; however, in soils with low infiltration rate and for crops sensitive to excess moisture in the rootzone, leaching at each irrigation may not be appropriate. Leaching can be done when soil moisture is low and water table is deep; it should precede the critical growing stage; at low evapotranspiration demand; at night, during high humidity, in cooler weather and; at the end of cropping season, as appropriate to area. The drainage lowers water table, provides adequate leaching, minimizes upward water flux, and thus controls salinity buildup. Provided the subsurface is permeable and relief is adequate, natural

34

Handbook of Plant and Crop Stress

drainage may work; however, experience shows that such ideal conditions do not prevail in saline areas, and therefore, a drainage system is always required. Based on site condition, nature of the problem, and available resources, a suitable drainage system (surface or subsurface) can be selected. Surface drainage allows runoff excess water before entering to soil and subsurface drainage is used to control water table at safer depth, consisting of open ditches or tile drains or perforated plastic pipes, mole drainage, and vertical drainage (pumping water) when the deep horizons have an adequate hydraulic conductivity. 2.5.2.1  Modern Irrigation Methods In arid and semiarid zones, the major constraints are limited quantities of good quality water and to increase its efficiency; and exploitation of unsuitable brackish/saline water for irrigation. Therefore, a suitable irrigation method is to be selected without invoking soil salinity hazards. Each irrigation system develops salinity at a specific soil zone that is to be carefully monitored. Surface irrigation includes flood, basin, border, and furrow methods. At the end of each irrigation cycle, soil dries out concentrating the salts, which adversely affects the crop yield. Frequent irrigation may lower the salinity, but increase the wastage of water; the alternatives to improve the efficiency of water are the drip, subsurface, or sprinkler irrigation. This shift from conventional surface irrigation to modern irrigation is costly and requires assurance on better crop adaptability. A good sprinkler irrigation must meet the requirements of the crop for water (ET). It often allows efficient and economic use of water and reduces deep percolation losses. If water application through sprinkler is in close agreement with crop needs (ET and leaching), drainage and high water table problem can be greatly reduced, which in turn should improve salinity control. The use of high salinity water may lead to leaf burn and, therefore, the quality of water must match with the leaf burn tolerance of plants. Under sprinkler irrigation, the net salinity built up is at subsurface. The drip (trickle) irrigation supplies the required quantity of water to the crop almost on a daily basis. The poor quality water used in drip irrigation may yield better due to continuous high moisture contents and daily replenishment of water loss by ET. Drip has priority over sprinkler as the latter may cause leaf burn, defoliation of sensitive species, which is not the case with drip irrigation. Maximum salt accumulation is outside the edges of the area wetted by emitters (Plate 2.5). 100

45

90

40

80

35

70

30

60

25 20

10

20

5

10 0 5 10 15 20 Distance (cm)

120

0

Drip emitter

100

40 30

75 cm 140

50

15

0

50 cm

Distance (cm)

25 cm

Distance (cm)

Distance (cm)

50

80 Salt accumulation

60 40

Drip emitter

20

0 10 20 Distance (cm)

Plate 2.5  Drip irrigation and salt accumulation (salinity map).

0

0 10 20 Distance (cm)

Soil Salinity Development, Classification, Assessment, and Management

35

The daily irrigation continuously moves the moisture down to keep the salts under control. Plants may get shock due to high salinity when rainfall, as the rain water pushes the salts from edges to central rootzone; it is advisable to not shut the drip irrigation during rainfall to push the salts continuously toward the edges of the area wetted by emitters. Subsurface drip irrigation (SDI) compared with other irrigation systems SDI reduce water losses to evaporation, deep percolation, and completely eliminate surface runoff (Phene, 1990), increase crop marketable yield and quality (Ayers et al., 1999), and can result in high nutrient use efficiency (Thompson et al., 2002). Saline irrigation water can be used with SDI, while maintaining yields and improving water use efficiency compared to surface irrigation (Tingwu et al., 2003; Cahn and Ajwa, 2005) because SDI can result in suitable root-zone salinity. The limitation of SDI is that salts continuously build up at surface through capillary action above the buried drip lines during growing season (Oron et al., 1999) and therefore the concept of LR does not work under SDI; however, salts above buried drip (surface salinity) can be managed by supplementing with sprinkler irrigation (Thompson, 2010). This approach may be costly, but a compromise.

2.5.3  Chemical Method Chemical methods are used to reclaim sodic soils. To have successful crops on sodic soils, ESP of the soil must be below threshold (1 for Na+: Pi for ANTR1-mediated transport has been suggested. There are reports indicating an electrogenic transport for other Pi transporters (Bacconi et al., 2005), but not specifically for the SLC17 family, to which ANTR1 belongs. Nevertheless, the observation that mutants lacking this protein display higher levels of NPQ than the wild type (Karlsson et al., 2010) points out to a possible electrogenic mechanism for the thylakoid Pi transporter as well. Thus, it appears that to maintain steady and balanced electrochemical gradient across the thylakoid membrane, electrogenic transporters may modulate the activity of the xanthophyll cycle (see Chapter 16). To summarize, the thylakoid transporters may have multiple roles during light stress, which make them very important targets for plant breeding.

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O’Halloran, T.V. and V.C. Culotta. 2000. Metallochaperones, an intracellular shuttle service for metal ions. J. Biol. Chem. 275:25057–25060. Oxborough, K. and P. Horton. 1988. A study of the regulation and function of energy-dependent quenching in pea chloroplasts. Biochim. Biophys. Acta 934:135–143. Palmieri, F., B. Rieder, A. Ventrella et al. 2009. Molecular identification and functional characterisation of Arabidopsis thaliana mitochondrial and chloroplastic NAD+ carrier proteins. J. Biol. Chem. 84:31249–31259. Picault, N., M. Hodges, L. Palmieri, and F. Palmieri. 2004. The growing family of mitochondrial carriers in Arabidopsis. Trends Plant Sci. 9:138–146. PlantsT: Functional Genomics of Plant transporters. http://plantst.genomics.purdue.edu/ (accessed October 30, 2009). Pohlmeyer, K., J. Soll, T. Steinkamp, S. Hinnah, and R. Wagner. 1997. Isolation and characterization of an amino acid-selective channel protein present in the chloroplastic outer envelope membrane. Proc. Natl. Acad. Sci. USA 94:9504–9509. Pohlmeyer, K., J. Soll, R. Grimm, K. Hill, and R. Wagner. 1998. A high-conductance solute channel in the chloroplastic outer envelope from pea. Plant Cell 10:1207–1216. Pottosin, I.I. and Schönknecht, G. 1995. Patch clamp study of the voltage-dependent anion channel in the thylakoid membrane. J. Membr. Biol. 148:143–156. Pottosin, I.I. and Schönknecht, G. 1996. Ion channel permeable for divalent and monovalent cations in native spinach thylakoid membrane. J. Membr. Biol. 152:223–233. Powles, S.B. 1984. Photoinhibition of photosynthesis induced by visible light. Annu. Rev. Plant Physiol. 35:15–44. Rausch, C. and M. Bucher. 2002. Molecular mechanisms of phosphate transport in plants. Planta 216:23–37. Rausch, C., P. N. Zimmermann, N. Amrhein, and M. Bucher. 2004. Expression analysis suggests novel roles for the plastidic phosphate transporter Pht2;1 in auto- and heterotrophic tissues in potato and Arabidopsis. Plant J. 39:13–28. Reiser, J., N.L. Linka, L. Lemke, W. Jeblick, and H.E. Neuhaus. 2004. Molecular physiological analysis of the two plastidic ATP/ADP transporters from Arabidopsis. Plant Physiol. 136:3524–3536. Rensing, S.A., D. Lang, A.D. Zimmer et al. 2008. The Physcomitrella genome reveals evolutionary insights into the conquest of land by plants. Science 319:64–69. Retzel, E.F., J.E. Johnson, J.A. Crow, A.F. Lamblin, and C.E. Paule. 2007. Legume resources: MtDB and Medicago.Org. Methods Mol. Biol. 406:261–274. Rolland, N., M. Ferro, D. Seigneurin-Berny, J. Garin, R. Douce, and J. Joyard. 2003. Proteomics of chloroplast envelope membranes. Photosynth. Res. 78:205–230. Roth, C., G. Menzel, J.M. Petétot, S. Rochat-Hacker, and Y. Poirier. 2005. Characterization of a protein of the plastid inner envelope having homology to animal inorganic phosphate, chloride and organic-anion transporters. Planta 218:406–416. Ruban, A.V. 2009. Plants in light. Commun. Integr. Biol. 2:50–55. Ruban, A.V. and P. Horton. 1994. Spectroscopy of non-photochemical and photochemical quenching of chlorophyll fluorescence in leaves; evidence for a role of the light harvesting complex of photosystem II in the regulation of energy dissipation. Photosynth. Res. 40:181–190. Ruiz Pavón, L., F. Lundh, B. Lundin, A. Mishra, B.L. Persson, and C. Spetea. 2008. Arabidopsis ANTR1 is a thylakoid Na+-dependent phosphate transporter: functional characterization in Escherichia coli. J. Biol. Chem. 283:13520–13527. Ruiz Pavón, L., P.M. Karlsson, J. Carlsson, D. Samyn, B. Persson, B.L. Persson, and C. Spetea. 2010. Functionally Important Amino Acids in the Arabidopsis Thylakoid Phosphate Transporter: Homology Modeling and Site-directed Mutagenesis. Biochemistry. June 22, DOI: 10.1021/bi/00239j. Schneider, A., R.E. Häusler, U. Kolukisaoglu et al. 2002. An Arabidopsis thaliana knock-out mutant of the chloroplast triose phosphate/phosphate translocator is severely compromised only when starch synthesis, but not starch mobilisation is abolished. Plant J. 32:685–699. Schönknecht, G., R. Hedrich, W. Junge, and K. Raschke. 1988. A voltage-dependent chloride channel in the photosynthetic membrane of a higher plant. Nature 336:589–592. Schröder, W.P. and T. Kieselbach. 2003. Update on chloroplast proteomics. Photosynth. Res. 78:181–193. Segalla, A., I. Szabo, P. Costantini, and G.M. Giacometti. 2005. Study of the effect of ion channel modulators on photosynthetic oxygen evolution. J. Chem. Inf. Model. 245:1691–1700. Seigneurin-Berny, D., A. Gravot, P. Auroy et al. 2006. HMA1, a new Cu-ATPase of the chloroplast envelope, is essential for growth under adverse light conditions. J. Biol. Chem. 281:2882–2892.

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Shikanai, T., P. Müller-Moulé, Y. Munekage, K.K. Niyogi, and M. Pilon. 2003. PAA1, a P-type ATPase of Arabidopsis, functions in copper transport in chloroplasts. Plant Cell 15:1333–1346. Shingles, R., M. North, and R.E. McCarty. 2001. Direct measurement of ferrous ion transport across membranes using a sensitive fluorometric assay. Anal. Biochem. 296:106–113. Shingles, R., M. North, and R.E. McCarty. 2002. Ferrous ion transport across chloroplast inner envelope membranes. Plant Physiol. 128:1022–1030. Silva, P., E. Thompson, S. Bailey, et al. 2003. FtsH is involved in the early stages of repair of photosystem II in Arabidopsis sp. PCC 6803. Plant Cell 15:2152–2164. Simillion, C., K. Vandepoele, M.C. Van Montagu, M. Zabeau, and Y. Van de Peer. 2002. The hidden duplication past of Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 99:13627–13632. Song, C.P., Y. Guo, Q. Qiu, G. Lambert, D.W. Galbraith, A. Jagendorf, and J.K. Zhu. 2004. A probable Na+(K+)/ H+ exchanger on the chloroplast envelope functions in pH homeostasis and chloroplast development in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 101:10211–10216. Spetea, C. and S. Thuswaldner. 2008. Update in nucleotide-dependent processes in plant chloroplasts. In Plant Cell Compartments—Selected Topics, ed B. Schoefs, pp 105–149. Kerala, India: Research Signpost. Spetea, C., T. Hundal, F. Lohmann, and B. Andersson. 1999. GTP bound to chloroplast thylakoid membranes is required for light-induced, multienzyme degradation of the photosystem II D1 protein. Proc. Natl. Acad. Sci. USA 96:6547–6552. Spetea, C., T. Hundal, B. Lundin, M. Heddad, I. Adamska, and B. Andersson. 2004. Multiple evidence for nucleotide metabolism in the chloroplast thylakoid lumen. Proc. Natl. Acad. Sci. USA 101:1409–1414. Sterck, L., S. Rombauts, K. Vandepoele, P. Rouzé, and Y. Van de Peer. 2007. How many genes are there in plants (… and why are they there)? Curr. Opin. Plant Biol. 10:199–203. Sugiura, M., M.N. Georgescu, and M. Takahashi. 2007. A nitrite transporter associated with nitrite uptake by higher plant chloroplasts. Plant Cell Physiol. 48:1022–1035. TAIR: The Arabidopsis Information Resource. http://www.tair.org (accessed October 30, 2009). Takahashi, S. and N. Murata. 2008. How do environmental stresses accelerate photoinhibition? Trends Plant Sci. 13:178–182. Takahashi, T., N. Inoue-Kashino, S. Ozawa, Y. Takahashi, Y. Kashino, and K. Satoh. 2009. Photosystem II complex in vivo is a monomer. J. Biol. Chem. 284:15598–15606. TCDB: Transport classification database. http://www.tcdb.org/ (accessed October 30, 2009). Teng, Y.-S., Y.-s. Su, L.-J. Chen, Y.J. Lee, I. Hwang, and H.-m. Li. 2006. Tic21 is an essential translocon component for protein translocation across the chloroplast inner envelope membrane. Plant Cell 18:2247–2257. Tester, M. and M.R. Blatt. 1989. Direct measurement of K channels in thylakoid membranes by incorporation of vesicles into planar lipid bilayers. Plant Physiol. 91:249–252. Thuswaldner, S., J.O. Lagerstedt, M. Rojas-Stütz et al. 2007. Identification, expression, and functional analyses of a thylakoid ATP/ADP carrier from Arabidopsis. J. Biol. Chem. 282:8848–8859. Tikkanen, M., M. Nurmi, S. Kangasjärvi, and E.M. Aro. 2008. Core protein phosphorylation facilitates the repair of photodamaged photosystem II at high light. Biochim. Biophys. Acta 1777:1432–1437. Tobacco Genome Initiative. http:// www.tobaccogenome.org (accessed October 30, 2009). TransportDB: Transporter Protein Analysis Database. http://www.membranetransport.org/ (accessed October 30, 2009). Tsunekawa, K., T. Shijuku, M. Hayashimoto et al. 2009. Identification and characterization of the Na+/H+ antiporter Nhas3 from the thylakoid membrane of Synechocystis sp. PCC 6803. J. Biol Chem. 284:16513–16521. Tuskan, G.A., S. Difazio, S. Jansson et al. 2006. The genome of black cottonwood, Populus trichocarpa. Science 313:1596–1604. Uehlein, N., C. Lovisolo, F. Siefritz, and R. Kaldenhoff. 2003. The tobacco aquaporin NtAQP1 is a membrane CO2 pore with physiological functions. Nature 425:734–737. Uehlein, N., B. Otto, D.T. Hanson, M. Fischer, N. McDowell, and R. Kaldenhoff. 2008. Function of Nicotiana tabacum aquaporins as chloroplast gas pores challenges the concept of membrane CO2 permeability. Plant Cell 20:648–657. van den Wijngaard, P.W.J. and W.J. Vredenberg. 1999. The envelope anion channel involved in chloroplast protein import is associated with Tic110. J. Biol. Chem. 274:25201–25204. van Wijk, K.J. 2004. Plastid proteomics. Plant Physiol. Biochem. 42:963–977. Vass, I., S. Styring, T. Hundal, A. Koivuniemi, E. Aro, and B. Andersson. 1992. Reversible and irreversible intermediates during photoinhibition of photosystem II: Stable reduced QA species promote chlorophyll triplet formation. Proc. Natl. Acad. Sci. USA 89:1408–1412.

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Walters, R.G., F. Shephard, J.J. Rogers, S.A. Rolfe, and P. Horton. 2003. Identification of mutants of Arabidopsis defective in acclimation of photosynthesis to the light environment. Plant Physiol. 131:472–481. Watanabe, M., M. Iwai, R. Narikawa, and M. Ikeuchi. 2009. Is the photosystem II complex a monomer or a dimer? Plant Cell Physiol. 50:1674–1680. Weber, A.P., R. Schwacke, and U.I. Flügge. 2005. Solute transporters of the plastid envelope membrane. Annu. Rev. Plant Biol. 56:133–164. Winkler H.H. and H.E. Neuhaus. 1999. Non-mitochondrial ATP transport. Trends Biochem. Sci. 24:64–68. Wudick, M.M., D.T. Luu, and C. Maurel. 2009. A look inside: Localization patterns and functions of intracellular plant aquaporins. New Phytol. 184:289–302. Yamamoto, H.Y., T.O. Nakayama, and C.O. Chichester. 1962. Studies on the light and dark interconversions of leaf xanthophylls. Arch. Biochem. Biophys. 97:168–173. Yin, L., B. Lundin, M. Bertrand, M. Nurmi, K. Solymosi, S. Kangasjäsvi, E. M. Aro, B. Schoefs, and C. Spetea. 2009. Role of thylakoid ATP/ADP carrier in photoinhibition and photoprotection of photosystem II in Arabidopsis. Plant Physiol. 153:666–677. Zybailov, B., H. Rutschow, G. Friso, A. Rudella, O. Emanuelsson, Q. Sun, and K.J. van Wijk. 2008. Sorting signals, N-terminal modifications and abundance of the chloroplast proteome. PLoS One 3:e1994.

Pigment 15 Photosynthetic Apparatus in Northern Plants Tamara Golovko, Olga Dymova, Yakov Yatsco, and Galina Tabalenkova Contents 15.1 Introduction........................................................................................................................... 391 15.2 Photosynthetic Pigment Contents in Northern Plants........................................................... 392 15.2.1 Pigment Complex of the Subpolar Ural Mountains Plants........................................ 392 15.2.2 Pigment Complex of South Tyman Plants................................................................. 396 15.2.3 Pigment Complex of Meadow and Forest Plants in the Middle Vychegda Basin..... 398 15.3 Seasonal Changes in Leaf Pigment Contents and Xanthophylls Cycle Activity in Evergreen Coniferous Species...........................................................................................400 15.4 Summary and Conclusions....................................................................................................402 Acknowledgment............................................................................................................................403 References.......................................................................................................................................403

15.1  Introduction In nature, plants frequently experience a wide range of stresses. An investigation of functional plasticity is necessary to understand responses to environmental conditions, the distribution of the species, and to predict the dynamics of vegetation under the global changes of climate. The indices of photosynthetic apparatus, such as the composition, the content, and the ratio of pigments are considered to be the most informative to characterize the functional plant state (Lubimenko 1963, Zalenskii 1977, Maslova and Popova 1993, Pyankov and Mokronosov 1993, Bazzaz 1996, Dymova and Golovko 2007, Golovko et al. 2007). Chlorophylls and carotenoids are required for photosynthesis. Chlorophylls are necessary for the capture of light energy and as primary electron donors. Carotenoids play crucial roles in both light harvesting and energy dissipation for the protection of photosynthetic structures. So far, the spectral characteristics and the biosynthesis of photosynthetic pigments have been studied, the concept of the antenna complex and the reaction centers has been developed, and the fundamental mechanisms of photosynthesis have been revealed. At the same time, the diversity and ecological conditions of habitation of plant species make the investigation of the pigments’ role in the stability and regulation of the photosynthetic apparatus activity the topical problem. A qualitative content and a quantitative composition of pigments and their ratio changes are the important and responsive characteristics of the physiological state of plants and their photosynthetic apparatus. However, current information about the pigment system of plants from different botanical and geographical zones is rare and contradictory (Lukyanova et al. 1986, Popova et al. 1989, Kornushenko and Solovjova 1992, Maslova and Popova 1993, Golovko et al. 2007). Northeast European Russia is a unique region in continental Europe. Here, plant growth is restricted by lack of warmth, short vegetative period, and poor soils. Photosynthetic apparatus, including pigment complex, prove additional stress in these severe conditions. 391

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We advance the idea of increasing the role of pigments in tolerance and in the productivity of photosynthesis of plants in the taiga cold climate environments. The results presented in this chapter provide proof for this idea.

15.2  Photosynthetic Pigment Contents in Northern Plants We studied the pigment complex of more than 100 plant species inhabiting three different sites in the European North-East of Russia: (1) the Subpolar Ural Mountains (65°22′ N, 60°46′ E), (2) the South Tyman (62°45′ N, 55°49′ E), and (3) the Middle Vychegda basin (61°38′ N, 50°43′ E). The main climatic traits of these regions are shown in Table 15.1. The Subpolar Ural Mountains (the extremely north taiga subzone) is characterized by the most severe climate and a short vegetative period. The climate in the Middle Vychegda (the middle taiga subzone) is warmer, but the growing season in this region does not exceed 100–110 days. The mean annual air temperature only slightly exceeds 1°C. Though the region of the South Tyman belongs to the middle taiga subzone, it differs from the Middle Vychegda by the temperature regime and the duration of the vegetative period. The list of examined plant species in each region is shown in Table 15.2. All 121 plant species have been investigated and most of them are boreal herbaceous plants and reflect the structure of the floristic complexes in the studied regions. Leaf samples for pigment analyses were collected from 20 to 30 plants of each species from the beginning–middle of July (2004–2008). Analyses were carried out on mature healthy current-year leaves (herbs) or current-year shoots (Empetrum nigrum and E. hermaphroditum, Lycopodium clavatum and L. annotinum, Diphasium complanatum). The second-year needles were collected from evergreen conifers (Abies sibirica, Juniperus communis, Picea obovata, Pinus sibirica). The majority of herbaceous plants flowered during the sampling. Leaf chlorophyll (Chl) and carotenoid (Car) contents were measured by UV-1700 spectrophotometer (“Shimadzu”, Japan) in acetone extracts at 662 (Chl a), 644 (Chl b), and 470 nm (total Car). The chlorophyll portion in light-harvesting chlorophyll (LHC) (LHC-Chl) was calculated, assuming that the total Chl b was located in LHC and the Chl a/b ratio in this complex was equal to 1.2 (Lichtenthaler 1987). Separations and quantifications of Car were done by reversed-phase highperformance liquid chromatography (HPLC) according to Gilmore and Yamamoto (1991).

15.2.1  Pigment Complex of the Subpolar Ural Mountains Plants The differences in the accumulation of photosynthetic pigments were revealed between species (Figure 15.1). The concentration of Chl and Car varied in the ranges of 1.5–14 and 0.5–5 mg/g dry weight (DW), respectively. The legume plants (Astragalus norvegicus, A. frigidus, Hedysarum arcticum)

Table 15.1 Mean Climatic Traits of Different Places in the European North-East Traits Mean annual air temperature, °C Mean July air temperature, °C Total temperature above +5°C The duration of growing season (above +5°C), days The duration of active growth period (above +10°C), days Mean annual precipitation, mm

Subpolar Ural Mountains

The South Tyman

The Middle Vychegda Basin

−4.8 +13 1070 105–110

−1.5 +15 1550 133

+1 +17 1800 150

60–70

80–90

100–110

600–685

660–750

650–765

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Table 15.2 List of the Species from Different Places in the European North-East No.

Subpolar Ural Mountains

The South Tyman

The Middle Taiga Subzone

1

2

3

4

 1

13

(H, B) Cystopteris dickieana R. Sim.

Aconitum septentrionale Koelle. (H, B) Antennaria dioica (L.) Gaertn. (H, B) Aster sibiricus L. (H, A) Astragalus danicus Retz. (H, P) Calamagrostis epigeios (L.) Roth (H, B) Caltha palustris L. (H, B) Cortusa matthioli L. (H, B) Cotoneaster melanocarpa Lodd. (SH, B) Crepis sibirica L. (H, B) Cypripedium calceolus L. (SH, B) Dactylorhiza fuchsii (Druce) Soo (H, B) Dendranthema zawadskii (Herbich) Tzvel. (H, R) Diphasium complanatum (L.) Rothm.

Abies sibirica Ledeb. (T, B) Achillea millefolium L. (H, B) Aconitum septentrionale Koelle (H, B) Alchemilla sp. (H, P) Alisma plantago-aquatica L. (H, B) Antennaria dioica (L.) Gaertn. (H, B) Bistorta major S.F. Gray (H, B) Bromopsis inermis Leyss. (H, B) Butomus umbellatus L. (H, B) Calla palustris L. (H, B) Chenopodium album L.

12

Achillea nigrescens (E. Mey) Rydb (H, B) Alchemilla murbeckiana Buser (H, B) Amoria repens (L.) C. Presl (H, B) Arctous alpina (L.) Niedz. (SH, A + AA) Artemisia tilesii Ledeb. (H, A + AA) Astragalus frigidus (L.) A. Gray (H, A) Astragalus norvegicus Grauer (H, A + AA) Atragene sibirica L. (SH, B) Betula nana L. (SH, A) Bartsia alpina L. (H, A + AA) Calamagrostis purpurea (Trin.) Trin. (H, B) Carex aquatilis Wahlenb.

 2  3  4  5  6  7  8  9 10 11

14 15

16 17 18 19 20

(H, A + AA) Diapensia lapponica L. (SH, A + AA) Empetrum hermaphroditum (Lange) (SH, A) Hedysarum arcticum B. Fedtsh. (H, A + AA) Larix sibirica Ledeb. (T, B) Ledum decumbens L. (H, A) Lycopodium clavatum L. (H, A) Lycopodium annotinum L. (H, B)

(H, B) Comarum palustre L.

(C-M, B) Dryas octopetala L. (SH, AA) Dryopteris filix-mas (L.) Schott.

(H, B) Deschampsia cespitosa (L.) Beauv (H, B) Dryopteris filix-mass (L.) Schott. (F, B) Elytrigia repens (L.) Nevski

(F, B) Equisetum palustre L. (H, B) Epipactis atrorubens (Hoffm.) Besser. (H, B) Geum rivale L. (H, B) Gymnadenia conopsea (L.) R.Br. (H, B) Gymnocarpium robertianum (Hoffm.) Newm. (SH, B)

(H, B) Empetrum nigrum L. (SH, A) Filipendula ulmaria (L.) Maxim (H, B) Galium boreale L. (H, B) Gymnocarpium dryopteris (L.) (H, B) Hylotelephium triphyllum (Haw.) Holub (H, B) (continued)

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Table 15.2 (continued) List of the Species from Different Places in the European North-East No. 1 21 22

23 24 25 26

27 28 29 30 31

32 33 34 35 36 37 38 39 40 41 42

Subpolar Ural Mountains

The South Tyman

The Middle Taiga Subzone

2

3

4

Pedicularis verticillata L. (H, A + AA) Pentaphylloides fruticosa (L.) O. Schwarz. (SH, B) Phyllodoce caerulea (L.) Bab. (SH, A + AA) Pyrola rotundifolia L. (H, B) Rhodiola rosea L. (H, A) Rosa acicularis Lindley.

Juniperus communis L. (SH, B) Lathyrus pratensis L.

Hypericum maculatum Crantz (H, B) Juniperus communis L.

(H, B) Lathyrus vernus (L.) Bernh. (H, B) Lycopodium annotinum L. (C-M, B) Melica nutans L. (H, B) Paeonia anomala L.

(SH, B) Rubus chamaemorus L. (H, A) Salix dasyclados Wimm. (T, B) Salix nummullaria L. (SH, A + AA) Salix reticulata L. (SH, A + AA) Tanacetum bipinnatum (L.) Sch. Bip. (H, A) Vaccinium myrtillus L. (SH, B) Vaccinium uliginosum L. (SH, B) Valeriana wolgensis Kazak. (H, B) Woodsia glabella R.Br. (H, A + AA)

(H, B) Paris quadrifolia L. (H, B) Parnassia palustris L. (H, B) Pedicularis verticillata L. (H, AA) Petasites radiatus (J.F. Gmel.) Holub. (H, A) Pinguicula vulgaris L.

(SH, B) Ledum palustre L. (SH, B) Leontodon autumnalis L. (T, B) Lycopodium clavatum L. (C-M, B) Maianthemum bifolium (L.) F.W. Schmidt (H, B) Oxalis acetosella L. (H, B) Paris quadrifolia L. (H, B) Petasites spurious (Retz.) (H, A) Pimpinella saxifraga L. (H, B) Picea obovata Ledeb

(H, A) Pinus sibirica Du Tour (T, B) Plantago media L. (H, B) Polygonum viviparum L. (H, AA) Pyrola rotundifolia L. (H, B) Sanguisorba officinalis L. (H, B) Saussurea alpina (L.) DC. (H, AA) Saxifraga hirculus L. (H, A) Thymus talijevii Klok. et Shost. (SH, B) Tofieldia pusilla L. (H, AA) Tussilago farfara L. (H, B) Vaccinum uliginosum L. (SH, A)

(T, B) Pinus sibirica Du Tour (T, B) Plantago major L. (T, B) Polygonum aviculare L. (H, B) Pyrola rotundifolia L. (H, B) Rorippa amphibia (L.) Bess (H, B) Rosa majalis Herrm. (SH, B) Rubus saxatilis L. (H, B) Rubus chamaemorus L. (H, A) Taraxacum officinale Wigg. (H, B) Thalictrum simplex L. (H, B) Trifolium medium L. (H, B)

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Table 15.2 (continued) List of the Species from Different Places in the European North-East No. 1 43

Subpolar Ural Mountains

The South Tyman

The Middle Taiga Subzone

2

3

4

Valeriana capitata Pallas. (H, B) Valeriana wolgensis Kazak. (H, B) Vicia cracca L. (H, B) Vicia sylvatica L. (H, B) Woodsia glabella R.Br. (SH, AA)

44 45 46 47

Trifolium pratense L. (H, B) Veronica longifolia L. (H, B) Vaccinium myrtillus L. (SH, B) Vaccinium uliginosum L. (SH, A) Vaccinium vitis-idaea L. (SH, A)

Notes: Life-form groups: H—herb, T—tree, SH—shrub; C-M—club-moss; latitudinal groups: A—arctic, AA—arctic and alpine, B—boreal species; P—prairie, R—rocky species. Latin names of species given by Cherepanov (1995). Species presented with note of its life form and geographical status.

18 16

Pigments, mg/g dry weight

14 12 10 8 6 4 2 0

14 19 17 20 33 4 15 26 1 23 31 27 18 9 35 2 32 22 29 30 11 10 28 12 13 34 24 5 25 8 21 3 16 7 6 Species

Figure 15.1  Chlorophyll (dark symbols) and carotenoid (light symbols) contents in the leaves of the Subpolar Ural Mountains plants. For species names view according to the numbers in Table 15.2.

were  characterized by a higher green pigment content. Conifer (Larix sibirica), dwarf shrubs (Empetrum hermaphroditum, Phyllodoce caerulea, Ledum decumbens, Vaccinium uliginosum), club mosses (Lycopodium clavatum and L. annotinum), and herbs (Diapensia lapponica) had a very low Chl content. In the leaves of more than 60% of the examined species, the content of green pigments consisted of 3–6 mg/g DW. These concentrations can be considered relatively low. The Chl a/b ratio varied from 2 to 3.5, but it was higher (3.8–4.0) in Bartsia alpina, Salix dasyclados, Astragalus frigidus, and Hedysarum arcticum. In most of the species, Chl belonging to the light-harvesting complex (LHC-Chl) consisted of 55%–65% of the total. In B. alpina, A. frigidus, H. arcticum, and woody S. dasyclados leaves, the LHC-Chl values were the lowest (43%–45%).

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Handbook of Plant and Crop Stress 18 Chlorophylls, mg/g dry weight

16

18

r = 0.6

16 14

14

12

12

12

10

10

10

8

8

8

6

6

6

4

4

4

2

2

2

0

2

4

6

0

0 (B)

2

4

r = 0.8

16

14

0 (A)

18

r = 0.9

6

0

0 (C)

2

4

6

Carotenoids, mg/g dry weight

Figure 15.2  The relationship between chlorophylls and carotenoids in the leaves of the Subpolar Ural Mountains (A), the South Tyman (B), and the Middle Vychegda (C) plants.

The leaves of Arctous alpine and Pyrola rotundifolia were characterized by the highest LHC-Chl level (70%–80%). There are positive correlations found between the yellow and the green pigment concentrations (Figure 15.2A). The majority of the examined species had the Car content of 0.9–1.7 mg/g DW. Diapensia lapponica, Arctous alpine, Phyllodoce caerulea, Achillea nigrescens, Lycopodium clavatum, L. annotinum, and Vaccinum uliginosum were distinguished by a low accumulation of yellow pigments (0.6–0.8 mg/g DW). Some of the legumes (Hedysarum arcticum, Astragalus frigidus) and Pedicularis verticcilata had a high Car content, 4.5 mg/g DW. The Chl/Car ratios were 2.5–3.0 in most of the species, but these values were higher (4–5) in Carex aquatilis, Calamagrostis purpurea, and Arctous alpine. Plants with different Chl and Car accumulation levels were revealed among the groups that differed by life forms (herbs, ferns, trees and dwarf shrubs, and mosses). In the herbaceous plants group, there were species with an extremely low (Achillea nigrescens) and a high (Astragalus frigidus) content of photosynthetic pigments. However, overall perennial herbaceous plants, especially leguminous, contained more Chl and Car than trees and dwarf shrubs (Figure 15.3A). Among shrubs, Salix species, Betula nana and Pentaphylloides fruticosa, had a comparatively high Chl content (2–5 mg/g DW). Ferns had the same Chl level. Mosses were characterized by the lowest pigment accumulation. A comparison of plants from different latitudinal groups (Figure 15.4A) showed that arctic, arcticalpine, and hypo-arctic species contained similar amounts of photosynthetic pigments as that of the boreal species. In the arctic and arctic-alpine species, the ratio of green and yellow pigments was lower (2.9) as compared to boreal species (3.2). This indicates a relatively high Car in the pool of photosynthetic pigments of arctic and arctic-alpine species on the Subpolar Ural Mountains. In general, the study carried out on the Subpolar Ural Mountains showed that the contents of the photosynthetic pigments depend more on the plant species and its life form, whereas the Chl/Car ratio depends on the geographical group. It should be noted that the pigment complex was characterized by a relatively low Chl content.

15.2.2  Pigment Complex of South Tyman Plants Among the 47 species of South Tyman plants examined, 74% belong to the boreal latitudinal group. The other species are included in the arctic, the arctic-alpine, and the hypo-arctic groups. These plant species were mostly found in rocky floristic complexes.

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8

8

8

6

6

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Pigments, mg/g dry weight

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0 (A)

H

F

T, SH C-M

0 (B)

H

F T, SH C-M Life-form groups

0 (C)

H

F

T, SH C-M

Pigments, mg/g dry weight

Figure 15.3  Chlorophyll (dark symbols) and carotenoid (light symbols) contents in the leaves of the Subpolar Ural Mountains (A), the South Tyman (B), the Middle Vychegda (C). Life-form groups: H—herbs, T—trees, SH—shrubs, F—ferns, and C-M—club-mosses. 10

10

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6

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4

4

4

2

2

2

0 (A)

B

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AA

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B

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Latitudinal groups

0

B

A

(C)

Figure 15.4  Chlorophyll (dark symbols) and carotenoid (light symbols) contents in the leaves of the Subpolar Ural Mountains (A), the South Tyman (B), and the Middle Vychegda (C). Latitudinal groups: B—boreal, A—hypo-arctic, AA—arctic and alpine.

The South Tyman plants differed appreciably by the green pigment content. The Chl concentrations of these plant species varied from 1.5 to 14 mg/g DW (Figure 15.5). Conifer (Pinus sibirica), shrubs (Vaccinum uliginosum) and some herbs (Pedicularis vercillata, Dendranthema zawadskii) had comparatively low pigment content. Several species, including Trilliaceae (Paris quadrifolia), Orchidaceae (Cypripedium calceolus), Asteracea (Crepis sibirica, Petasites radiatus), Rosaceae (Geum rivale), and Fabaceae (Viccia cracca and V. sylvatica, Lathyrus pratensis) were characterized by high Chl and Car contents. The Chl and Car ratios were 2.4–3.1 in most of these species. These values were higher (3.6–3.9) in Gymnadenia conopsea, Pedicularis verticillata, and Thymus taljievii plants. The majority of species had LHC-Chl levels equal to 50%–65% of total Chl. Among the examined species, only in the leaves of Pinguicula vulgaris, LHC-Chl was 75%. The high percentage of Chl in LHC can increase the absorption of light energy and compensate a relatively low level of the green pigment in Pinguicula leaves. It should be noted that this plant was characterized by various types of nutrient deficiencies. It is an insectivorous plant. As for the Subpolar Ural Mountains plants, a strong positive relationship between the content of the green and the yellow pigments with that of the South Tyman plants was revealed (Figure 15.2B).

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18 16

Pigments, mg/g dry weight

14 12 10 8 6 4 2 0

42 32 39 24 29 21 38 12 31 2 13 26 25 16 47 40 20 36 35 15 8 19 14 3 11 5 44 7 23 17 28 37 33 6 34 41 4 30 22 43 18 45 9 46 10 1 27

Species

Figure 15.5  Chlorophyll (dark symbols) and carotenoid (light symbols) contents in the leaves of the South Tyman plants. For species names see according to the numbers in Table 15.2.

Although the Car concentration varied strongly, from 0.3 to 2.5 mg/g DW, the ratio Chl/Car was rather similar for most of the plants, i.e., equal to 4–5. The herbaceous plants were characterized by higher Chl and Car contents than ferns, trees, and shrubs. The mosses had the lowest concentration of photosynthetic pigments (Figure 15.3B). The comparison of the plants from different latitudinal groups showed that arctic and hypo-arctic species accumulated lower amounts of pigment than the boreal plants (Figure 15.4B). Thus, arcticalpine species were characterized by a low value of Chl/Car ratio (3.7) as compared to boreal plants (4.1).

15.2.3  Pigment Complex of Meadow and Forest Plants in the Middle Vychegda Basin Forty-seven species inhabiting the meadows and forests were studied. Our data showed (Figure 15.6A) that the leaf Chl content was relatively high (more than 4–5 mg/g DW) in a majority of these plants. In Paris quadrifolia, Calla palustris, and Aconitum septentrionale, the Chl content was about 14 mg/g DW. The LHC-Chl level was varied significantly, from 44% to 74%. More LHC-Chl was accumulated in the forest herbs compared to the rest of the other species examined in this area. Although the species were distinguished by the Chl and Car concentrations, there was a positive correlation between the green and the yellow pigment accumulations. The content of the yellow pigments was four- to eightfold less than that of the green pigments. Lycopodium clavatum, Vaccinum vitis-ideae, V. myrtillus, Abies sibirica, and Pinus sibirica contained a comparatively small amount of Car (0.4–0.5 mg/g DW). But, Thalictrum simplex, Alisma plantago-aquatica, and Aconitum septentrionale accumulated five times more yellow pigments than green pigments. In the Middle Vychegda flora, like the other regions, it was observed that the herbs were characterized by the highest accumulation of photosynthetic pigments (Figure 15.3C). The Chl and Car contents were 1.5–2-fold lower in trees and mosses. The comparison of the plants from different

399

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Pigments, mg/g dry weight

14 12 10 8 6 4 2 0

1 32 22 47 29 31 46 15 25 6 5 34 23 2 8 45 16 20 24 4 40 13 11 35 19 30 21 33 14 37 38 36 18 41 17 39 42 44 12 27 7 9 43 26 28 10 3

Species

Figure 15.6  Chlorophylls (dark symbols) and carotenoids (light symbols) content in the leaves of the Middle Vychegda basin plants. For species names see according to the numbers in Table 15.2.

latitudinal groups showed that hypo-arctic species accumulated 1.3 times lower photosynthetic pigments than the boreal species (Figure 15.4C). So, our data have shown that most of the examined plants from the Subpolar Ural Mountains and the South Tyman regions were characterized by a relatively higher content of the yellow pigments (Figure 15.7). The herbs accumulated 2–3 times more pigments than trees and mosses. Photosynthetic pigment pools in the leaves of the plants inhabited in the Middle Vychegda basin were larger as compared to plants on the Subpolar Ural Mountains. 6

Chlorophyll/carotenoids

5 4 3 2 1 0

Sub polar Ural

South Tyman

The Middle Vychegda

Figure 15.7  The ratio of chlorophylls to carotenoids in the leaves of the plants from different regions.

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15.3  Seasonal Changes in Leaf Pigment Contents and Xanthophylls Cycle Activity in Evergreen Coniferous Species

Pigments, mg/g dry weight

The evergreen conifers are the main woody species in the taiga zone and they occupy about 80% of the woodland of the Komi Republic (Virgin spruce forests … 2006). The retention and maintenance of the pigment complex’s functional activity in the annual cycle are important for the evergreen species of the boreal zone, which adapt to the long-term cold period. Low-temperature conditions during winter can inhibit CO2-exchange and carbon photosynthetic metabolism, but low temperatures do not affect the ability of chlorophyll to absorb light. Thus, winter conditions can cause a severe imbalance between light absorption and its utilization via photosynthesis. In such conditions, plants use photoprotective mechanisms to deal with the excess light absorbed by chlorophyll. The key mechanism includes the xanthophyll cycle-dependent thermal energy dissipation of excess light within the light-harvesting complexes. It is shown that the de-epoxidation of the xanthophyll cycle pigments was significantly higher in evergreen plants during winter (Garcia-Plazaola et al. 2003, Verhoeven et al. 2005). We studied seasonal changes in the photosynthetic pigment content in three conifers: Abies sibirica, Picea obovata, and Juniperus communis. The concentrations of Chl and Car were relatively low, 2.4–4.3 × 0.45–1.0 mg/g DW, respectively (Figure 15.8). In winter, the reduction of the photosynthetic pigment pool was maximal in Juniperus communis needles. In Picea obovata and Abies sibirica, the pool of green pigments is more stable during the year. Essential seasonal differences in the chlorophyll a/b, the Chl, and the Car ratio were not found (Table 15.3). In spring and summer, all species had a tendency to a higher LHC-Chl level. 5 4 3 2 1 0

III

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XII

III

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IV

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Figure 15.8  Seasonal changes in the chlorophyll (dark symbols) and carotenoid (light symbols) contents in Picea abies (A), Abies sibirica (B), and Juniperus communis (C) needles. (From Yatsco, Y.N. et al., Botan. Z., 94(12), 1812, 2009.)

Table 15.3 Chlorophyll and Carotenoid Composition of Evergreen Conifers Needles Sampled in Spring (III—March), Summer (VII—July), and Winter (XII—December), n = 5 Chlorophyll a/b Species Abies sibirica Picea abies Juniperus communis

LHC-Chl, %

Chlorophyll/Carotenoids

III

VII

XII

III

VII

XII

III

VII

XII

2.4 ± 0.1 2.4 ± 0.1 2.8 ± 0.1

2.5 ± 0.1 2.5 ± 0.3 2.8 ± 0.1

2.7 ± 0.1 2.9 ± 0.1 2.9 ± 0.1

65 66 60

64 63 57

59 57 56

4.6 ± 0.1 4.5 ± 0.3 4.5 ± 0.1

5.7 ± 0.1 4.5 ± 0.4 4.2 ± 0.1

4.6 ± 0.1 4.4 ± 0.1 4.1 ± 0.1

Source: Yatsco, Y.N. et al., Botan. Z., 94(12), 1812, 2009. Note: LHC-Chl—Chlorophyll belonging to light-harvesting complex, n = 5.

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Figure 15.9  Seasonal changes in the carotenoid composition (as a percent of total carotenoid content) in Picea obovata (A), Abies sibirica (B), and Juniperus communis (C) needles. 1—zeaxanthin, 2—neoxanthin, 3—antheraxanthin, 4—lutein, 5—violaxanthin, and 6—β-carotene.

The carotenoid composition for all species was presented mainly by xanthophylls (90%) during the whole year (Figure 15.9). The portion of β-carotene consisted of 10%–20% of Car content. Among the xanthophylls, the level of lutein was the highest and reached 70%. Neoxanthin (7%–10%) and violaxanthin (3%–15%) were presented constantly in relatively small amounts. In the studied plants, violaxanthin was the predominant component of the xanthophylls cycle, whereas two other components of the xanthophyll cycle (zeaxanthin and anteraxanthin), were noted mainly during winter and spring, and none identified in summer. The analyses of the seasonal changes of the individual carotenoids showed an increase of β-carotene and violaxanthin levels in summer, whereas antheraxanthin and zeaxanthin were practically absent at this time. A low amount of antheraxanthin was found in autumn. Antheraxanthin levels in the carotenoid pools of Picea obovata and Abies sibirica needles were 7%–8% in winter and at the beginning of spring. There was 15% (of total carotenoid content) of antheraxanthin in Juniperus communis in winter. The level of antheraxanthin (up to 5%) was preserved in Juniperus needles until May. Zeaxanthin appeared in the beginning of winter, and its level increased in spring. Zeaxanthin disappeared fully in summer. The same seasonal pattern of zeaxanthin was revealed for Picea obovata and Abies sibirica. The de-epoxidation state (DEPS) was characterized by the ratio between de-epoxidized and the epoxidized forms of the xanthophyll cycle components. According to Schindler and Lichtenthaler (1996), DEPS can be estimated from the equation, DEPS = (zeaxanthin + 0.5 antheraxanthin)/ (zeaxanthin + antheraxanthin + violaxanthin). Evaluation of DEPS showed that the de-epoxidation of the xanthophyll cycle pigments was high in all the examined species in winter and the early spring (Table 15.4). The DEPS values decreased 2–3 times in May. The de-epoxidation of the xanthophyll cycle pigments was almost completely suppressed in summer (July). The repair of deepoxidation reaction (by 30%) was observed in Picea obovata needles in autumn (September), in the other two species—in December. It should be noted that for this period, Picea obovata was already characterized by maximum DEPS values. We noted already, that Picea obovata needles were distinguished by the greatest stability of the pigment pool during the year among the studied species. In Abies sibirica and Juniperus communis needles, a portion of the pigments in winter and spring had been destroyed. Several investigators have reported the loss of chlorophyll in the needles of the conifers, in winter (Ottander et al. 1995, Ensminger et al. 2004, Martz et al. 2007). Information about pigment complex preservation was

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Table 15.4 Seasonal Changes in Deep-Oxidation State of Xanthophyll Cycle Pigments in Evergreen Conifer Needles, %, n = 5 Months March May July September December

Picea obovata

Abies sibirica

Juniperus communis

54 12 0 13 53

46 23 2 0 30

60 21 0 3 38

given by Lukyanova et al. (1986). It seems that the different opinions about the character of the seasonal changes in pigment composition in conifers depend on the species traits and the climatic conditions of the habitats. Winter and spring loss of pigment was repaired in summer and autumn. The structural and functional changes of mesophyll at the initial stage of the hardening in autumn are important for the preservation of the photosynthetic apparatus. In the needles of Picea obovata, the disappearance of the starch grains, the change of form, size and localization of the chloroplasts are shown (Ladanova and Tuzhilkina 1992). The absorption of light energy was reduced due to the relocation of chloroplasts to the central part of the cell and grouping around the nucleus. This fact can be regarded as the adaptive reaction that is directed to the reduction of light absorption in winter and spring when the photosynthetic consumption of excitation energy is blocked.

15.4  Summary and Conclusions The contents and the ratio of photosynthetic pigments in plant leaves, growing in the Subpolar Ural Mountains, the South Tyman, and the Middle Vychegda basin, were investigated. We found that the level of the carotenoid accumulation in the plant leaves from all the studied regions correlated closely with the chlorophyll content. In comparison of our data with the results reported by Maslova and Popova (1993) for other regions, it was found that the Subpolar Ural Mountains plants are closer to the alpine plants of the East Pamirs, the South Tyman plants—to the arctic plants of Taimir, and the middle Vychegda plants—to the species of the temperate zone on the Chl/Car index. Carotenoids exhibit both light-harvesting and photoprotective functions (Demmig-Adams 1990, Rmiki et al. 1999, Cuttriss and Pogson 2004). Therefore, the relatively high level of the yellow pigments in the leaves of the Northern plants, especially arctic species, can be regarded as the adaptive reaction that is directed to an increase in the stability of the pigment complex and to prevent its photodynamic destruction in the cold climate. The pigment complex of the species from the middle taiga subzone was characterized by a higher Chl/Car ratio and the lower yellow pigment contents were in contrast to the Subpolar Ural Mountains plants. This confirms the important role of the carotenoids in the stability of the pigment complex of the extremely north taiga subzone plants. It should be noted that carotenoids not only have a protective role, but also absorb light in the near ultraviolet (UV) as well as the visible region. Carotenoids are bound, together with Chl, to proteins and participate in light harvesting. As components of the antenna complex, they provide more effective use of solar radiation during the short north summer. Pigments, especially carotenoids, perform an essential photoprotective role in evergreen conifers’ photosynthetic apparatus. Preservation and the long-term functioning of the photosynthetic apparatus allow conifers to occupy the temperate and subarctic Northern hemisphere, where freezing temperatures occur regularly and cold acclimation processes are essential to enable plants to withstand periods of very low temperatures (Levitt 1980). Conifers retain their needles for several

Photosynthetic Pigment Apparatus in Northern Plants

403

years and so their photosynthetic apparatus must survive severe freezing periods, often combined with high light, especially in early spring. The interaction of light and low temperature can result in the photo-inhibition of photosynthesis in nature (Martin et al. 1978, Ottander and Oquist 1991). Our data showed that the pool of carotenoids in conifers during the vegetative period consists of β-carotene, lutein, and violaxanthin. In pigment apparatus of the conifers, the high level of lutein remained fairly constant during the year. It is possible that the lutein content is related to the structural role of this xanthophyll, which is the integral component of the peripheral unit of LHC II, and on its possible antioxidant function (Kuhlbrandt et al. 1994). Accumulation of antheraxanthin and zeaxanthin in the carotenoid spectrum was observed in winter and at the beginning of spring under freezing stress combined simultaneously with high light. According to (Demmig-Adams 1990, Eskling et al. 1997) zeaxanthin is the exclusive xanthophyll that is accumulated under excess light by deep oxidation of the existing violaxanthin in the xanthophyll cycle. It is widely thought to play a photoprotective role by the dissipation of excessive light energy as heat (Niyogi 1999). Previous research has shown that zeaxanthin can quench the 1Chl* state directly through the process of nonphotochemical quenching (NPQ) (Muller et al. 2001). Antheraxanthin, another xanthophyll, has a similar function of photoprotection with zeaxanthin, and it can replace zeaxanthin in the absence of zeaxanthin (Goss et al. 1998). So, our data show that the primary photoprotective mechanism of the three evergreen conifers growing in the cold climate is the increase in the degree of the de-epoxidation state of the xanthophyll cycle and the accumulation of zeaxanthin. It provides a harmless dissipation of excess excitation energy in the photochemical system. Lutein, another xanthophyll, is important for photoprotection in conifers too. As a whole, information on pigment apparatus of different botanical and ecological groups of Northern plants was completed and summarized. The significant differentiation of species on the green and the yellow pigment contents was found. An increase of the relative content of carotenoids in row boreal—hypo-arctic—arcto-alpine species was found. New data on the light-dependent changes of xanthophyll cycle pigments (violaxanthin, antheraxanthin, and zeaxanthin) on the Northern conifers are found. The role of carotenoids in the tolerance of photosynthetic apparatus and protection against photo-oxidative damage in leaves of plants from the Northern regions is shown. It was concluded that the relatively high content of carotenoids plays a major role in the stability of the photosynthetic apparatus in cold climatic conditions. The results obtained extend our knowledge about mechanisms of adaptation of pigment apparatus and photosynthetic function realization in plants under cold climate environments.

Acknowledgment This research was supported by the Russian Foundation for Basic Research (Grant Nos. 04-0448255 and 07-04-00436).

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Schindler, C. and H.K. Lichtenthaler. 1996. Photosynthetic CO2-assimilation, chlorophyll fluorescence and zeaxanthin accumulation in field grown maple trees in the course of a sunny and cloudy day. J. Plant Physiol. 148:399–412. Verhoeven, A.S., A. Swanberg, M. Thao, and J. Whiteman. 2005. Seasonal changes in leaf antioxidant systems and xanthophyll cycle characteristics in Taxus media growing in sun and shade environments. Physiol. Plant. 123:428–434. Yatsco, Y.N., O.V. Dymova, and T.K. Golovko. 2009. Pigment complex of ever- and wintergreen plants in the middle taiga subzone of the European north-east. Botan. Z. 94(12):1812–1820. Zalenskii, O.V. 1977. Ecologofiziologicheskie aspect izucheniya fotosinteza (Ecological and physiological aspects in studies of photosynthesis). Thirty seventh Timiryazev Lecture, Leningrad, Russia: Nauka.

of the 16 Modifications Carotenoid Metabolism in Plastids: A Response to Stress Conditions Pascale Moulin, Yves Lemoine, and Benoît Schoefs Contents 16.1 16.2 16.3 16.4

Introduction...........................................................................................................................408 Properties of Carotenoids in Photosynthetic Organisms.......................................................408 Diversity of Stresses on Carotenoid Biosynthesis.................................................................408 The Violaxanthin Cycle: A Short-Term Photoprotective Mechanism...................................409 16.4.1 Violaxanthin De-Epoxidase......................................................................................409 16.4.2 Zeaxanthin Epoxidase............................................................................................... 411 16.4.3 Second Xanthophyll Cycle in Higher Plants: The Lutein-5,6-Epoxide Cycle........... 412 16.4.4 Specific Xanthophyll Cycle Involved in Diatoms, Xanthophytes, Dinophytes, and Haptophytes: The Diadinoxanthin Cycle............................................................ 413 16.4.5 Xanthophyll Cycles and Evolution of Plants and Algae............................................ 413 16.4.6 The Xanthophyll Cycle: The Mechanistic of the Cycle and Its Implications in the Chloroplast Physiology.................................................................................... 414 16.4.6.1 Role of Antenna Proteins............................................................................ 415 16.4.6.2 Change of Light Harvesting Complex Properties....................................... 416 16.4.6.3 Role for Membrane Lipids.......................................................................... 417 16.4.6.4 Xanthophyll Cycle Pool of Pigments Is Dynamic...................................... 417 16.5 Secondary Carotenoid and Apocarotenoid Biosynthesis in Algae and Higher Plants.......... 418 16.5.1 Secondary Carotenoids in Microalgae...................................................................... 418 16.5.1.1 Dunaliella sp.: A Model for the Study of β-Carotene Biosynthesis........... 418 16.5.1.2 Haematococcus pluvialis: A Model to Study Astaxanthin Biosynthesis......................................................................................... 419 16.5.2 Root Colonization by Arbuscular Mycorrhizal Fungi Triggers the Production of Secondary Carotenoids and Apocarotenoids........................................................ 420 16.5.2.1 Biosynthesis of Apocarotenoids.................................................................. 420 16.5.2.2 Roles and Functions of Secondary Apocarotenoids................................... 421 Conclusions and Perspectives......................................................................................................... 422 Abbreviations.................................................................................................................................. 422 References....................................................................................................................................... 423

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16.1  Introduction Since the emergence of life on Earth, the food chains depend on the use of solar energy by photosynthetic organisms. One characteristic feature of these organisms resides in their global lack of mobility, even thought microalgae can move in the water column. Consequently, this type of organisms cannot run away from the alterations in their biotic and/or abiotic environments, including deficiencies or excesses of light or nutrients. To cope with these alterations, photosynthetic cells activate different types of defense mechanisms, including reorientations of the plastid metabolism. Because the rapidity and efficiency of the responses differ among species or even plant cultivars, this aptitude to adapt to unfavorable conditions constitutes a major factor for their survival (Külheim et al. 2002). For instance, sun-grown leaves exhibit a greater ability to activate photoprotective mechanism, such as the xanthophyll cycle, than shade-grown plants when both types of plants are exposed to high light (Demmig-Adams and Adams 1994, 1995, 2006). In this chapter, that is an update of the data presented in the second edition of the Handbook of Plant and Crop Stress (Rmiki et al. 1999), we have reviewed the data dealing with the changes in the carotenoid (Car) and apoCar (Apocar) metabolisms in algae and plants as a response to stress.

16.2  Properties of Carotenoids in Photosynthetic Organisms Cars are C40-polyisoprenic compounds characterized by a large number of conjugated double bonds (n > 7). More than 600 Cars have been identified to date and several new ones are reported annually. Conjugated double bonds allow Cars to absorb light in the near-ultraviolet as well as in the visible region. In photosynthetic organisms, Cars are bound, together with chlorophylls (Chls), to proteins and participate to light harvesting and to energy transfer to the photosynthetic reaction centers. This last process resides on a very efficient singlet–singlet energy transfer from Cars to Chls and needs a precise arrangement of pigment molecules in the light-harvesting complexes (LHCs). Cars are also recognized to be essential for the survival of illuminated plants, since their numerous conjugated double bonds are able to quench the Chl triplet state and also to scavenge singlet oxygen and the other reactive oxygen species (ROS), which are abundantly produced during photoinhibition. This photoprotective function is generally achieved via triplet–triplet energy transfer. In addition to these well-established functions, Cars of higher plant plastids might play important structural roles in stabilizing the lipid phase of the thylakoid membranes through modification of the membrane fluidity (for a review, see Havaux 1998).

16.3  Diversity of Stresses on Carotenoid Biosynthesis Various factors (e.g., nutrient deficiency, excess light, drought, and chilling) are known to have consequences on the photosynthetic apparatus, and photoinhibition may be often observed under these environmental conditions. Actually, in their natural environment, plants are constantly confronted with reconciling an excessive energy supply with the demands of the photosynthetic carbon reduction cycle for the products of electron transport, ATP and NADPH (Osmond 1981). When light absorption by LHC exceeds both the capacity to use the photosynthetic NADPH and ATP for carbohydrate synthesis and the capacity of energy dissipation mechanisms, photosynthesis is progressively inhibited (i.e., photoinhibition phenomenon). This means that even low light levels may become excessive if combined with chilling and then may result in photoinhibition in crops or in algae. The consequences at the Car level of any stress may be multiple (for a review, see Young and Britton 1990). To cope with the absorption of excessive light and its consequences, the photosynthetic organisms have evolved a series of short-term (10–30 min) and long-term photoprotective mechanisms (Horton and Ruban 1992). Among these mechanisms, the thermal dissipation of excess absorbed light energy at the photosystem (PS) II, the so-called nonphotochemical quenching (NPQ), is believed to play a key role in regulating the light harvesting and in the prevention of photooxidative damages.

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16.4  The Violaxanthin Cycle: A Short-Term Photoprotective Mechanism Basically, the photosynthetic apparatus constitutes an optimized biological device to store the energy of light into molecules. Because the photosynthetic apparatus manipulates oxygen and light, the interaction between singlet Chl molecules and triplet oxygen is strictly avoided by a variety of photoprotective Cars bound to the proteins of the photosynthetic apparatus (Frank and Cogdell 1996), because the interaction yields to the formation of singlet oxygen, which can damage proteins, pigments, and lipids in the photosynthetic apparatus (Niyogi 1999, Asada 2006). When the capacity to harvest the light energy is higher than the capacity to use it, the actual set of photoprotective Cars is no longer able to efficiently dissipate the absorbed energy and the excess of absorbed light should be dissipated through other pathways. Early research identified a dissipation process induced by the lowering of the lumen pH, a condition triggered by high-light conditions (for reviews, see Krause and Weis 1991, Demmig-Adams and Adams 1996, Rmiki et al. 1999). A connection was subsequently made between energy dissipation and the Cars of the xanthophyll cycle (Demmig et al. 1987, Demmig-Adams 1990). The role of zeaxanthin (Zea) as a general antioxidant has been confirmed using the npq2 mutant that accumulates Zea (Niyogi et al. 1998) and shows a higher NPQ level than in the wild type (Niyogi et al. 1998, Havaux et al. 2000, Kalituho et al. 2007). Studies of Arabidopsis plants containing nearly three times the amount of xanthophyll cycle Cars present in the wild type have given further details on the antioxidant function of these pigment conversions involving their binding to proteins of the LHC family (Johnson et al. 2007). With the development of a high-pH gradient across the thylakoids, two-steps mono de-epoxidation reactions of the Violaxanthin (Vio) into Zea with antheraxanthin (Ant) as an intermediate are catalyzed by the Vio de-epoxidase (VDE) (EC 1.10.99.3) (Figure 16.1). The Vio molecules are regenerated through two light-independent epoxidation steps that are catalyzed by the Zea epoxidase (ZEP) (EC 1.14.13.90). The transformation of Vio to Zea proceeds in about 10–30 min (Sierfermann-Harms 1977) whereas the conversion of Zea to Vio is 5–10 times slower (Härtel et al. 1996, Marin et al. 1996). Both VDE and ZEP have a similar basic tertiary structure, maybe because they share a common Ant substrate and belong to the lipocalin family of proteins (Bugos et al. 1998; Hieber et al. 2000, 2002, Wang et al. 2008; for a review, see Jahns et al. 2009). Lipocalins are characterized by a similar tertiary structure and similar functions (Grzyb et al. 2006). These enzymes are supposed to contain eight antiparallel β-sheets and three highly conserved short consensus repeat motifs (Flower et al. 2000). The motif I is composed of the first of the eight β-sheets and a short fragment of the preceding α-helix. Motif II is composed of the loop between β-sheets number 6 and 7 and parts of the end of β-sheet number 6 and the beginning of β-sheets number 7. The motif III is composed of the end of β-sheet number 8 and part of the C-terminal α-helix, including the loop between both fragments. VDE and ZEP are conserved in two and only one short consensus repeat motif, respectively (Akerstrom et al. 2000), and differ by the number of amino acid residues between the motif I and II (Bugos et al. 1998). The 3D structure of lipocalins is characterized by the deep conic hollow, formed by the β-sheets. The deep conic hollow is necessary for substrate binding (Newcomer et al. 1984, Holden et al. 1987).

16.4.1  Violaxanthin De-Epoxidase The first cDNA sequence of VDE was obtained from romaine lettuce (Bugos and Yamamoto, 1996). The VDE sequences homologous to VDE from plants were identified in the genome of diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum (Bacillariophyceae). No VDE sequence was found in the red algae genome library even if some red seaweeds are able to produce Ant (Rmiki et al. 1996) or Vio (Coesel et al. 2008). For instance, Gracilaria gracilis and G. multipartita are devoid of Vio, and a xanthophyll cycle restricted to the interconversions of Ant to Zea was observed (Rmiki et al. 1996; Garcia-Plazaola et al. 2007). A sequence constituting a novel gene,

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The violaxanthin cycle Violaxanthin HO

O

HO

Epoxidation OH Diadinoxanthin HO

O

De-epoxidation

De-epoxidation

Epoxidation OH

HO

Diatoxanthin

O

OH

OH Lutein-epoxide HO

Epoxidation

O

De-epoxidation

Epoxidation OH

OH Lutein HO

HO

Figure 16.1  The different xanthophyll cycles. Under stress conditions, Vio, Ddx, or Lut-epoxide are de-epoxidized into Zea, Dtx, or Lut, respectively. After suppression of the stress conditions, the de-epoxidated xanthophylls are epoxidized.

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Zeaxanthin

The lutein-epoxide cycle

O

De-epoxidation Antheraxanthin

The diadinoxanthin cycle

OH

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named VDE-related or VDR, was identified in the genome of Chlamydomonas reinhardtii as sharing a low sequence similarity with Arabidopsis thaliana VDE. VDE-related homologs were found in higher plants and in two diatom genomes (Coesel et al. 2008). VDE is a nucleus-encoded protein. The plastid import mechanism involves a bipartite transit peptide located at the N-terminal of the preprotein sequence. The first part of the transit peptide allows the targeting of the protein to the chloroplast stroma, whereas the second part drives the thylakoid-targeting. These results confirm the location of mature VDE protein (43 kDa) in the lumen of the thylakoids (Bugos et al. 1999; for a review, see Rmiki et al. 1999). Depending on the pH, the mature VDE, that is present in very small amounts, occurs either as a soluble protein (neutral pH) or as a tightly bound protein at the lumenal surface of the thylakoid when the lumen acidifies (Eskling et al. 1997). The mutant npq1 is defective in VDE (Niyogi et al. 1998). The comparison of the VDE sequences showed similarities at two domains. The first one is a cystein-rich N-terminal domain, probably organized in α-helixes, whereas the second one is a glutamic acid-rich C-terminal domain, composed of long α-helixes (Hieber et al. 2000). This last domain would serve for the pH-dependent binding. Actually, when it is partially protonated, the binding of VDE protein to the thylakoid membrane is increased (Hieber et al. 2002, Coesel et al. 2008). In VDE, the lipocalin hollow fits with the length of Vio molecule and would explain why there is strict specificity of the enzyme for 3-OH, 5-6-epoxy Cars in configuration 3R, 5S, and 6R (Yamamoto 1979, Grotz et al. 1999). VDE requires ascorbate, under its acidic form, as a cosubstrate (Yamamoto 1979, Eskling et al. 1997, Jahns et  al. 2009). The protonated ascorbate content of the lumen is an endogenous proton and electron donor and it has been shown to activate the VDE activity in vivo in Arabidopsis (Yamamoto 1979, Neubauer and Yamamoto 1994, Müller-Moulé et al. 2002). The optimum ascorbate concentration for VDE activity is pH-dependent, that is, at pH 4.5–5.5, the enzyme becomes saturated at 10–20 mM, whereas at pH 6, the saturation level is not reached before 100 mM (Bratt et al. 1995). Ascorbate, reduced in the stroma by the glutathione cycle, which consumes NADPH and glutathione, may cross the thylakoid membrane and regulate the activity of PSII (Foyer et al. 1989). The existence of a membrane transporter for negatively charged ascorbate in exchange for dehydroascorbate was postulated (Bratt et al. 1995, Eskling et al. 1997). The VDE activity is reversibly inhibited by dithiothreitol through the reduction of the disulfide bonds in the enzyme if the inhibitor is added at pH 5.2–5.7 (Yamamoto and Kamite 1972, Bugos et al. 1999, Coesel et al. 2008).

16.4.2  Zeaxanthin Epoxidase The first ZEP cDNA, originally named as ABA2, was isolated from tobacco (Nicotiana plumbaginifolia) using the insertional mutagenesis technique (Marin et  al. 1996, Audran et  al. 1998, Wang et al. 2008). Later, this cDNA was used to allow the isolation of other ZEP sequences from other dicotyledon plants like pepper (Capsicum annuum), tomato (Lycopersicon esculentum), Arabidopsis, and apricot (Prunus armeniaca) (Hieber et  al. 2000), monocotyledon plants, green algae, diatoms, and Tetrahymena thermophila—a ciliate organism (Coesel et al. 2008). The mutant npq2 of A. thaliana is devoid in ZEP (Niyogi et al. 1998). ZEP preprotein contains a chloroplast transit peptide that is cleaved during the targeting into the chloroplast stroma (Audran et  al. 1998). The comparison of ZEP mature protein sequences indicated that, in addition to the lipocalin typical domain, the sequences also share a flavoprotein monooxygenase consensus domain similar to the prokaryotic aromatic-substrate monooxygenases (Hieber et al. 2000). The sequence has a phosphopeptide-binding domain, which could be involved in protein–protein interactions (Coesel et al. 2008, Wang et al. 2008). The ZEP enzyme, that is active at the stromal side of thylakoids, with an optimum pH around 7.5, requires FAD, O2, NADPH, and “ferredoxin-like” reductives as cosubstrates for the epoxidation reaction (Hager 1975, Siefermann and Yamamoto 1975a, Siefermann and Yamamoto 1975b, Siefermann and Yamamoto 1975,

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Wang et al. 2008). So far, no ZEP protein could be purified from organisms. Actually, the regulation of ZEP activity is not well understood but it has been shown that downregulation may occur under extreme photooxidative stress conditions in the short- (Jahns 1995, Reinhold et al. 2008) or long term (Adams et al. 2002). Even if the ZEP tomato gene was expressed preferentially in chlorophyllous tissue, the ZEP transcripts were also detected in flowers, roots, and fruits (Wang et al. 2008). In the nonchlorophyllous tissues of Arabidopsis, Zea epoxidation is involved in the abscissic acid biosynthesis (Audran et al. 2001). Diatoms that do not produce this phytohormone also use ZEP to synthesize fucoxanthin, their major light-harvesting Car, is realized at the expense of Vio (Lohr and Wilhelm 2001).

16.4.3  Second Xanthophyll Cycle in Higher Plants: The Lutein-5,6-Epoxide Cycle In maturing tomato fruit (Lycopersicum esculentum Mill. cv Moneymaker), besides a typical Vio cycle, a second cycle of lesser amplitude was observed between Lutein (Lut) and Lut monoepoxide (Rabinowitch et al. 1975) (Figure 16.1). More recently, operation of this new type of xanthophyll cycle was observed in two parasitic angiosperms, that are, Cuscuta reflexa (Bungard et al. 1999) and Amyema miquelii (Matsubara et al. 2001) but not in the roots of other parasitic organisms such as Arceuthobium sp. and Phoradendron sp. (Ladygin 2008). This cycle was later found in maturing green fruits of tomato and in leaves of eight Quercus species (Garcia-Plazaola et al. 2002a). A deepoxidase is involved in this cycle allowing, under a strong light, the conversion of Lut-5,6-epoxide into Lut, whereas under low light, the reverse reaction goes back but at a very slow rate. One important question that remains is to determine which enzymes are involved in the Lut-Lut-epoxide cycle. In the one hand, no enzyme implicated in the Lut-5,6-epoxyde cycle was clearly identified (Giuliano et  al. 2008) but in the other hand, VDE could catalyze, in addition to the Vio-de-epoxidation, the conversion of Lut-5,6-epoxide into Lut in vitro (Grotz et al. 1999, Goss 2003, Ladygin 2008). In addition, the β-ring of Lut may be a substrate for ZEP (Hieber et al. 2000). Thus, the Lut-epoxide cycle is probably driven by the same set of enzymes that the Vio cycle. The species in which the Lut-epoxide cycle has been studied range into three groups according to their Lut-epoxide cycle pool of pigments (for more details, see Garcia-Plazaola et al. 2007): (i) species such as Quercus subpyrenaica (Abadia et al. 1996), Q. ilex (Llorens et al. 2002), and Viscum album (Matsubara et  al. 2003) have very low amounts (1–10 mmol mol−1 Chl) and Lut-epoxide/VioAntZea ratio < 0.1; (ii) species such as Q. robur (Garcia-Plazaola et  al. 2002a), the mistletoe A. miquelii Thiegh (Matsubara et al. 2003), and Acacia melanoxylon (Watson et al. 2004) contain high amounts of Lut-epoxide and the Lut-epoxide/VioAntZea ratio ranges between 0.1 and 1; and (iii) leaves from shade plants such as the tropical tree Inga sapindoides (Matsubara et al. 2005), Laurus nobilis, and Umbellaria californica (Estaban et al. 2007) have a high pool of Lut-epoxide, up to 78 mmol mol−1 Chl with a Lut-epoxide/VioAntZea > 1. The frequent Lut-epoxide accumulation in shade leaves relies on a slow epoxidation of Lut by ZEP and on the absence of VDE activity in such conditions. The extremely slow conversion of Lut to Lut-epoxide has been suggested to result from a low affinity of ZEP for Lut (Pogson and Rissler 2000) or it could be due to the low availability of this substrate, which is mainly restricted to the intraprotein Lut sites of Lhcb (Morosinotto et al. 2002, Dekker and Boekema 2005). In their review on the occurrence and possible functions of the Lut-epoxide cycle, Garcia-Plazaola et  al. (2007) proposed a model describing the relationships between the Lut-epoxide and Vio cycles. In deep shade, both Vio and Lut epoxide may occupy the Vio1 site while the Lut2 site may be occupied by Lut-epoxide instead of Lut, giving rise to very efficient light-harvesting PSII antenna. After exposure to strong light, the displacement of Lut-epoxide by Lut in Vio1, Lut2, and most Lut1 sites gives efficient energy dissipation centers. A greater pool of Lut derived from Lut-epoxide de-epoxidation as compared with Ant and Zea derived from Vio offers the possibility of a rapid and strong engagement of NPQ. Photoprotection may then be stabilized within hours of exposure to strong light by simultaneous engagement of both cycles, the slowly reversible conversion of Lut being proposed to “lock-in” a

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primary mechanism of photoprotection (Matsubara et al. 2005). Thus, the Lut-epoxide cycle could serve as an additional, more slowly relaxing mechanism, to accelerate and sustain the development of NPQ (Garcia-Plazaola et al. 2003).

16.4.4  S pecific Xanthophyll Cycle Involved in Diatoms, Xanthophytes, Dinophytes, and Haptophytes: The Diadinoxanthin Cycle The diadinoxanthin (Ddx) cycle involves the Ddx de-epoxidase (DDE), which de-epoxidises the epoxy-xanthophyll Ddx, whereas the Dtx epoxidase (DEP) catalyzes the back reaction (Wilhelm et al. 2006, Garcia-Plazaola et al. 2007) (Figure 16.1). Several publications have reported on the detection and the regulation of DDE and DEP activities in diatoms (Jakob et al. 2001, Goss et al. 2006, Kroth 2007). Homologous sequences to DDE and DEP genes were found in the genome database of two diatoms, T. pseudonana and P. tricornutum, and were named VDE-like because they are distantly related to the plant VDE. VDE-like enzyme could be located differently than the VDE protein because the C-terminal region, which could be important for binding the protein to the thylakoid membrane, is uncharged. Coesel et al. (2008) have proposed that VDE-like enzyme could be implicated in the Ddx cycle of these diatoms. In the same way, the ZEP gene product is implicated in the reverse reaction. Compared with the enzymes involved in the Vio cycle, much less is known about the Ddx cycle enzymes from algae. The DDE, homologous of VDE, exhibits a different pH-dependance for its activation, which is shifted by at least 0.7 pH unit toward higher pH values (Jakob et al. 2001). The affinity of DDE for the cosubstrate ascorbate is three to four times higher than in the case of VDE (Grouneva et al. 2006). DEP, which catalyzes the back conversion of Dtx to Ddx is almost inhibited under high-light conditions (Mewes and Richter 2002) and can be also inhibited by cadmium (Bertrand et al. 2001, for reviews see Bertrand and Poirier 2005, Poirier et al. 2008; see also the chapter by Solymosi and Bertrand). Lohr and Wilhelm (1999) have shown that besides the Ddx cycle, some diatoms may also display a Vio cycle, even if the pool size of pigments concerned with the Vio cycle is rather low. Another xanthophyll cycle has been proposed to operate in green algae, involving L-siphonaxanthin, a xanthophyll present in the siphonaceous genera, in Ulva olivescens (Levavasseur 1981) and in a few Prasinophyceae (Latasa et al. 2004). In Codium fragile and Cladophora opaca, Vershinin and Kamnev (1996) observed a reversible light-induced de-esterification of siphonaxanthin dodecenoate ester (wrongly identified as siphonein B (Britton et al. 2004)), with siphonaxanthin accumulation in high light and siphonaxanthin dodecenoate ester in low light. More recently, Raniello et al. (2006) described another xanthophyll cycle operating in addition to the Vio cycle in the shallowest populations of the invasive alga Caulerpa racemosa var. cylindracea. Interconversions between Lut and siphoxaxanthin were shown to occur along the day with Lut and siphonaxanthin highest content under high- and low irradiances, respectively. No variations of the siphonaxanthin dodecenoate ester content could be observed by these authors. Thus, besides the conventional Vio and Ddx cycles, which have been exhaustively studied along the last 40 years, several other xanthophyll cycles have been described more recently, which often operate in parallel with the Vio cycle and participate to photoprotection.

16.4.5  Xanthophyll Cycles and Evolution of Plants and Algae As no homolog of VDE protein have been found in prokaryotes, it could be possible that this protein represents an ancient eukaryotic innovation (Coesel et al. 2008). Actually, cyanobacteria cells seem to be totally devoid of any xanthophyll cycle (Lemoine et al. 1993), even if many taxons, such as Spirulina, have a high Vio content. However, the lipocalin protein membership of VDE and ZEP suggests that they may have evolved from lipocalin genes of cyanobacterial ancestors that developed an endosymbiotic relationship with a eukaryotic host cell to form the chloroplast of algae and

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higher plants (Durnford et al. 1999, Palmer 2003, Charron et al. 2005). The presence of Vio and its precursors Ant and Zea was frequently observed among Rhodophyte species (Schubert et  al. 2006) and operation of a total Vio cycle was proved in G. birdiae (Ursi et al. 2003). Even if the only Rhodophyte species in which the Vio cycle was reported belong to the Gracilariales (Rmiki et al. 1996, Ursi et al. 2003), the Vio cycle pigments were also detected in other orders such as the Ceramiales and the Corallinales (Schubert et al. 2006). These observations are important on an evolutive point of view because they suggest that the epoxy-Cars involved in the Vio cycle were present early in the evolution of red algae, before their diversification and before the divergence of the Rhodophyta and Chlorophyta from the ancestral chloroplast (Durnford et al. 1999). A truncation of the Vio cycle was observed in the Prasinophycean alga Mantoniella squamata Manton and Parke (Goss et al. 1998). In this primitive green alga, the Vio cycle is limited to the Vio-Ant interconversions because of an extremely slow second de-epoxidation step from Ant to Zea due to a reduced affinity of Mantoniella VDE for Ant and a much faster epoxidation rate in the algae than in higher plants (Frommolt et al. 2001). This implies that the role of Zea in qE, the pH or energy component of NPQ (Müller et al. 2001) can be replaced by Ant in this alga. Formation of Zea can, however, be obtained by artificially low-pH treatment (Goss et al. 1998). Lut-epoxide is widespread among photosynthetic organisms and plant tissues. Its absence in algae discards an ancestral origin. Furthermore, it seems that Lut-epoxide concentration is not phylogenetically determined and would depend on ecological constraints as no correlation was found between its presence and recurrent mutations in ZEP (Esteban et al. 2009). Altogether, the xanthophyll cycle operation seems involved in nearly all photosynthetic eukaryotes (terrestrial and marine) in the photoprotection of chloroplast membranes, with the possible exception of the Cryptophyceae class of algae (Stransky and Hager 1970; Rmiki et al. 1999, Ladygin 2008).

16.4.6  The Xanthophyll Cycle: The Mechanistic of the Cycle and Its Implications in the Chloroplast Physiology The lumen pH is higher than 7.0 and VDE is supposed to be mobile within this compartment (Hager and Holocher 1994). When the light absorption by LHC exceeds both the possibility for its utilization by the transducers and the capacity of energy dissipation mechanisms, lumen acidification takes place. When the pH value is below 6.5, VDE binds to the thylakoid membrane and is activated (Sierfermann and Yamamoto 1975b, Hager and Holocher 1994). The process occurs in a highly cooperative manner, the pH dependence of Vio de-expoxidation exhibiting a cooperativity for protons of about 5.5 and an inflexion point around pH 6 (Pfündel and Dilley 1993, Jahns and Heyde 1999; for a review, see Horton et al. 2008). VDE activity is also regulated by the proton concentration. The highest activity has been observed for pH values lower than 5.8. The acidity also controls the equilibrium between the basic and acidic forms of the cosubstrate ascorbate (Bratt et al. 1995, Eskling et al. 1997). Anyway, the lumen pH cannot fall below 5, otherwise the electron transport at the cytochrome b6/ f complex is inhibited (Horton et al. 2008). Once Ant produced, VDE transfers an electron and a proton from ascorbate to the epoxy group located at the other side of Ant molecule, creating Zea, water, and dehydroascorbate. This implies that the xanthophyll cycle includes a flip-flop movement of the monoepoxidated xanthophyll in the thylakoid membrane. Altogether, the kinetics of the de-epoxidation is likely to be limited by the xanthophyll diffusion within the membrane (Macko et  al. 2002). Indeed, the sorting of the Vio molecules from the proteins and their diffusion within the lipid phase to VDE for conversion to Ant exhibit a four to six times higher rate constant than the conversion of Ant to Zea (Yamamoto and Higashi 1978, Härtel et al. 1996), and thus represents the rate-limiting step of de-epoxidation (for a review, see Jahns et al. 2009). Despite the fact that the expression of the gene encoding VDE has been shown to vary in response to light intensity (Bugos et al. 1999), the control of VDE activity through the modification of the gene expression seems minor (Macko et al. 2002, Deng et al. 2003).

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The regulation of the epoxidation reactions is limited and does not involve a control by the stromal pH (Takeguchi and Yamamoto 1968, Siefermann and Yamamoto 1975a). ZEP can be downregulated after exposure of plants to high light (Jahns and Miehe 1996, Verhoeven et  al. 1996, Reinhold et  al. 2008) or under overwintering conditions (Öquist and Huner 2003). The biochemical basis of the regulation is so far unclear but would involve ZEP phosphorylation (Xu et al. 1999; for a review, see Jahns et al. 2009). A linear relationship has been found between the amount of Zea produced and the capacity for energy dissipation. This dissipation is reflected in the NPQ. NPQ is formed by several components and is therefore kinetically heterogeneous (Horton and Hague 1988). In moderate excess light, that is, in absence of accumulated damaged PSII reaction centers, NPQ is found to form and relax with two major components, reflecting events taking place in the antenna of PSII. These two components are qE (Müller et al. 2001) and the slowly reversible component of NPQ, namely, qI (Jahns and Miehe 1996, Verhoeven et al. 1996). In the mutant npq1, which is defective in VDE, most of qE is inhibited (Niyogi et al. 1998), while the double mutant npq1 lut2 (the mutant lut2 is unable to synthesize Lut (Pogson et al. 1998)) is devoid in qE, suggesting a role for Lut in qE (Niyogi et al. 2001) or that alteration in the Lut content could indirectly affect qE by disturbing the assembly and structure of the PSII antenna (Niyogi et al. 1997, Pogson et al. 1998, Lokstein et al. 2002). Thus, the Zea molecules produced through the xanthophyll cycle play a crucial role in the NPQ efficiency. Besides the roles of the xanthophyll cycle in the qE component of NPQ, there are evidences for the involvement of Zea in (i) the photoprotection of the photosynthetic membranes toward stress-induced peroxidations (Havaux and Niyogi 1999, Havaux et al. 2000, Baroli et al. 2003, Havaux et al. 2007, Johnson et al. 2007). Indeed, it was demonstrated that photoinduced peroxidative damage in leaves is highly increased if the Vio cycle operation is inhibited by the addition of dithiothreitol (Sarry et al. 1994); (ii) the control of thylakoid membrane fluidity because a light-induced decrease in membrane fluidity occurs in thylakoid membranes (Gruszecki and Strzalka 1991, Havaux and Gruszecki 1993) when Vio is allowed to convert in Zea under stress, and thus the thermostability of the thylakoid membranes increases (for a review, see Gruszecki and Strzalka 2005). The LHCII prepared from illuminated leaves was shown to be poorer in xanthophyll cycle pigments than the LHCII prepared from dark-adapted leaves (Havaux and Gruszecki 1993). A diffusive displacement of Zea from the Chl–protein (CP) complexes to the surrounding lipid domain (Gruszecki 1995, Havaux and Tardy 1995) has been suggested to increase membrane rigidity; (iii) the regulation of the cyclic electron flow around PSII. In fact, when the Vio content is high, the cyclic flow of electrons around PSII is reduced (Gruszecki et al. 1995); (iv) the removal of active oxygen species formed during a stress through Zea epoxidation (Lichtenthaler and Schindler 1992, Schubert et al. 1994). The Vio formed could be again de-epoxidated. In such a case, the epoxidation could be nonenzymatic (Schubert et al. 1994). Altogether, the operation of the xanthophyll cycle is strongly dependent on the antenna, the properties of the thylakoids, and on an active Car biosynthetic pathway. These aspects are detailed in the next paragraphs. 16.4.6.1  Role of Antenna Proteins When the proteins of the photosynthetic apparatus are separated by nondenaturing electrophoresis, Vio is found as a rather high proportion in the free pigment fraction, indicating that Vio is very weakly bound to these proteins (Lee and Thornber 1995). On the other hand, Vio is present at the periphery of the LHC (Kühlbrandt et al. 1994). Thus, the lumen acidification would favor Vio liberation from Vio-binding site in the different peripheral antenna proteins. Thus, the peripheral and weak binding of Vio are therefore two conditions allowing its involvement in the xanthophyll cycle (Demmig-Adams and Adams 1996), whereas Zea would have to return into the LHC to quench the energy (for a review, see Havaux 1998). The resolution of the crystal structure of LHCII from spinach and pea and the discovery of Vio binding to it (Liu et al. 2004) allowed to get more information on the participation of pigment–protein

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complexes in NPQ. Most evidence is given that PSII is dimeric in the appressed grana parts of the thylakoid membranes of green plants. This aggregated state may be stabilized by protein phosphorylation (Kruse et al. 1997), binding with phosphatidylglycerol (Kruse et al. 2000) and with extrinsic proteins (Boekema et al. 2000). PSII antennae associated with dimeric PSII core complexes constitute PSII–LHCII supercomplexes containing two to four copies of trimeric LHCII complexes (LHCb1–3 proteins) per PSII core dimer, together with three “minor” antenna complexes: LHCb4 (CP29), LHCb5 (CP26), and LHCb6 (CP24). Each protein binds at least one Vio molecule in the dark-adapted state (for a review, Dekker and Boekema 2005). Most of the Vio molecules are bound by the trimeric antenna complex LHCII at the peripheral Vio1 site (Liu et  al. 2004), while the “minor” monomeric complexes CP29, CP26, and CP24 bind one to two Vio molecules (Ruban et al. 2002), one of which may be at the internal Lut2 site that binds Lut in LHCII (Morosinotto et al. 2002, Wehner et al. 2006). Upon exposure to excess light, VDE leads to the appearance of Ant and Zea. Vio binding from site Vio1 is pH dependent and it is released to provide a source of substrate for VDE. Vio is also liberated from the minor complexes providing empty Car-binding sites for newly formed Zea (Morosinotto et al. 2003). The liberated Vio molecules are dissolved in the thylakoid membrane lipids. Their de-epoxidation by the lumenal enzyme VDE needs the binding of VDE to the thylakoid membranes to provide access to its substrate. Monomeric antennae, especially CP26 and CP24, show high binding rates and these proteins are probably one of the sites where Zea plays its function (Morosinotto et al. 2003). Some Zea binding to the peripheral LHCI antenna of PSI was also observed (Morosinotto et al. 2002), suggesting a PSI response to the environmental stress conditions. 16.4.6.2  Change of Light Harvesting Complex Properties The detailed relationship between qE and the operation of the xanthophyll cycle is still unknown. Actually, the biophysical mechanism of energy quenching in qE remains controversial because two mechanisms, although nonexclusive, have been proposed. The first model implies a direct role of Zea in the antenna via the formation of a quenching complex with a short fluorescence lifetime (Gilmore et al. 1995). Zea operates in the quenching through a charge-transfer mechanism (Holt et al. 2005) in the minor LHC complexes associated with PSII, that is, CP29 (Lhcb4), CP26 (Lhcb5), and CP24 (Lhcb6) (Ahn et al. 2008, Avenson et al. 2008, 2009). The charge-transfer mechanism involves an energy transfer from closely coupled Chl and Zea molecules that transiently produce pigment ions, a Chl anion and a Zea cation, respectively (Frank et al. 1994). The subsequent charge recombination dissipates the excitation energy as heat. In the second model, Zea is not required for qE (Rees et al. 1989, Horton et al. 1996, Havir et al. 1997) but is involved as an indirect allosteric regulator (Crouchman et al. 2006, for a review, see Horton et al. 2008) of the pH sensitivity of qE (Noctor et al. 1991). In this model, qE arises from a conformational change (Horton et al. 1991) in the peripheral, trimeric antenna of PSII (Pascal et al. 2005) and its mechanism involves an energy transfer from Chl to Lut1 (Ruban et al. 2007). Evidences that these events are of a cooperative nature, that is, involving interactions between subunits of PSII antenna have been presented (Noctor et al. 1991, Ruban et al. 2001). The interactions between LHCII proteins are influenced by divalent cations. The influence of the changes in their concentration of the curve qE versus lumen pH supports the cooperative nature of qE (Noctor et al. 1993). According to the model, the LHCII can exist under four states (Horton et  al. 1991; for a review, see Horton et al. 2008). During the transition State I to State III, the lumen acidifies rapidly and LHC proteins are protonated. The intensity of the lumen acidification is, however, insufficient to saturate qE. The transition State III and State IV occurs while VDE takes place and the qE slowly builds up. When the light source is turned off, qE relaxes following the collapse of the ΔpH. If the light source is again switched on, qE increases very rapidly to its maximum capacity because the Zea molecules formed during the previous illumination keep the photosynthetic apparatus “light activated”. In this case, the system shifts rapidly from State II to State IV, allowing photosynthesis and energy dissipation through qE to function at its maximal rates (Horton et al. 1991; for reviews,

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see Horton et  al. 1996, Horton et  al. 2008). Using the double mutant npq1 lut2, Li et  al. (2009) obtained evidences for the first mechanism and also for the involvement of Lut in qE. Among the additional factors involved in both mechanisms, PsbS, a small Lhc-like protein, was shown to have a key role in the dissipation of excess energy in higher plants (Li et al. 2000) and there is evidence that its level is correlated with the extent of NPQ (Li et al. 2002). Upon lowering of the lumenal pH, a rapid response, prior to xanthophyll cycle operation, called quenching 1 by Morosinotto et al. (2003) was shown to involve protonation of luminal exposed acidic residues of PsbS. More recently, PsbS has been proposed to form a quenching complex together with LHCII and Zea (Bonente et al. 2008). Thus, PsbS would act as a sensor of lumen acidification that triggers conformational changes in the PSII antenna such as LHC aggregation (Garab et al. 1988, Croft and Yerkes 1994, Horton et al. 1994, Ruban and Horton 1994, Walters et al. 1994, Ruban et al. 1997), triggering a more efficient Chl deexcitation (Ahn et al. 2008, Avenson et al. 2008, Horton et al. 2008). The aggregation may be stimulated by the addition of Zea and inhibited by Vio (Ruban et al. 1997). The action of the pigments would be mediated by their difference in hydophobicity, that is, Zea is much more hydrophobic than Vio (Ruban et al. 1993, Darko et al. 2000). This would fit with the fact that the transmembrane organization of the xanthophyll cycle, with VDE in the thylakoid lumen and ZEP located on the stromal side, implies a certain freedom of movement of the xanthophyll cycle pigments in the membrane lipid to allow contact between the enzymes and the head group of the xanthophylls. A role for Elips and Ohps, two groups of PsbS-related stress-induced proteins, was suggested in the xanthophyll cycle-associated photoprotection (Demmig-Adams et al. 2006, Zarter et al. 2006). 16.4.6.3  Role for Membrane Lipids Activity in vitro of VDE was shown to require MGDG, the main lipid of thylakoid membranes (Yamamoto and Higashi 1978), and the essential role of this lipid for VDE activity in liposomes was further confirmed more recently (Goss et al. 2006). Because Vio molecules are liberated in the lipid phase of membranes, VDE needs to bind to the membrane for its activity. It was proposed that VDE would attach to MGDG-enriched membrane domains where de-epoxidation would occur (Latowski et al. 2002). In liposomes composed of phosphatidylcholine and MGDG, as in native thylakoid membranes, inverted hexagonal structures (H II) have been described (Quinn and Williams, 1983). In such liposomes, VDE activity depends on the rate of lateral diffusion to MGDG domains and thus on the MGDG/Vio ratio (Latowski et al. 2002). The presence of (HII) phases in the MGDG domains facilitates the flip-flop movement of the Ant molecule to allow VDE access to the second epoxy group and conversion of Ant to Zea. An updated model for the mechanism of xanthophyll conversions was recently proposed by Jahns et al. (2009). In this model, the presence of HII domains and Vio molecules free in MGDG phases are required. They also propose that the transition to the light-adapted state is accompanied by aggregation of LHCII complexes, which facilitates the liberation of Vio molecules from the external Vio1-binding sites. After binding of VDE to the MGDG phases, Vio is de-epoxided into Zea. The decreased solubility of Zea in MGDG compared with phosphatidylcholine facilitates reentrance of Zea in the bilayer phase of the membranes where the antenna proteins are located. The binding of Zea to the minor antenna proteins of PSII (Lhcb4–6) could facilitate the dissipation of energy via a Zea cation radical. 16.4.6.4  Xanthophyll Cycle Pool of Pigments Is Dynamic So far, we described the xanthophyll cycle as a device catalyzing pigment interconversions. There are more and more results suggesting that, in fact, the amount of pigments in the xanthophyll cycle pool depends on the environmental conditions but is also adjusted quantitatively as a function of the stress intensity (Esteban et al. 2007). For instance, in the two Pheophyceae, Pelvetia caniculata and Laminaria saccharina, the amounts of Zea accumulated through the operation of the xanthophyll cycle operation after a light stress are very different on a Chl a basis. Such a difference has been shown to be one of the main factors responsible for the specific distributions of these two species at opposite levels on the seashore (Harker et al. 1999). Duckweed (Lemna minor) plants grown under

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high light present a higher xanthophyll cycle pool of pigments and also a higher proportion of transformable Vio than the plants grown under low light. When plants were transferred to a high-light environment, a quick adjustment of the Car composition was observed. It mostly concerns the pigments involved in the xanthophyll cycle. This increase in pigments results of a de novo synthesis of Cars because in the presence of norflurazon, an inhibitor of carotenogenesis, this increase is inhibited. The extent of this light-induced synthesis is proportional to the light treatment and also on the operation of the xanthophyll cycle. Accordingly, the inhibition of the xanthophyll cycle by dithiothreitol also abolished the light-induced carotenogenesis (Garcia-Plazaola et al. 2002b).

16.5  Secondary Carotenoid and Apocarotenoid Biosynthesis in Algae and Higher Plants Cars are found in all photosynthetic organisms and in some bacteria and fungi (for a review, see Almeida and Cerda-Olmedo 2008, Lemoine et al. 2008). While primary Cars constitute functional components of the photosynthetic apparatus, the secondary Cars are accumulated in oil droplets in the cytoplasm or in the plastids. Car synthesis has been investigated since the mid-1960s and the genes for almost all the enzymes involved in this biosynthesis have been cloned and analyzed in various organisms (Ye et al. 2008). Two distinct pathways may produce the isoprenoid precursor, isopentenyl diphosphate (IPP): the cytosolic mevalonate pathway and plastidic nonmevalonate or methylerythritol 4-phosphate (MEP) pathway (Das et al. 2007). The MEP pathway seems to be the favored way to synthesize Cars in higher plants (DellaPenna and Pogson 2006) and the acetate/mevalonate pathway was shown to be absent in chlorophytes (Schwender et al. 2001). The condensation of glyceraldehyde-3-phosphate and pyruvate gives the 5-carbon compound IPP and its isomer dimethylallyl pyrophosphate. A series of condensation reactions of one dimethylallyl pyrophosphate molecule and three IPP molecules gives the 20-carbon geranyl diphosphate, which produce the 40-carbon Car precursor phytoene. Successive desaturation reactions catalyzed by phytoene desaturase and ζ-carotene desaturase yield lycopene. Lycopene is cyclized into β-carotene, a nonoxygenated Car, by the lycopene β-cyclase (LCY-B). The oxygenated derivatives of carotenes are designated as xanthophylls—the key components of the LHCs (DellaPenna and Pogson 2006). The Car metabolism is not limited to the chloroplast in photosynthetic organisms since β-carotene oxidation into astaxanthin occurs in the cytosol of Haematococcus pluvialis, and it has been shown in Marchantia polymorpha that the complete synthesis of Cars occurs in cytoplasmic oil bodies (Suire et al. 2000). Xanthophylls can also be metabolized further into Apocars.

16.5.1  Secondary Carotenoids in Microalgae Despite the fact that several green microalgal species such as H. pluvialis, Dunaliella sp., Neochloris wimmeri, Scenedesmus vacuolatus, Scotiellopsis oocystiformis, Chlorella zofingiensis, and Protosiphon botryoides are able to accumulate secondary Cars under stressful environmental conditions (Kopecky et al. 2000, Orosa et al. 2000), most of the studies have been performed on Dunaliella sp. and H. pluvialis, probably because these two taxons are used for the commercial production of Cars (e.g., Zhang et al. 1999, Das et al. 2007). 16.5.1.1  Dunaliella sp.: A Model for the Study of 𝛃-Carotene Biosynthesis Dunaliella salina, a halotolerant alga, accumulates large amounts of β-carotene (up to 10% of the dry algal biomass) under high light intensity, high salinity, high temperature, heavy metal stress, and/or insufficient nitrogen and phosphate conditions (Ramos et  al. 2008). The other Cars synthesized are α-carotene, Lut, Zea, cryptoxanthin, and neoxanthin. The cell color of Dunaliella changed from green to orange-red following Car accumulation (Lamers et  al. 2008). Because D. salina accumulates high amounts of β-carotene under stress conditions, the culture of this alga

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is the most important process for natural production of this Car. This alga thus represents a useful model to understand the mechanism of Car formation (Ye et al. 2008). Enzymes and intermediates in the nonmevalonate pathway are seldomly analyzed, but more information would be obtained after the release of the genome sequence now under sequencing on the DOE Joint Genome Institute Web site (http://www.jgi.doe.gov/). In biflagellate and motile cells and especially under carotenogenic conditions, the β-carotene was accumulated as lipid globules in the interthylakoid spaces of the cell’s single chloroplast. This membrane-free lipid globule exclusively contained more than half of β-carotene, neutral lipids, and a small amount of a protein (38 kDa) called the carotene globule protein that could stabilize the lipid globule within the chloroplast (Hadi et al. 2008, Ye et al. 2008). Car synthesis in Dunaliella has recently been shown to proceed via the MEP pathway (CapaRobles et al. 2009). Key sequences of the carotenogenesis production were isolated from a few species of Dunaliella taxon and sequenced: 1-deoxyxylulose-5-phosphate synthase (DXS), responsible of the first step in the biosynthesis of IPP, phytoene synthase, phytoene desaturase, and LYC, which catalyzes cyclization at both ends of the linear lycopene to form cyclic β-carotene (Yan et al. 2005, Sanchez-Estudillo et al. 2006, Zhu et al. 2008). During environmental stress, the generation of ROS, which damage proteins, carbohydrates, and DNA, triggers the accumulation of internal antioxidants such as β-carotene in Dunaliella (Murthy et al. 2005, Ye et al. 2008). The 9-cis stereoisomer is more efficient than all-trans-β-carotene in scavenging of ROS (Ye et al. 2008). These properties of the Cars explained in part why the medical science used these molecules against many kinds of human diseases (Handelman 2001, HigueraCiapara et al. 2006). Triacylglycerol biosynthesis and the carotenogenesis were shown to be interdependent in Dunaliella (Rabbani et al. 1998), newly formed lipid droplets being necessary for the accumulation of β-carotene. Lipid metabolism is also affected by the increase of the volume of Dunaliella cells because they are surrounded by a thin elastic membrane, and the membrane composition changes under stress conditions (Murthy et al. 2005, Ye et al. 2008). 16.5.1.2  Haematococcus pluvialis: A Model to Study Astaxanthin Biosynthesis The main factors inducing astaxanthin accumulation in H. pluvialis are low nitrate, high light, and high temperature (Lemoine et al. 2008). Several genes, such those coding IPP isomerase, phytoene synthase (PSY), phytoene desaturase (PDS), lycopene β-cyclase (LCY-B), and β-carotene ketolase, involved in the Car biosynthesis are activated under stress conditions. Astaxanthin biosynthesis is the most studied secondary Car and is produced by α-protobacteria, fungi, and plants (Tao et al. 2006, Martin et al. 2008). Astaxanthin is synthesized from β-carotene but the biosynthesis pathways in bacteria, fungi, algae, and plants differ (Martin et al. 2008). In H. pluvialis, the first astaxanthin molecules are synthesized at the expense of the β-carotene molecules taken from the photosynthetic apparatus. To sustain the large accumulation of astaxanthin molecules, a de novo β-carotene synthesis is triggered when the β-carotene pool, present before the stress, is almost exhausted (Schoefs et al. 2001). Regardless of their origin, the β-carotene molecules are exported in the cytoplasm in lipid vesicles where they are transformed into astaxanthin that are, in turn, esterified with fatty acid molecules (for a review, see Lemoine et al. 2008). The astaxanthin biosynthesis route is now quite well defined: β-carotene is transformed into echinenone after ketolation with either canthaxanthine or hydroxy-echinenone as a first intermediate and further hydroxylation of adonirubin (Schoefs et al. 2001). The use of a pharmacological approach has shown that the other routes via adonixanthin are poorly performed (Fraser et al. 1998; Hirschberg 1998, Schoefs et al. 2001). There is at least a β-carotene hydrolase belonging to the cytochrome P-450 monooxygenase protein superfamily that is involved in this pathway (Schoefs et al. 2001). In the fungus Xanthophyllomyces dendrorhous, the conversion of β-carotene into astaxanthin could be catalyzed by a single multifunction β-carotene oxidase (Ojima et  al. 2006) by the combined activity of a β-carotene hydroxylase and a β-carotene ketolase working successively on different intermediates (Martin et al. 2008).

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Some Car could be produced through chemosynthetic pathway but they are devoid of stereoisomers. To produce Cars, researchers used engineered microorganisms such as E. coli and yeast or transgenic plants such as Oryza sativa employing transformation with carotenogenic genes (Ye et al. 2008). The carotenogenesis genes of D. salina and H. pluvialis may promote massive accumulation of Cars in heterologous expression systems. The genome of D. salina could be used as a tool to identify and find the sequences of enzymes such as the isomerase involved in the formation of Cars with the active configuration.

16.5.2  Root Colonization by Arbuscular Mycorrhizal Fungi Triggers the Production of Secondary Carotenoids and Apocarotenoids Most of land plants form mutualistic association or symbiosis with soil-borne fungi. The arbuscular mycorrhizal symbiosis is the most frequently encountered since it concerns ca. 80% of the plants and 90% of crop plants. In arbuscular mycorrhizal symbiosis, the fungus colonizes root cortical cells forming highly branched structures, the so-called arbuscule, that facilitates the uptake of the soil nutrients such as phosphate, nitrate, or water, while the plant partner provides the fungus with photosynthetically synthesized carbohydrates (up 20% of plant-fixed carbon) (Parniske 2008). The formation of the arbuscules leads to a complex and profound reorganization of the subcellular structures of the cortical cells including a massive proliferation of the plastids of the colonized root cortical cells. These plastids are connected to each other by stromules (Köhler et al. 1997, Tobin 1997; for a review, see Fester 2008), forming a dynamic network that is formed and degradated with the formation and the senescence of the arbuscules (Fester et al. 2001, Strack and Fester 2006; for reviews, see Bonfante-Fasolo 1984, Gianinazzi-Pearson 1996). In root plastids, different sets of enzymes catalyze numerous biochemical processes such as those leading to the production of fatty acids, amino acids, purines and pyrimidines, nitrogen assimilation, and isoprenoids (Miernyk 1985, Daher et  al. 2010). The activities of these pathways are enhanced during mycorrhization suggesting a crucial role for the plastids in the accumulation of Apocars and during the mycorrhization (Fester et al. 2001, 2007, Lohse et al. 2005). In particular, the cellular program driving the colonization contains lines specifying reorientation of the root plastid metabolism to the production of secondary metabolites including Cars and Apocars to a point that the white roots become yellow when colonized (Klingner et al. 1995, Fester et al. 2002a, Schliemann et al. 2006). The core structure of the yellow pigment contains an acyclic C14 polyene, namely, mycorradicin. This pigment is accompanied by a C13 cyclohexenone diglucoside (Fester et al. 2002a, Walter et al. 2007). Both Apocar types are actually present as mixture because they are differently esterified (Maier et al. 1995, Fester et al. 2002a) and their production results of the oxidative cleavage of a common Car precursor (Walter et al. 2000). The Apocars are massively deposited in the vacuole (Klingner et al. 1995) and probably in the cytoplasm too (Fester et al. 2002a). The accumulation of the yellow pigments mostly occur during the late phase of mycorrhization, that is, during arbuscule degradation, phase of the mycorrhization process during which the plastid network is disintegrated (Fester et al. 2001). 16.5.2.1  Biosynthesis of Apocarotenoids The activation of the AM-dependent MEP pathway has been demonstrated for Medicago truncatula (Lohse et al. 2005, Floss et al. 2008a). Northern blot experiments showed an increase of the transcript levels of the DXS2 and 1-deoxy-xylulose-5-phosphate reductoisomerase, the first enzymes of the plastids-located MEP pathway, in AM roots from wheat, maize, rice, and barley (Fester et al. 2007, Walter et al. 2000, Walter et al. 2000, 2002, 2007). Transcript levels of the genes coding the enzymes involved in the Car pathway such as PDS and ζ-carotene desaturase increase in the AM roots of Nicotiana tabacum (Strack and Fester 2006) but no Car accumulates because they are further metabolized into Apocars through the action of Car cleavage dioxygenase (CCD) enzymes, the production of which is induced by colonization by AM fungi (Strack and Fester 2006).

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Root plastid

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Unknown C40 substrate CCD7 C27 + C13

C27 Cytoplasm

CCD1 C14 + C13

Plastid envelopes C13 Control of arbuscule development Tonoplast

Vacuole storage

Figure 16.2  Scheme presenting the biochemical pathways of mycorradicine (C14) and cyclohexenone (C13) in response of mycorrhization.

CCD activities exhibit a high degree of regiospecificity and stereospecificity. They give rise to a variety of products with different biological functions as Apocar aromatic volatiles of flower scents, fruit aroma, phytohormones such as abscissic acid, and strigolactone (Akiyama 2007, Garcia-Garrido et al. 2009). Using a very elegant approach, Floss et al. (2008a) demonstrated that the CCD enzyme that uses the C40 Car precursor is CCD7. It has been shown that CCD7 cleaves Cars asymmetrically, producing C13 and C27 fragments (Bouvier et al. 2003, Rubio et al. 2008) (Figure 16.2). Another candidate for the catalysis of the reaction would be CCD4, but expression data have shown that this enzyme is not produced in roots (Ohmiya et al. 2006, Floss et al. 2008a, Rubio et al. 2008). This reaction takes place in the plastids, and the products are exported to the cytoplasm where they are further metabolized by the CCD1 enzyme into C13 cyclohexenone and C14 mycorradicin (Floss et al. 2008a, Vogel et al. 2008) (Figure 16.2). This scheme fits with the subcellular localization of the enzymatic activities. CCD1 that is able to directly split up a C40 into C13 and C14 (Schwartz et al. 2001) is the only CCD to be localized outside plastids (Bouvier et al. 2003, Tan et al. 2003, Auldridge et al. 2006) and, consequently, it has no access to the C40 precursor. Thus, it is its second catalytic activity (cleavage at the 5,6/5′,6′ positions) that is at work in the cytoplasm (Figure 16.2). These results highlight that, to really understand the functioning of a biochemical process such as the production of Apocars, it is crucial to consider the subcellular localization(s) of the enzymatic activities (for reviews, see Schoefs 2008, Seddas et al. 2009). The Car fragments are partly esterified and deposited as small lipophilic droplets in the vacuoles of root cortical cells (Strack and Fester 2006). Traces of ζ-carotene were detected and no accumulation of other detectable intermediates suggested that the biosynthesis of the accumulating Apocars appeared to proceed rapidly (Fester et al. 2007). Very importantly, the accumulation of these secondary Apocars is restricted to the colonized cells because the expression of the DXS2 gene increases only in these cells (Floss et al. 2008b), reinforcing the idea that plastids play a crucial role in the metabolism of these cells. Another type of secondary Apocars, such as the strigolactone family of molecules, is produced by plant roots (Akiyama 2007, Garcia-Garrido et  al. 2009). Their synthesis would also involve CCD7 but in this case, the C27 product would be cleaved into a C9 and a C18 products through the catalytic action of CCD8 (Schwartz et al. 2004; Alder et al. 2008) (Figure 16.2). 16.5.2.2  Roles and Functions of Secondary Apocarotenoids Strigolactones, a novel class of plant hormone, are released usually in trace amounts from the roots on phosphate deficiency but in sufficient amount to be detected by new potential hosts (Strack and Fester 2006, Garcia-Garrido et al. 2009, Seddas et al. 2009). Strigolactone stimulates spore

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germination in some AM fungi (Parniske 2008) and induces a continued hyphal growth and a profuse branching of hyphae, and could also activate the respiration of the arbuscular mycorrhizal fungi (for a review, see Seddas et al. 2009). Abscisic acid, another plant hormone, is an Apocar. A possible role of the abscisic acid in AM roots was suggested because the level of this phytohormone increases during the fungal symbiosis in Zea mays and Glycine max (Strack and Fester 2006, Parniske 2008, Seddas et al. 2009). The accumulated Apocars C13 and C14 may have structural functions and could provide efficient protection against oxidative stress (ROS scavenging) as the general antioxidant function of Cars accumulated under environmental stress (Fester et al. 2002b, Floss et al. 2008a). ROS could act as second messengers inducing the Car biosynthesis and/or are required for signaling fungal cell death (Strack and Fester 2006). Mycorradicin might play a role in the detoxification of H2O2 generated during the arbuscule disintegration. Apocars may protect membranes from oxidative damage, limiting oxygen penetration to the hydrophobic membrane core (Strack and Fester 2006). In plants having CCD1 gene silenced, the amount of C13 cyclohexenone decreased strongly, whereas the amount of senescent or dead arbuscules increased (Floss et al. 2008b). Therefore, it was proposed that cyclohexenone is involved in the control of the duration of the functionality of arbuscules (Walter et al. 2007, Floss et al. 2008b). This conclusion fits with the fact that exogenous blumenin shows a negative influence on AM (Fester et al. 1999, Walter et al. 2007). In contrast, cyclohexenone derivatives (other C13) were not responsible for the systemic suppression of mycorrhization in precolonized barley plants (Vierheilig et al. 2000), and mycorradicin did not suppress an elicitor-induced oxidative burst reaction in N. tabacum and Medicago sativa cell cultures (Schröder et al. 2001).

Conclusions and Perspectives Cars and Apocars are commonly concerned in biotic and abiotic stresses. They participate efficiently to the various plant responses and in these sense, they constitute a crucial element of the plant survival strategies. An essential function of Cars is to prevent the chloroplasts against harmful photooxidative reactions either through a direct dissipation of the excess of energy as heat or by participating in the formation of the NPQ through their involvement in the xanthophyll cycle. Besides these contributions to plant physiology, Cars and Apocars act as storage compounds, allowing stressed algae to overcome the disruption of the photosynthetic activity triggered by the stress. Many progresses that have been performed in the elucidation of the metabolic pathways leading to the production of the Cars, and physiological studies have suggested that previously described catabolic pathways, might actually be metabolic and/or regulation pathways. Thus, they do not solely correspond to degradation of useless molecules but they could produce crucial actors of plants physiology such as strigolactones and cyclohexenones. This has opened completely new avenues for research in different fields of plant sciences. Altogether, the central role of different types of plastids (chloroplasts and root plastids) in plant life is strongly reinforced.

Abbreviations AMS Ant Apocar Car CCD Chl CP Ddx DDE DEP

Arbuscular mycorrhizal symbiosis Antheraxanthin Apocarotenoid Carotenoid Carotenoid cleaving dioxygenase Chlorophyll Chlorophyll-protein Diadinoxanthin Diadinoxanthin de-epoxidase Diatoxanthin epoxidase

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Dtx DXS2 IPP LCY Lcy-B LHC Lut MEP MVA NPQ PDS PS PSY qE qI VDE Vio Zea ZEP

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Diatoxanthin 1-deoxy-D-xylulose 5-phosphate synthase Isopenthenyl diphosphate Lycopene cyclase Lycopene β-cyclase Light-harvesting complex Lutein Methylerythritol 4-phosphate Mevalonate pathway Nonphotochemical quenching Phytoene desaturase Photosystem Phytoene synthase High-energy state quenching Photoinhibition-related quenching Violaxanthin de-epoxidase Violaxanthin Zeaxanthin Zea epoxidase

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Strack, D. and Fester, T. 2006. Isoprenoid metabolism and plastic reorganization in arbuscular mycorrhizal roots. New Phytol. 172:22–34. Stransky, H. and Hager, A. 1970. The carotenoid pattern and the occurrence of the light-induced xanthophyll cycle in various classes of algae. VI. Chemosystematic study. Arch. Mikrobiol. 73:315–323. Suire, C., Bouvier, F., Backhaus, R.A., Bégu, D., Bonneu, M., and Camara, B. 2000. Cellular localization of isoprenoid biosynthetic enzymes in Marchantia polymorpha. Uncovering a new role of oil bodies. Plant Physiol. 124:971–978. Takeguchi, C.A. and Yamamoto, H.Y. 1968. Light-induced 18O2-uptake by epoxy xanthophylls in New Zealand spinach leaves (Tetragonia expansa). Biochim. Biophys. Acta 153:459–465. Tao, L.A., Wilczek, J., Odom, J.M., and Cheng, Q. 2006. Engineering a β-carotene ketolase for astaxanthin production. Metabol. Eng. 8:523–531. Tan, B.C., Joseph, L.M., Deng, W.T., Liu, L., Li, Q.B., Cline, K., and McCarty, D.R. 2003. Molecular characterization of the Arabidopsis 9-cis epoxycarotenoid dioxygenase gene family. Plant J. 35:44–56. Tobin, E.M. 1997. Renewing an old view of chloroplasts. Trends Plant Sci. 2:405–406. Ursi, S., Pedersen, M., Plastino, E., and Snoeijs, P. 2003. Intraspecific variation of photosynthesis, respiration and photoprotective carotenoids in Gracilaria birdiae (Gracilariales: Rhodophyta). Mar. Biol. 142:997–1007. Verhoeven, A.S., Adams, W.W., and Demmig-Adams, B. 1996. Close relationship between the state of xanthophyll cycle pigments and photosystem II efficiency during recovery from winter stress. Physiol. Plant. 96:567–576. Vershinin, A.O. and Kamnev, A.N. 1996. Xanthophyll cycle in marine macroalgae. Botanica Marina 39:421–425. Vierheilig, H., Maier, W., Wyss, U., Samson, J., Strack, D., and Piché, Y. 2000. Cyclohexenone derivative- and phosphate-levels in split-root systems and their role in the systemic expression of mycorrhization in precolonized barley plants. J. Plant Physiol. 157:593–599. Vogel, J.T., Tan, B.C., McCarty, D.R., and Klee, H.J. 2008. The carotenoid cleavage dioxygenase 1 enzyme has broad substrate specificity, cleaving multiple carotenoids at two different bond positions. J. Biol. Chem. 283:11364–11373. Walter, M.H., Fester, T., and Strack, D. 2000. Arbuscular mycorrhizal fungi induce the non-mevalonate methylerythritol phosphate pathway isogenes of isoprenoid biosynthesis correlated with accumulation of the “yellow pigment” and other apocarotenoids. Plant J. 21:571–578. Walter, M.H., Hans, J., and Strack, D. 2002. Two distantly related genes encoding 1-deoxy-D-xylulose 5-phosphate synthases: Differential regulation in shoots and apocarotenoid-accumulating mycorrhizal roots. Plant J. 31:243–254. Walter, M.H., Floss, D.S., Hans, J., Fester, T., and Strack, D. 2007. Apocarotenoid biosynthesis in arbuscular mycorrhizal roots: Contributions from methylerythritol phosphate pathway isogenes and tools for its manipulation. Phytochemistry 68:130–138. Walters, R.G., Ruban, A.V., and Horton, P. 1994. Higher plant light-harvesting complexes LHCIIa and LHCIIc are bound by dicyclohexylcarbodiimide during inhibition of energy dissipation. Eur. J. Biochem. 226:1063–1069. Wang, N., Fang, W., Han, H., Sui, N., Li, B., and Meng, Q.W. 2008. Overexpression of zeaxanthin epoxidase gene enhances the sensitivity of tomato PSII photoinhibition to high light and chilling stress. Physiol. Plant. 132:384–396. Watson, T.L., Close, D.C., Davidson, N.J., and Davies, N.W. 2004. Pigment dynamics during cold-induced photoinhibition of Acacia melanoxylon. Funct. Plant Biol. 31:481–489. Wehner, A., Grasses, T., and Jahns, P. 2006. De-epoxidation of violaxanthin in the minor antenna proteins of photosystem II, LHCB4, LHCB5, and LHCB6. J. Biol. Chem. 281:21924–21933. Wilhelm, C., Büchel, C., Fisahn, J., Goss, R., Jakob, T., Laroche, J., Lavaud, J., Lohr, M., Riebesell, U., Stehfest, K., Valentin, K., and Kroth, P.G. 2006. The regulation of carbon and nutrient assimilation in diatoms is significantly different from green algae. Protist 157:91–124. Xu, C.C., Jeon, Y.A., Hwang, H.J., and Lee, C.H. 1999. Suppression of zeaxanthin epoxidation by chloroplast phosphatase inhibitors in rice leaves. Plant Sci. 146:27–34. Yamamoto, H.Y. 1979. Biochemistry of violaxanthin cycle in higher-plants. Pure Appl. Chem. 51:639–648. Yamamoto, H.Y. and Higashi, R.M. 1978. Violaxanthin de-epoxidase: Lipid composition and substrate specificity. Arch. Biochem. Biophys. 190:514–522. Yamamoto, H.Y. and Kamite, L. 1972. The effects of dithiothreitol in violaxanthin de-epoxidation and absorbance changes in the 500-nm region. Biochim. Biophys. Acta 267:538–543.

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Yan, Y., Zhu, Y.H., Jiang, J.G., and Song, D.L. 2005. Cloning and sequence analysis of the phytoene synthase gene from a unicellular chlorophyte, Dunaliella salina. J. Agric Food Chem. 53:1466–1469. Ye, Z.W., Jiang, J.G., and Wu, G.H. 2008. Biosynthesis and regulation of carotenoids in Dunaliella: Progresses and prospects. Biotechnol. Adv. 26:352–360. Young, A.J. and Britton, G. 1990. Carotenoids and stress. In Stress Responses in Plants: Adaptation and Acclimation Mechanisms, eds., R.G. Alscher and J.R. Cumming, pp. 87–112. WileyLiss, New York. Zarter, C.R., Demmig-Adams, B., Ebbert, V., Adamska, I., and Adams, W.W. 2006. Photosynthetic capacity and light harvesting efficiency during the winter-to-spring transition in subalpine conifers. New Phytol. 172:283–292. Zhang, K.W., Gong, W.D., and Chen, F. 1999. Dynamics and stability analysis of the growth and astaxanthin production system of Haematococcus pluvialis. J. Ind. Microbiol. Biotechnol. 23:133–137. Zhu, Y.H., Jiang, J.G., and Chen, Q. 2008. Characterization of cDNA of lycopene β-cyclase responsible for a high level of beta-carotene accumulation in Dunaliella salina. Biochem. Cell Biol. 86:285–292.

17 Thermoluminescence Study of Photosystem II Activity in Resurrection Plant Haberlea rhodopensis during Desiccation Liliana T. Maslenkova, Violeta N. Peeva, Yuliana K. Markovska, and Yuzeir Zeinalov Contents 17.1 Introduction........................................................................................................................... 435 17.2 Strategies of Resurrection Angiosperm Haberlea rhodopensis to Preserve Cell Integrity.................................................................................................................... 436 17.3 Functional Features of Photosystem II in Haberlea rhodopensis Leaves Studied by Thermoluminescence.......................................................................................... 438 17.3.1 Peculiarities of Thermoluminescence Emission from Haberlea rhodopensis Leaves...................................................................................................................... 438 17.3.2 Changes in Thermoluminescence Characteristics during Dark Desiccation and Rehydration......................................................................................................... 439 17.4 Photosystem II Reactions in Chloroplasts Membranes Isolated from Fully Hydrated and Dehydrated Haberlea rhodopensis Leaves..................................................................... 441 17.5 Conclusion.............................................................................................................................444 References.......................................................................................................................................444

17.1  Introduction Desiccation-tolerant (poikilohydric) plants represent a unique group of organisms, which are able to withstand loss of water to an air-dry state and to survive extended periods of severe water deficit [1,2]. Poikilohydry is a relatively common phenomenon in nonvascular taxa such as lichens, algae, and bryophytes, but only 74 pteridophytes and 145 angiosperms belong to these so-called resurrection plants [3]. In a desiccated state, their physiological functions, including photosynthetic activity completely ceased, but during rehydration this activity can be fully restored, with different rates in homoiochlorophyllous (HDT) and in poikilochlorophyllous (PDT) desiccation-tolerant plants [4,5]. Various aspects of the desiccation tolerance in vascular plants have received considerable attention, the latest efforts being focused on clarifying the physiological and molecular basis of this phenomenon [6–20]. However, until now the exact mechanisms which preserve the highly sensitive photosynthetic system in HDT plants during desiccation, and the characteristics of the recoverable photosynthetic system in the desiccated stage are still not well understood. The complete reconstitution of chloroplast structure and functional activity in resurrection plants on rehydration suggests some peculiarities of thylakoid membrane and/or chloroplast stroma composition [21,22], which make these plants a very suitable model system for investigation of photosystem II (PSII) complex perturbations and its adaptive plasticity in the course of desiccation and rehydration. 435

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During our studies of Haberlea rhodopensis Friv., using a highly sensitive thermoluminescence (TL) technique, we observed some peculiarities of PSII redox reactions that can reflect some specific adaptive characteristics of the photosynthetic system, related to desiccation tolerance of this resurrection plant. In addition to multiple mechanisms for chloroplast integrity preservation, the observed stabilization of charge storage in PSII complex together with a strong reduction of the total number of PSII centers without any changes in their energy status, can explain the fast recovery of the photosynthetic activity after desiccation.

17.2  Strategies of Resurrection Angiosperm Haberlea rhodopensis to Preserve Cell Integrity The Balkan endemic Haberlea rhodopensis Friv. (Gesneriaceae) is a unique species of European flora, (spread in Bulgaria and Northeastern Greece), belonging to a very small group of desiccationtolerant vascular flowering plants (Figure 17.1C). The current distribution of the plants is restricted to the northern rocky slopes of gorges and canyons, mainly of foothills, sometimes reaching the alpine belts. Its natural habitats are characterized by high mean annual temperatures (of about 20°C), frequent mist and dew fall in the early morning hours and in the evening, and high periodic rainfalls totaling about 600 mm annually. Besides episodic droughts and high temperatures, pronounced short and long-term changes in irradiance may be the major environmental constraints affecting water balance and photosynthetic activity of these rosette plants. Homoiochlorophyllous desiccation-tolerant (HDT) plants evolve various morphological and physiological mechanisms to preserve their intrachloroplastic membrane system and photosynthetic apparatus in an easily and rapidly recoverable form, which can be ready to function in a normal way immediately after rehydration, following the desiccated stage [5]. During drying, a controlled folding of Haberlea rhodopensis leaves occurs (Figure 17.1A) with the abaxial surface exposure to light thought to be an important protective strategy in HDT plants to preserve their chloroplasts from photoinhibitory damages [23–25]. Simultaneously, a phenomenon of leaf surface decrease (more than 60% compared to fully hydrated leaves), accompanied by a reduction in specific leaf area, was also observed for H. rhodopensis leaves, dehydrated to about 30% relative water content (RWC) [26]. These typical resurrection plant reactions, which minimize mechanical damage, are due to cell volume reduction in conformity with cell walls folding (Figure 17.2B). The lack of significant differences in the electrolyte leakage between fully hydrated, desiccated, and rehydrated H. rhodopensis leaves confirms the preservation of membrane integrity (Table 17.1). In contrast to desiccationtolerant species, the observed leaf surface decrease in mesophytic spinach during desiccation was only 20%, which disturbed membrane integrity and induced irreversible injuries of the cell membranes, leading to further substantial increase in leakage upon rehydration of the leaves (Table 17.1). We also found clear differences in the level of MDA, H2O2, and proline accumulation between H. rhodopensis and spinach leaves, confirming the symptoms of injury in non-tolerant plants.

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FIGURE 17.1  Morphological changes in the resurrection plant Haberlea rhodopensis Friv. (Gesneriaceae). (A) Desiccated plant. (B) Fully hydrated plant. (C) Haberlea rhodopensis plants in their natural habitat.

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FIGURE 17.2  Light microscope micrograph cross-sections from detached leaves of the homoiochlorophyllous resurrection plant Haberlea rhodopensis treated in the dark. (A) The fully hydrated leaf shows extended oval cells with a large central vacuole and peripheral location of the chloroplasts. (B) Dehydrated to 20% RWC leaf. The cells become smaller with irregular shape, the central vacuole have shrunk or even disappeared, with a formation of several small vacuoles, the chloroplasts are scattered in the cytoplasm. (C) During rehydration for 12 h, the cells have notably restored their normal shape and plastid location. Bars = 20 μm (left) and 100 μm (right).

TABLE 17.1 Changes in Electrolyte Leakage, Malondialdehyde, Hydrogen Peroxide, and Proline Content in Haberlea rhodopensis and Spinach Leaves during Dehydration and Rehydration (R)

Species Haberlea Haberlea Haberlea (R) Spinach Spinach Spinach (R)

RWC (%)

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95 20 75 95 20 30

298 ± 56 532 ± 25 302 ± 35 1076 ± 206 4490 ± 421 7201 ± 114

0.240 ± 0.01 0.251 ± 0.01 0.197 ± 0.01 0.352 ± 0.02 0.400 ± 0.01 0.577 ± 0.01

55.75 ± 2.38 29.05 ± 0.78 60.13 ± 0.85 59.50 ± 0.40 47.63 ± 0.38 108.43 ± 0.21

0.420 ± 0.06 0.543 ± 0.04 0.147 ± 0.04 1.541 ± 0.06 10.009 ± 0.88 2.794 ± 1.10

The protection against desiccation damage in angiosperms is linked to the accumulation of carbohydrates, various compatible solutes and specific proteins as well as some changes in lipid composition. The analysis of mainly carbohydrates in the leaves of H. rhodopensis showed dextrans, sucrose, fructose, glucose, and starch [27]. During drying, starch content declined and large quantities of sucrose [27,28] was accumulated. Similarly, the lipid and sterol compositions of leaves of H. rhodopensis changed significantly during drying [29]. The most substantial changes were observed at 50% water deficit, where linolenic

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acid concentration diminished, but the campesterol/sitosterol ratio increased, causing membrane stabilization. The authors suggested that the highest proportion of DGDG (a bilayer-forming lipid) may play a role in the control of ionic permeability in the H. rhodopensis chloroplasts. An increased DGDG/MGDG ratio upon dehydration was also found after water stress in a tolerant variety of Vigna unguiculata [30], in a drought-tolerant cultivar of wheat [31], in Ramonda serbica [29,32], and in Sporobolus stapfianus [33]. Different arrangements of MGDGs and DGDGs within the thylakoid membranes [34] and changes in their proportions correlate with its physical properties.

17.3  Functional Features of Photosystem II in Haberlea rhodopensis Leaves Studied by Thermoluminescence 17.3.1  Peculiarities of Thermoluminescence Emission from Haberlea rhodopensis Leaves

Thermoluminescence (a.u.)

H. rhodopensis belongs to the group of homoiochlorophyllous poikilohydric dicotyledons, which upon desiccation preserve above 80% of the chlorophyll, and its photosynthetic apparatus is able to recover very fast [35,36]. Moreover, H. rhodopensis has the rare ability of its resurrection occurring in detached leaves (and even a small leaf disk). Young, fully expanded leaves, from the middle of rosettes, of similar size and appearance were used in the measurements. In order to distinguish the direct effect of water loss on photosynthetic activity and to avoid photoinhibition, the dehydration of detached leaves was carried out under dark conditions. For the purpose of comparison, experiments with leaves of the desiccationsensitive mesophytic plant Spinacia oleraceae L. (Chenopodiaceae) were also done. TL glow curve parameters were used to access the functional features of PSII. TL proved to be a very sensitive and reliable biophysical method for investigation of the functioning of both PSII donor and acceptor side components (see Refs. [37–39] for review). TL signals have been assigned to result from the thermal-activated recombination of the trapped electrons and stabilized positive equivalents on the reduced quinone acceptors (QA or QB) and on the S2 (or S3) oxidation state of the water-splitting complex, respectively. Figure 17.3 shows TL curves of H. rhodopensis leaves in comparison to those from spinach. Excitation of dark-adapted spinach leaves with a single flash (F), generating a S2QB− charge pair, induced a B band peaking at around 32°C (Figure 17.3A), which was

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FIGURE 17.3  TL from fully hydrated dark-adapted Haberlea rhodopensis (solid line) and spinach (dashed line) leaves excited with one (A) and two (B) saturating flashes at 5°C. (C) Leaves infiltrated with 20 μM DCMU and excited at −10°C with one saturating flash. Leaf disks with a diameter of 10 mm were used in the experiments.

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usually observed in the higher plants [40]. The most striking feature of the TL emission observed in the H. rhodopensis leaves was the upshift of the B-peak emission temperature to about 45°C. Similarly, different emission temperatures were registered when two (S2(3)QB−) flashes were given (Figure 17.3B). The B band position at higher temperature is indicative of more stably stored S2/3QB− charge pairs in the resurrection plant [41]. With intact, photosynthetically active cells, such a high TL B band emission temperature had been reported for other types of stress-tolerant organisms, namely thermophilic cyanobacterium or desiccation-tolerant ferns and lichen [22,42,43]. The high emission temperature of the TL B band from H. rhodopensis leaves could be attributed to some changes in the properties of redox partners on the donor or on acceptor side of PSII, or both. One way to test the contribution of QB− is to monitor TL after infiltration of the leaves with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which specifically inhibits the electron transport between the primary (QA) and the secondary (QB) quinone acceptors. Recordings on Figure 17.3C show that DCMU treatment of spinach leaves leads to a significant downshift in B  band position, concomitantly with a decrease in its amplitude and the appearance of a new, so-called Q band, peaking at around 0°C, which is thought to originate from S2QA− charge recombination [40]. In 20 μM DCMU treated H. rhodopensis leaves disks the Q band also appeared at approximately the same temperature. Since the S states are the common pole for positive charges of the B and Q band, the distinct differences of B-peak temperature position in H. rhodopensis and spinach leaves suggest major alterations in the redox property of QB− in the resurrection plant. Surprisingly, a part of B band was still clearly expressed even at higher inhibitor concentrations. These results show that some PSII reaction centers in H. rhodopensis leaves, with more stable stored S2(3)QB− charge pairs, are unsusceptible to DCMU, therefore, can possibly indicate some modifications of the redox properties of the quinone acceptor QB (especially in D1 core protein). In accordance with this suggestion are the data of Ohad et al. [44] and Hideg et al. [45], considering the observed incomplete suppression of the B band by DCMU after high light and UV-B irradiation as a proof for acceptor side modifications. Analogical deeper stabilization of S2(3)QB− charge recombination as a result of induced mutations in D1 protein have been also reported [46–49]. The already described specific lipid and sterol composition of H. rhodopensis leaves and the presence of different protective compounds in chloroplasts stroma may contribute to these modifications.

17.3.2  Changes in Thermoluminescence Characteristics during Dark Desiccation and Rehydration Under conditions of severe or prolonged water deficit, most plants are desiccation-intolerant (homoiohydric) and react to stress by the suspension of metabolism and irreversible damage to membrane structures and internal organization. Our data of changes in TL B band parameters from dehydrated spinach leaves [50] are in agreement with the respective desiccation sensitivity of this mesophytic plant. Severe dehydration of the leaves inhibits the number of operating centers, but leads predominantly to a well-expressed downshift of B band position close to Q band position. This observation is indicative for the destabilization of PSII centers as it was also shown in TL study on desiccating barley leaves [51]. It may be concluded that in mesophytic plants subjected to severe dehydration the electron transport between primary (QA) and secondary (QB) quinone acceptors is inhibited, and damaged oxygen-evolving complexes occur. Such PSII centers do not restore their photochemical activity during rehydration. Even more pronounced differences became in agreement with the results from Table 17.1, showing that damage sustained during dehydration become particularly detrimental after full metabolic activity have set in after spinach leaves rehydration. The most important result emerging from TL studies of H. rhodopensis leaves is that severe dehydration of resurrection plants affects mainly the number of PSII reaction centers, judging from significant decrease of B band amplitude (Figure 17.4, left panels) without any changes in the energetic state of the remaining operative centers. After rehydration of desiccated H. rhodopensis leaves, the number and the oscillation pattern of operating PSII centers were nearly completely

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FIGURE 17.4  TL curves of fully hydrated, dehydrated, and rehydrated Haberlea rhodopensis leaves after excitation by one saturating flash without DCMU, or in the presence of 20 μM DCMU (A, C, E). TL B band oscillations as a function of flash number for corresponding state of dehydration (B, D, F). Amplitudes were normalized at the second flash. The fully hydrated leaves were dehydrated in the dark to 60% RWC (A, B), 40% RWC (C, D), 20% RWC (E, F) and rehydrated in moist filter paper for 24 h. Leaf disks with a diameter of 10 mm were used in the experiments.

restored (Figure 17.4, right panels). This process was very rapid and rehydration for only 2 h restored more than 80% of the initial B band amplitude. The effect of desiccation and subsequent rehydration on the redox functioning of PSII donor and acceptor side redox components of H. rhodopensis leaves was also assessed by the changes in the main TL bands emitted at illumination with continuous white light during cooling the leaf disks from room temperature to –20°C. Under these experimental conditions a complex glow curve with well-resolved TL bands at about 0°C and 45°C, corresponding to Q and B bands [52] was obtained. Representative TL curve pattern from the leaves of fully hydrated H. rhodopensis plants is shown in Figure 17.5A. The traces in Figure 17.5B and C reveal that increasing dehydration resulted in clear changes in the overall intensity of TL signals and redistribution of the TL emission between the existing Q and B bands with practically unchanged peak temperatures. In desiccated leaves, the amplitude of the TL B

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17.4  Photosystem II Reactions in Chloroplasts Membranes Isolated from Fully Hydrated and Dehydrated Haberlea rhodopensis Leaves

A Thermoluminescence (a.u.)

band (S2(3)QB−) sharply decreased and mainly a charge recombination related to S2QA− (Q-peak) takes place (Figure 17.5C). After rehydration TL glow curve pattern resembles that of the control (fully hydrated) leaves (Figure 17.5D), which means electron transport between the primary and secondary electron acceptors was reversibly modified. Analogical changes in the amplitude and oscillation pattern of the main TL B and Q bands, obtained during flash illumination [53], suppose that some changes in the kinetic characteristics of S2 and S3 states of PSII donor side during desiccation cannot be excluded. We suggested that the increased contribution of S2QA− charge recombination in dehydrated H. rhodopensis leaves served to protect QB site from over excitation. There are data that the increased population of QA− enhances the probability for non-radiative energy dissipation and represents an effective mechanism of protection [48].

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FIGURE 17.5  TL curves of Haberlea rhodopensis dark-adapted leaves after illumination by continuous white light of 150  μmol m−2 s−1 from room temperature to −20°C for 1 min. (A) Fully hydrated leaves 95% RWC and leaf disks infiltrated with 20  μM DCMU (in dots). (B) Dehydrated leaves, 50% RWC. (C) Desiccated leaves, 10% RWC. (D) Rehydrated leaves for 24 h. Leaf disks with a diameter 10 mm were used in the experiments.

Answering the question whether the unique TL properties of the chlorophyll molecules of H. rhodopensis leaves were determined by some structural peculiarities required TL to be measured on isolated thylakoids. The obtained results show that the isolated photosynthesizing membranes from resurrection plant retain to a great extent the TL pattern of intact leaves thus indicating they were intrinsic features of PSII complex of H. rhodopensis. The illumination of dark-adapted chloroplast suspensions isolated from fully hydrated H. rhodopensis leaves by continuous white light revealed a glow curve with a B band temperature maximum positioned at about 40°C (Figure 17.6A). The respective maximum in spinach chloroplast membranes appeared at much lower temperature of 26°C. The addition of DCMU to chloroplast suspension downshifted the B band emission maximum with formation of Q band at lower temperature (Figure 17.6C and D), but a residual B band like those in the leaves infiltrated with the electron-transport inhibitor was also observed, confirming the lower affinity of QB binding from PSII acceptor side in H. rhodopensis chloroplasts to DCMU. TL emission pattern of chloroplasts isolated from desiccated to 20% RWC H. rhodopensis leaves was identical to those isolated from fully hydrated plants (Figure 17.6A and C). The same maximal temperature position of B- and Q bands obtained in the both varieties of chloroplasts evidenced for a stable energetic state of recombination pairs, moreover, the reduction in overall TL intensity was found to be negligible. This is an indication that the chloroplasts isolated from desiccated leaves have been completely rehydrated when setting in resuspension medium and their functional activity was fully recovered in conformity with the preserved membrane integrity (Table 17.1). A significant downshift in temperature maximum and the decrease in the intensity of the respective TL bands in chloroplasts isolated from desiccated spinach leaves (Figure 17.6B and D) are a consequence of the membrane injuries occurred during severe stress.

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FIGURE 17.6  TL emissions of Haberlea rhodopensis (A, C) and spinach (B, D) chloroplasts excited by continuous white light of 150 μmol m−2 s−1 at −20°C for 1 min. The chloroplasts were isolated from fully hydrated leaves (solid lines) and leaves dehydrated to 20% RWC (dashed lines). Dark-adapted chloroplasts of 1 mg Chl mL−1 were used in the experiments.

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Changes in B band parameters (Figure 17.7A and C) and TL oscillation pattern (Figure 17.7B and D) of desiccated H. rhodopensis and spinach chloroplasts illuminated with saturated flashes compared to respective hydrated controls demonstrated once more that PSII reaction centers functioning was preserved in a great extent in resurrection plant. Another reliable approach to study the properties of PSII complex in H. rhodopensis chloroplasts was to compare the kinetics of oxygen-evolving reactions with those of spinach membranes. The obtained results (Figure 17.8) demonstrated significant differences in the degree of inhibition of oxygen flash yields and the amplitude of initial oxygen burst between resurrection and mesophytic

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10 20 30 40 50 Time (s)

20

FIGURE 17.8  Oxygen flash yields sequences of chloroplasts isolated from fully hydrated (A) and dehydrated to 20% RWC (C) Haberlea rhodopensis leaves. Fully hydrated (B) and dehydrated to 20% RWC (D) spinach leaves. Insert: Oxygen evolution burst induced by 135 W m−2 continuous white light. Dark-adapted chloroplasts of 0.3 mg Chl mL−1 were used. The experiments were performed by a polarographic oxygen rate electrode with short (10 μs) saturating (4 J) flash sequences or continuous white light.

TABLE 17.2 Changes in the Kinetic Parameters of Oxygen-Evolving Reactions of Chloroplasts Isolated from Fully Hydrated and Desiccated Haberlea rhodopensis and Spinach Leaves, according to Kok’s Model Species

RWC (%)

S0 + S1 (a.u.)

Misses (𝛂)

Double Hits (𝛃)

Haberlea Haberlea Spinach Spinach

95 20 95 20

238.7 ± 6 213.1 ± 3 265.3 ± 11 54.2 ± 3

0.151 ± 0.013 0.161 ± 0.008 0.137 ± 0.005 0.321 ± 0.008

0.031 ± 0.004 0.027 ± 0.001 0.029 ± 0.002 0.020 ± 0.002

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plants. The damping of oscillations of control chloroplasts and the chloroplasts, isolated from desiccated H. rhodopensis and spinach leaves was in accordance to the changes in the respective kinetic parameters, calculated by Kok’s model [54]. As shown in Table 17.2, the desiccation of H. rhodopensis leaves do not change significantly the initial distribution of oxygen-evolving centers in S0 and S1 states and the values of misses (α) and double hits (β). Alterations in the values of these parameters and in the shape of oxygen induction curve in desiccated spinach chloroplasts are indicative for membrane damage in the desiccation-intolerant plant.

17.5  Conclusion The homoiochlorophyllous resurrection plant Haberlea rhodopensis Friv., demonstrated a deeper stabilization of PSII charge pairs, evidenced by an unusually high temperature maximum of the main TL B peak in the leaves and isolated thylakoid membranes. In addition, a part of these centers was less susceptible to DCMU, an inhibitor of electron transport. These features, as well as the strong reduction of the number of active PSII centers performing S2(3)QB− charge separation during desiccation without any changes in the energetics of the charge recombination in the rest operating centers were considered to indicate modifications of the redox properties of QB, related to desiccation tolerance of H. rhodopensis. It is reasonable to suggest that these modifications favor S2QA− charge recombination under desiccation. The increased population of QA− enhances the probability for non-radiative energy dissipation and can represent an effective mechanism of protection during unfavorable environmental conditions.

References

1. Gaff, D.F. and N.D. Hallam. 1974. Resurrecting desiccated plants. R. Soc. N. Z. Bull. 12: 389–393. 2. Bewley, J.D. 1979. Physiological aspects of desiccation tolerance. Annu. Rev. Plant Physiol. 30: 195–238. 3. Proctor, M.C.F. and V.C. Pence. 2002. Vegetative tissues: Bryophytes, vascular resurrection plants, and vegetative propogules. In Desiccation and Survival in Plants: Drying without Dying, eds. M. Black and H.W. Pritchard, pp. 207–237. Wallingford, Oxon, U.K.: CABI Publishing. 4. Hambler, D.J. 1961. A poikilohydrous poikilochlorophyllous angiosperm from Africa. Nature 191: 1415–1416. 5. Tuba, Z., Proctor, M.C.F., and Zs. Csintalan. 1998. Ecophysiological responses of homoiochlorophyllous and poikilochlorophyllous desiccation tolerant plants: A comparison and ecological perspective. Plant Growth Regul. 24: 211–217. 6. Gaff, D.F. 1997. Mechanisms of desiccation—Tolerance in resurrection vascular plants. In Mechanisms of Environmental Stress Resistance in Plants, eds. A.S. Basra and R.K. Basra, pp. 43–58. London, U.K.: Harwood Academic Publishers. 7. Gaff, D.F. 1989. Responses of desiccation tolerant “resurrection” plants to water stress. In Structural and functional responses to environmental stresses, Eds., K.H. Kreeb, H. Richter, and T.M. Hinckley, pp. 225–268, The Hague, the Netherlands: SPB Academic Publishing. 8. Gaff, D.F. 1980. Protoplasmic tolerance of extreme water stress. In Adaptation of Plants to Water and High Temperature Stress, eds. N.C. Turner and P.J. Kramer, pp. 207–230. New York: Wiley. 9. Bewley, J.D. and J.E. Krochko. 1982. Desiccation tolerance. In Encyclopedia of Plant Physiology, New Series, eds. A. Pirson and M.H. Zimmermann, pp. 325–378. Berlin-Heidelberg, Germany: Springer-Verlag. 10. Stewart, G.R. 1989. Desiccation injury anhydrobiosis and survival. In Plants under Stress: Biochemistry, Physiology and Ecology and Their Application in Plant Improvement, eds. N.G. Jones, T.J. Flowers, and M.B. Jones, pp. 115–130. Cambridge, U.K.: Cambridge University Press. 11. Leopold, A.C. 1990. Coping with desiccation. In Stress Responses in Plants: Adaptation and Acclimation Mechanisms, ed. R.G. Alcher, pp. 36–56. New York: Wiley-Liss. 12. Proctor, M.C.F. 1990. The physiological basis of bryophyte production. Bot. J. Linn. Soc. 104: 61–77. 13. Bewley, J.D. and M.J. Oliver. 1992. Desiccation—tolerance in vegetative plant tissues and seed: Protein synthesis in relation to desiccation and a potential role for protection and repair mechanisms. In Water and Life: A Comparative Analysis of Water Relationships at the Organismic, Cellular and Molecular Levels, eds. C.B. Osmond and G. Somero, pp. 141–160. Berlin, Germany: Springer Verlag.

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14. Ingram, J. and D. Bartels. 1996. The molecular basis of dehydration tolerance in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 377–403. 15. Hartung, W., Shiller, P., and K.-J. Dietz. 1998. Physiology of poikilohydric plants. Prog. Bot. 59: 299–327. 16. Neale, A.D., Blomstedt, C.K., Bronson, P. et al. 2000. The isolation of genes from the resurrection grass Sporobolus stapfianus which are induced during severe drought stress. Plant Cell Environ. 23: 265–277. 17. Bartels, D. and F. Salamini. 2001. Desiccation tolerance in resurrection plant Craterostigma plantagineum. A contribution to the study of drought—Tolerance at the molecular level. Plant Physiol. 127: 1346–1353. 18. Ramanjulu, S. and D. Bartels. 2002. Drought- and desiccation-induced modulation of gene expression in plants. Plant Cell Environ. 25: 141–151. 19. Cooper, K. and J.M. Farrant. 2002. Recovery of the resurrection plant Craterostigma wilmsii from desiccation: Protection versus repair. J. Exp. Bot. 53: 1805–1813. 20. Vicré, M., Farrant, J.M., and A. Driouich. 2004. Insights into the cellular mechanisms of desiccation tolerance among angiosperm resurrection plant species. Plant Cell Environ. 27: 1329–1340. 21. Schwab, K.B., Schreiber, U., and U. Heber. 1989. Response of photosynthesis and respiration of resurrection plants to desiccation and rehydration. Planta 177: 217–227. 22. Maslenkova, L. and P. Homann. 2000. Stabilized S2 state in leaves of the desiccation tolerant resurrection fern Polipodium polipodioides. Compt. Rend. Bulg. Acad. Sci. 53: 99–102. 23. Ludlow, M.M. and S.B. Powles. 1988. Effects of photoinhibition induced by water stress on growth and yield of grain sorghum. Aust. J. Plant Physiol. 15: 179–194. 24. Vicré, M., Sherwin, H.W., Driouich, A., Jaffer, M.A., and J.M. Farrant. 1999. Cell wall characteristics and structure of hydrated and dry leaves of the resurrection plant Craterostigma wilmsii, a microscopical study. J. Plant Physiol. 155: 719–726. 25. Vicré, M., Lerouxel, O., Farrant, J., Lerouge, P., and A. Driouich. 2004. Composition and desiccationinduced alterations of the cell wall in the resurrection plant Craterostigma wilmsii. Physiol. Plant. 120: 229–239. 26. Peeva, V. and G. Cornic. 2009. Leaf photosynthesis of Haberlea rhodopensis before and during drought. Environ. Exp. Bot. 65: 310–318. 27. Markovska, Y. and G. Kimenov. 1998. Carbohydrates content during drought and rewatering of Haberlea rhodopensis Friv. and Ramonda serbica Panc., Compt. Rend. Acad. Bulg. Sci. 51: 91–94. 28. Muller, J., Sprenger, N., Bortlik, K., Boller, T., and A. Wiemken. 1997. Desiccation increases sucrose levels in Ramonda and Haberlea, two genera of resurrection plants in the Gesneriaceae. Physiol. Plant. 100: 153–158. 29. Stefanov, K., Markovska, Y., Kimenov, G., and S. Popov. 1992. Lipid and sterol changes in leaves of Haberlea rhodopensis and Ramonda serbica at transition from biosis into anabiosis and vice versa caused by water stress. Photochemistry 31: 2309–2314. 30. Monteiro de Paula, F., Pham Thi, A.T., Zuily Fodil, Y., Ferrari-Iliou, R., Vieira da Silva, J., and P. Mazliak. 1993. Effect of water stress on the biosynthesis and degradation of polyunsaturated lipid molecular species in leaves of Vigna unguiculata. Plant Physiol. Biochem. 31: 707–715. 31. Quartacci, M.F., Sgherri, C.L.M., Pinzino, C., and Navari-Izzo. 1994. Superoxide radical production in wheat plants differently sensitive to drought. Proc. R. Soc. Edinb. Sect. B Biol. Sci. 102: 287–290. 32. Stevanovic, B., Pham Thi, A.T., Monteiro de Paula, F., and J. Vieira da Silva. 1992. Effects of dehydration and rehydration on the polar lipid and fatty acid composition of Ramonda species. Can. J. Bot. 70: 107–113. 33. Quartacci, M.F., Forli, M., Rascio, N., Dalla Veccia, F., Bochiccio, A., and F. Navari-Izzo. 1997. Desiccation-tolerant Sporobolus staphianus: Lipid composition and cellular ultrastructure during dehydration and rehydration. J. Exp. Bot. 311: 1269–1279. 34. Quinn, P.J. and W.P. Williams. 1983. The structural role of lipids in photosynthetic membranes. Biochim. Biophys. Acta 737: 223–266. 35. Markovska, Y.K., Tsonev, Ts.D., Kimenov, G.P., and A.A. Tutekova. 1994. Physiological changes in higher poikilohydric plants—Haberlea rhodopensis Friv. and Ramonda serbica Panc. during drought and rewatering at different light regimes. J. Plant Physiol. 144: 100–108. 36. Georgieva, K., Maslenkova, L., Peeva, V., Markovska, Yu., Stefanov, D., and Z. Tuba. 2005. Comparative study on the changes in photosynthetic activity of the homoiochlorophyllous desiccation-tolerant Haberlea rhodopensis and desiccation-sensitive spinach leaves during desiccation and rehydration. Photosynth. Res. 85: 191–203.

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37. Sane, P.V. and A.W. Rutherford. 1986. Thermoluminescence from photosynthetic membranes. In Light Emission by Plants and Bacteria, eds. J.A. Govindjee and D.C. Fork, pp. 329–361. New York: Academic Press. 38. Vass, I. and Y. Inoue. 1992. Thermoluminescence in the study of photosystem II. In Topics in Photosynthesis, vol. II, The Photosystems: Structure, Function and Molecular Biology, ed. J. Barber, pp. 259–294. Amsterdam, the Netherlands: Elsevier. 39. Sane, P.V. 2004. Thermoluminescence. A technique for probing photosystem II. In Methods in Molecular Biology. Photosynthesis Research Protocols, ed. R. Carpentier, pp. 229–248. Totowa, NJ: Humana Press Inc. 40. Rutherford, A.W., Crofts, A.R., and Y. Inoue. 1982. Thermoluminescence as a probe of Photosystem II photochemistry. The origin of the flash-induced glow peaks. Biochim. Biophys. Acta 682: 457–465. 41. Rutherford, A.W., Renger, G., Koike, H., and Y. Ynoue. 1984. Thermoluminescence as a probe of photosystem II. The redox protonation states of the secondary acceptor quinone and O2-evolving system. Biochim. Biophys. Acta 682: 457–465. 42. Govindjee, J.A., Koike, H., and Y. Ynoue. 1985. Thermoluminescence and oxygen evolution from a thermophilic blue-green alga obtained after single-turnover light flashes. Photochem. Photobiol. 42: 579–585. 43. Sass, L., Csintalan, Z., Tuba, Z., and I. Vass. 1996. Thermoluminescence studies on the function of photosystem II in the desiccation tolerant lichen Cladonia convoluta. Photosynth. Res. 48: 205–212. 44. Ohad, I., Adir, N., Koike, H., Kyle, D.J., and Y. Inoue. 1990. Mechanism of Photoinhibition in vivo. A reversible light-induced conformational change of reaction center II is related to an irreversible modification of the D1 protein. J. Biochem. Chem. 265: 1972–1979. 45. Hideg, E., Sass, L., Barbato, R., and I. Vass. 1993. Inactivation of photosynthetic oxygen evolution by UV-B irradiation: A thermoluminescence study. Photosynth. Res. 38: 455–462. 46. Mäenpää, P., Miranda, T., Tyystjärvi, E. et al. 1995. A mutation in the D-de loop of D1 modifies the stability of the S2QA− and S2QB− states in Photosystem II. Plant Physiol. 107: 187–197. 47. Alfonso, M., Pueyo, J.J., Gaddour, K., Etienne, A.-L., Kirilovsky, D., and R. Picorel. 1996. Induced new mutation of D1 serine-268 in soybean photosynthetic cell cultures produced atrazine resistance, increased stability of S2QB− and S3QB− states, and increased sensitivity to light stress. Plant Physiol. 112: 1499–1508. 48. Vavilin, D.V. and W.F. Vermaas. 2000. Mutations in the CD-loop region of the D2 protein in Synechocystis sp. PCC 6803 modify charge recombination pathways in photosystem II in vivo. Biochemistry 39: 14831–14838. 49. Sane, P.V., Ivanov, A., Hurry, V., Huner, N., and G. Oquist. 2003. Changes in the redox potential of primary and secondary electron-accepting quinones in photosystem II confer increased resistance to photoinhibition in low-temperature-acclimated arabidopsis. Plant. Physiol. 132: 2144–2151. 50. Peeva, V. and L. Maslenkova. 2004. Thermoluminescence study of photosystem II activity in Haberlea rhodopensis and spinach leaves during desiccation. Plant. Biol. 6: 319–324. 51. Skotnica, J., Matouskova, M., Naus, J., Lazar, D., and L. Dvorak. 2000. Thermoluminescense and fluorescence study of changes in photosystem II photochemistry in desiccating barley leaves. Photosynth. Res. 65: 29–40. 52. Vass, I. and J.A. Govindjee. 1996. Thermoluminescence from the photosynthetic apparatus. Photosynth. Res. 48: 117–126. 53. Georgieva, K., Szigeti, Z., Savari, E. et al. 2007. Photosynthetic activity of homoiochlorophyllous desiccation tolerant plant Haberlea rhodopensis during dehydration and rehydration. Planta 225: 955–964. 54. Kok, B., Forbush, B., and M. McGloin. 1970. Cooperation of charges in photosynthetic O2 evolution. I. A linear four step mechanism. Photochem. Photobiol. 11: 457–475.

Metabolism 18 Carbon and Plant Stress Carlos M. Figueroa, Alberto A. Iglesias, and Florencio E. Podestá Contents 18.1 Introduction........................................................................................................................... 447 18.2 Carbon Partitioning in Plants................................................................................................448 18.3 Carbon Metabolism in the Cytosol and Plant Stress............................................................. 451 18.3.1 Organization of Plant Glycolysis............................................................................... 451 18.3.2 Metabolism of Sucrose and Polyols........................................................................... 453 18.4 Plants Carbohydrate Metabolism and Stress......................................................................... 454 18.4.1 Responses of Glycolytic Carbon Metabolism Enzymes to Stress............................. 454 18.4.1.1 Hexose-P Metabolism................................................................................. 454 18.4.1.2 PEP Metabolism......................................................................................... 455 18.4.2 Soluble Carbohydrates and Their Role against Stress............................................... 456 18.4.2.1 Osmotic Stress Adaptation.......................................................................... 456 18.4.2.2 Low-Temperature Stress............................................................................. 457 18.4.2.3 Drought Stress............................................................................................. 457 18.4.2.4 High-Salinity-Induced Stress...................................................................... 457 18.4.2.5 Hydroxyl-Radical Scavengers..................................................................... 458 18.5 Concluding Remarks............................................................................................................. 458 Acknowledgments........................................................................................................................... 458 References....................................................................................................................................... 458

18.1  Introduction Carbohydrates are the most abundant compounds in living organisms. However, their importance does not purely rely in abundance, as they play key roles in cell functionality. In plants, carbohydrates are utilized for the synthesis of various structural components, and they are used to transport carbon and energy between tissues (Iglesias and Podestá 2005, Smith 1999). The central role of carbohydrates in the energetic metabolism is highlighted in the glycolytic and the oxidative pentose-P pathways. However, these pathways have another important role, as they serve as source of intermediates for the synthesis of a large number of cellular components, such as lipids, nucleic acids, organic acids, and proteins. Also, carbohydrates are fundamental components of structural molecules like cellulose, the most abundant biomolecule. In addition, sugars can be combined with other compounds, like lipids or proteins, to produce glycolipids and glycoproteins, which play key roles related with cell structure and function (Iglesias and Podestá 2005, Smith 1999). On the other hand, carbohydrates are the major reserve and mobilization constituents in plants, where they can be found as starch, sucrose, fructans, and polyols (Iglesias and Podestá 2005, Loescher and Everard 2004, Smith 1999).

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The sessile lifestyle of plants imposes severe challenges to their ability to survive in a changing environment. Plants are affected by a variety of stresses including, but not limited to, water shortage, nutrients deficiency, presence of toxic compounds (natural or arising from contamination), and temperature extremes. A series of mechanisms have been developed by plants that allow the adjustment of the metabolic machinery to cope with unfavorable conditions, which in fact may be the prevailing situation in the wild. Carbon metabolism contributes in various ways with the adaptation to a stressful situation. Relocation of carbon skeletons for the synthesis of specific stress-related metabolites, excretion of organic acids to confront the presence of toxic metals, or to minimize Pi deficiency or alterations in the main course of metabolic pathways through alternative routes are some of the responses observed. Metabolic adaptations include the transient adjustment of constitutive processes or the induction of latent ones to accommodate to a new condition by fine-tuning metabolism. More extensive changes can even include the adaptation of the whole photosynthetic mechanism of CO2 assimilation as a result of a long-term challenge. This chapter deals with the main adjustments of plant primary carbon metabolism to the most common forms of abiotic stress, which endow these organisms with the capacity to survive in a world of permanently changing situations. The outstanding role of carbohydrates for carbon and energy metabolism forces to highlight their involvement as key components of cellular processes. We analyze the different fates of carbohydrates produced by photosynthetic assimilation in plants. The analysis emphasizes on how metabolic routes mainly operating in the cytosol are critical to direct carbohydrate fluxes to produce energy, reduce power, and transport or reserve molecules. The overall picture seeks to understand relationship between carbohydrates and plant physiology, and the capacity to cope with different stress conditions.

18.2  Carbon Partitioning in Plants Oxygenic photosynthesis is a process where the energy from light is utilized for carbohydrate production, thus playing a key role on Earth, because most organisms depend directly or indirectly on it to obtain energy. In the global photosynthetic process, the occurrence of two phases can be differentiated: (a) a light phase, where the electromagnetic sun energy is converted into chemical energy (ATP) and reducing power (NADPH) and (b) a synthetic phase, comprising the use of ATP and NADPH to fix atmospheric CO2 to primarily generate carbohydrates. As shown in Figure 18.1, carbon fixation occurs in the chloroplast of the photosynthetic plant cell mainly through the Benson– Calvin cycle (BCC), utilizing ATP and NADPH produced in the light phase. This renders triose-P and hexose-P that can be used within chloroplasts for the production of starch. Alternatively, photosynthates (mainly in the form of triose-P) can be exported to the cytosol to be used in glycolytic routes as well as to synthesize sucrose or polyols, the major compounds used for carbon translocation from source to sink tissues. In this way, photoassimilates are subjected to two levels of partitioning. At the intracellular level, they are partitioned between the chloroplast and the cytosol, being triose-P the key intermediaries interexchanged with Pi through a specific translocator of the plastid envelope (TPT in Figure 18.1). In the cytosol, carbohydrates are derived to different routes, including glycolysis and synthesis of soluble sugars that are accumulated in this subcellular compartment and also utilized for transport to other parts of the plant through the phloem. In most plants, the major soluble sugar synthesized is sucrose, although many species also produce sugar-alcohols (e.g., glucitol, abbreviated Gol, see Figure 18.1) as a main metabolite. The second level of partitioning takes place as carbohydrates are distributed to other tissues having heterotrophic characteristics, being sucrose (or a sugar-alcohol in certain species) the main metabolite transported between different tissues. Once in the non-photosynthetic cell, sucrose (or the sugar-alcohol) is derived to different metabolic routes with the establishment, once again, of a partitioning of photoassimilates between the cytosol and the plastid (Figure 18.2). As described above, carbohydrates are partitioned between a relatively stationary (starch) and a mobile (sucrose, polyols) form as major products of photosynthesis. It is important to understand the

449

Carbon Metabolism and Plant Stress Cytosol ST

Sucrose

Chloroplast

UDP

Glc1P

CO2

UDPGlc

Starch

RuBP

Fru6P PPi

ATP

Glc6P

NADP+ NADPH Triose-P Pi ATP 3PGA BCC

GT

Gol

Sucrose-P

SPSase

ADPGlcPPase

ADPGlc

To heterotrophic tissues

Pi

Pi TPT

NADH

ADP

ADP ATP OAA

Gol6P

A6PRase Pi ADP NADP+ NADPH Frul,6bisP

Triose-P NAD+

ATP

Glc6P

Ga3P NADP+ 1,3bisPGA

np-Ga3PDHase

NADPH 3PGA

HCO3– PEP

Pyr

ADP ATP

Figure 18.1  Schematic representation of carbon metabolism in plant photosynthetic cells. Key enzymes for carbon partitioning, the major photosynthetic products (starch, sucrose, and glucitol), and transporters (TPT, triose-P translocator; ST, sucrose transporter; GT, glucitol transporter) are highlighted. BCC is the abbreviation of Benson–Calvin cycle.

Cytosol Plastid Sucrose UDP SSase

ST From photosynthetic tissues

GT

Glc

Pi Glc1P

Fru UDPGlc PPi GolDHase UTP Glc1P NAD+ NADH

Gol

Glc6P Fru6P

OAA Pyr

H2O

Pi

Starch

ADPGlc

ADPGlcPPase

GPT

Glc6P Pi

Triose-P HCO3– PEP ADP ATP

Figure 18.2  Schematic representation of carbon metabolism in plant heterotrophic cells. Enzymes involved in sucrose, starch, and glucitol metabolism, photosynthates translocated from leaves (sucrose and glucitol), starch (the major storage compound), and the glucose-P translocator (GPT) are highlighted.

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processes of synthesis, partition (within one cell and between source and sink tissues), and storage of carbohydrates because these molecules are crucial for plant productivity. The control of these processes is affected by different factors, at systemic or cellular levels, which may modify carbon and energy demands of different tissues (Iglesias and Podestá 2005). Starch biosynthesis takes place in the chloroplast (Figure 18.1) and involves three enzymatic steps, sequentially catalyzed by (see Equations 18.1 through 18.3, respectively, in Figure 18.3) ADP-glucose pyrophosphorylase (ADPGlcPPase, EC 2.7.7.27), starch synthase (EC 2.4.1.21), and branching enzyme (EC 2.4.1.18) (Ballicora et al. 2003, 2004). This polyglucan is the major storage compound in higher plants, and in photosynthetic and heterotrophic tissues. It has been established that starch constitutes a transitory storage glucan in source tissues and its level varies during the photoperiod; while the long-term storage occurs in the plastids of heterotrophic cells in non-photosynthetic tissues, such as fruits, roots, and tubers (Figure 18.2) (Iglesias and Podestá 2005). Glc1P + ATP

ADPGlc + PPi

(18.1)

ADPGlc + (α-1,4-glucan)n

ADP + (α–1,4-glucan)n+1

(18.2)

Linear α-1,4-glucan chain

α-1,6-branched α-1,4-glucan chain

(18.3)

Fru6P

Glc6P

(18.4)

Glc6P

Glc1P

(18.5)

Fru6P + ATP

Fru1,6bisP + ADP

(18.6)

Fru6P + PPi

Fru1,6bisP + Pi

(18.7)

Ga3P + NAD++ Pi

1,3PGA + NADH + H+ 3PGA + NADPH + 2 H+

Ga3P + NADP+ + H2O PEP + HCO3– PEP + ADP

OAA + Pi Pyr + ATP

PEP + ADP + HCO3–

(18.8) (18.9) (18.10) (18.11)

OAA + ATP

(18.12)

UDPGlc + Fru6P

Sucrose-P + UDP

(18.13)

Sucrose-P + H2O

Sucrose + Pi

(18.14)

Sucrose + H2O Sucrose + UDP

Glc + Fru UDP-Glc + Fru

Glc6P + NADPH + H+ Gol6P + H2O Gol + NAD+

Gol6P + NADP+

Gol + Pi Fru + NADH + H+

(18.15) (18.16) (18.17) (18.18) (18.19)

Figure 18.3  Key reactions catalyzed by enzymes involved in carbohydrate metabolism and partitioning in plants.

Carbon Metabolism and Plant Stress

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18.3  Carbon Metabolism in the Cytosol and Plant Stress Many of the pathways of carbohydrate metabolism take place in the cytosol, where they are connected to other metabolic routes. In this compartment, there is a metabolic node constituted by fructose-6-P (Fru6P), glucose-6-P (Glc6P), and glucose-1-P (Glc1P). The conversion between Fru6P and Glc6P (see Figure 18.3, Equation 18.4) is catalyzed by a hexose-P isomerase (EC 5.3.1.9) while the reaction that converts Glc6P into Glc1P (Equation 18.5 in Figure 18.3) is catalyzed by a phosphoglucomutase (EC 5.4.2.2). In plants, the hexose-P pool can be utilized to produce many other metabolites, thus directing carbon skeletons to different metabolic fluxes (Smith 1999). The main fates for cytosolic hexose-P include glycolysis and synthesis of soluble sugars as sucrose and polyols. These central metabolic routes produce intermediate metabolites or final products that play critical roles in plants under physiological as well as stress conditions.

18.3.1  Organization of Plant Glycolysis Glycolysis is a ubiquitous metabolic pathway that converts glucose (Glc) into pyruvate (Pyr) in most organisms, producing 2 ATPs as a result (Givan 1999, Plaxton 1996, Podestá 2004). Glycolysis is just the first step of respiration, the fundamental process of intermediary metabolism. In plants, glycolysis is unique in that it possesses a series of differential features and its function goes beyond the provision of ATP and Pyr for mitochondrial respiration (Plaxton and Podestá 2006). One of these characteristics is the ability to use PPi instead of ATP as phosphoryl donor (Plaxton and Podestá 2006, Stitt 1998). Also, ancillary enzymes can be found that expand the classic 10 reactions pathway and lend a great degree of flexibility to metabolism (Plaxton 1996). Most important, the final product of cytosolic plant glycolysis is not necessarily Pyr. Instead, the cytosolic pool of phosphoenolpyruvate (PEP) represents a metabolic branch point from which carbon skeletons may follow different fates (Plaxton 1996, Plaxton and Podestá 2006). In addition, Glc is not the obligated starting metabolite for glycolysis: the photosynthetically generated hexose-P or triose-P pools can both contribute with carbon skeletons in the route toward PEP or Pyr (Dennis and Blakeley 2000). Finally, it must be mentioned that plants possess a second set of glycolytic enzymes in the plastid, with its own distinctive characteristics. All of these features can be efficiently exploited by plants to face different types of stress situations. Starting from the top, down along the conventional glycolytic pathway, the first distinguishing characteristic of the plant cell cytosolic glycolytic metabolism arises at the level of the conversion of Fru6P to Fru1,6bisP. As in most other organisms, it is a key regulatory step, but the phosphorylation reaction can be achieved by two different enzymes (see, respectively, Equations 18.6 and 18.7 in Figure 18.3): a classical but Fru2,6bisP-insensitive ATP-dependent phosphofructokinase (ATP-PFKase, EC 2.7.1.11) or a PPi-dependent enzyme that uses PPi as phosphoryl donor (PPiPFKase, EC 2.7.1.90) and is potently activated by Fru2,6bisP (Carnal and Black 1979, Dennis and Blakeley 2000, Givan 1999, Iglesias and Podestá 2005, Plaxton and Podestá 2006) (Figure 18.1). Plant PPi-PFKase is widespread in different plant species and tissues and its subunit composition and activity respond to environmental and developmental cues (Nielsen 1995, Plaxton and Podestá 2006, Podestá and Plaxton 1994a, Theodorou and Plaxton 1996, Trípodi and Podestá 1997). Current knowledge indicates that it most probably works in the glycolytic direction, despite the fact that it catalyzes a reversible reaction, controlling the balance between the triose-P and hexose-P pools and carbon partitioning among starch and sucrose (Hajirezaei et al. 1994, Plaxton 1996, Plaxton and Podestá 2006, Podestá and Plaxton 2003). Following conversion of Fru1,6bisP into triose-P, the first energy-conserving reaction takes place with the generation of ATP by the joint action of glyceraldehyde 3-P dehydrogenase (Ga3PDHase, EC 1.2.1.12) and phosphoglycerate kinase (EC 2.7.2.3). The first reaction (Equation 18.8 in Figure 18.3), the only oxidation that takes place during glycolysis, can be circumvented by a NADPdependent, non-phosphorylating glyceraldehyde 3-P dehydrogenase (np-Ga3PDHase, EC 1.2.1.9)

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that yields 3PGA and uses NADP rather than NAD as acceptor of the reducing equivalents (Bustos and Iglesias 2002, Iglesias et al. 2002) (see Figure 18.3, Equation 18.9). The reaction is irreversible in this case, and leads to a product that cannot be used to generate ATP. The contribution of this enzyme thus leads to a nil ATP yield. However, the importance of np-Ga3PDHase in plant cytosolic metabolism is beginning to be acknowledged, having been implicated in fruit development and energetic metabolism. A recent work (Bustos et al. 2008) points out that the np-enzyme is implicated in the response to oxidative stress in wheat leaves. Also, plants deficient in this enzyme were found to exhibit a reduced glycolytic capacity and increased levels of oxidative stress (Rius et al. 2006). From then on, two reactions lead to the production of PEP. As stated above, PEP occupies a central role in plant carbohydrate metabolism as a regulatory molecule, but it is also significant as a branchpoint metabolite. PEP is the substrate for a carboxylation reaction (see Figure 18.3, Equation 18.10), mediated by PEP carboxylase (EC 4.1.1.31), a regulatory enzyme that produces oxaloacetate (OAA) and Pi (Chollet et al. 1996, Iglesias et al. 1997) (Figure 18.1). PEP carboxylase exists in various isoforms and different levels in plant tissues. It plays a major role in anapleurosis replenishing intermediates of the tricarboxylic acid pathway, and thus links cytosolic carbon metabolism with respiration and N assimilation. In C4 and Crassulacean acid metabolism plants the enzyme is responsible for the primary CO2 fixation, starting the CO2 concentrating mechanism. PEP carboxylase is subjected to tight regulation by metabolites and by reversible phosphorylation on a serine residue (Bakrim et al. 2001, Chollet et al. 1996, Trípodi and Plaxton 2005). Malate is a strong inhibitor of PEP carboxylase, but its effect is dependent on pH and, most important, on the phosphorylation status of the enzyme. The phosphorylated form of PEP carboxylase is usually more active and less sensitive to malate, specially at pH values on the acidic side of the optimum, which is around pH 8.0 (Baur et al. 1992, Chollet et al. 1996, Hartwell et al. 1999, Moraes and Plaxton 2000, Trípodi and Plaxton 2005). Glc6P is an important allosteric effector, promoting activation of the enzyme and reducing malate sensitivity (Chollet et al. 1996, Plaxton 1996). The enzyme from C3 plants (i.e., banana fruit and castor bean germinating endosperm) is very sensitive to inhibition by aspartate and glutamate, highlighting the link between N and C metabolism mediated by PEP carboxylase (Law and Plaxton 1995, Trípodi and Plaxton 2005). Phosphorylation alleviates the effect of aspartate (Gregory et al. 2009, Law and Plaxton 1995, Trípodi and Plaxton 2005). The characterization of PEP carboxylase kinase has shown that its ability to phosphorylate the target enzyme is impaired in the presence of malate, while it performs better in the presence of PEP (Murmu and Plaxton 2007). PEP is also the substrate for the usual end of glycolysis in most organisms, the reaction catalyzed by Pyr kinase (PyrKase, EC 2.7.1.40) that produces Pyr and ATP (see Figure 18.3, Equation 18.11) and constitutes the second energy-conserving reaction. PyrKase has been studied in many plant tissues where its regulatory role of the whole glycolytic process has been recognized (Hu and Plaxton 1996, Lin et al. 1989, Moraes and Plaxton 2000, Plaxton 1989, Podestá and Plaxton 1991). In fact, the activity of this enzyme is instrumental in controlling the flux of the upper part of glycolysis by regulating the levels of PEP (Givan 1999, Plaxton 1996), as it will be further analyzed below. PyrKase regulation is organ specific in plants. The cytosolic PyrKase from germinating castor seed endosperm or cotyledons shows a pH-dependent response to several metabolite inhibitors (Podestá and Plaxton 1991, Podestá and Plaxton 1994b). A concerted decrease in pH and inhibitor concentrations, as could be caused by anoxia, will cause PyrKase activity to rise. Glutamate is an important inhibitor of the enzyme from several sources (Hu and Plaxton 1996, Lin et al. 1989, Plaxton 1996, Plaxton and Podestá 2006, Podestá and Plaxton 1994b), while aspartate activates PyrKase and relieves from the inhibitory effect of the former. The reciprocal effects of aspartate on PyrKase and PEP carboxylase provide an effective mechanism to balance both activities during active N assimilation. Thus, aspartate accumulation reduces the flux through PEP carboxylase while enhancing PyrKase activity in leaves, ripening banana fruit and castor bean endosperm (Plaxton and Podestá 2006, Smith et al. 2000, Turner et al. 2005, Turner and Plaxton 2000).

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A third enzyme also makes use of PEP. PEP carboxykinase (PEPCKase, EC 4.1.1.49) catalyzes the reversible carboxylation of PEP, using ADP as substrate as well, to yield OAA and ATP (Chen et al. 2004, Daley et al. 1977, Leegood and Walker 2003, Rylott et al. 2003, Walker and Leegood 1996, Wingler et al. 1999) (Figure 18.3, Equation 18.12). Although generally implicated in gluconeogenesis, the activity of PEPCKase is also crucial to determine the concentration of PEP. It has been shown that the enzyme is subjected to phosphorylation in many but not all plants (Leegood and Walker 2003). The study of PEPCKase has been hindered by the extreme sensitivity of the enzyme to proteolysis upon extraction (Martín et al. 2007). The truncated, but otherwise active enzyme exhibits few, if any, regulatory properties. The phosphorylation site lies within the excised peptide, which strongly suggest that whatever changes in the regulation of PEPCKase are brought about by phosphorylation are not evident on the proteolyzed enzyme (Leegood and Walker 2003). So far, studies with a preparation containing only the phospho or dephospho forms of PEPCKase are lacking, but the use of special reaction media designed to measure preferentially one form demonstrated that phosphorylation lowers PEPCKase activity (Leegood and Walker 2003). A strict coordination in the phosphorylation status of PEP carboxylase and PEPCKase is necessary as the two enzymes acting at the same time can give rise to a potential futile cycle. Levels of PEP thus depend on the coordination of the activities of at least three regulatory enzymes. The importance of this can be appreciated after examination of the role of PEP as a regulatory metabolite. In plants, the conversion of Fru6P to Fru1,6bisP does not represent the primary control point of glycolysis, in part due to the lack of activation of ATP-PFKase by Fru2,6bisP. PPi-PFKase, instead, is strongly activated by Fru2,6bisP (Kruger and Dennis 1987, Theodorou and Plaxton 1994, Trípodi and Podestá 1997). To date, all plant cytosolic ATPPFKases examined are inhibited by PEP (Givan 1999, Lee and Copeland 1996), and this metabolite also is an inhibitor of the synthesis of Fru2,6bisP (Plaxton and Podestá 2006). Levels of PEP, thus, control the flux through the upper part of glycolysis, reinforcing the role of PEP carboxylase and PyrKase as pacemaker enzymes of the whole pathway. This does not mean that control is completely delegated on these two enzymes; rather that the primary control depends on them. ATP-PFKase and PPi-PFKase are still relevant for the fine-tuning of carbon flow in the cytosol, with activities that depend on the levels of PEP but also on Pi concentration, as Pi activates ATPPFKase while it is a powerful inhibitor of the glycolytic reaction of PPi-PFKase (Plaxton and Podestá 2006, Podestá 2004). In any case, the study of the contribution of ATP-PFKase, PPiPFKase, PEP carboxylase, and PyrKase by the use of transgenic lines deficient in or overexpressing these enzymes is complicated by the great degree of metabolic flexibility that plants show. In many cases, successful creation of a transgenic plant does not result in an evident phenotype, or it displays it only under a particular condition (Grodzinski et al. 1999, Hajirezaei et al. 1994, Stitt and Sonnewald 1995).

18.3.2  Metabolism of Sucrose and Polyols In leaves, during the light period, sucrose is produced from triose-P translocated from the chloroplast trough the TPT in exchange for Pi (Figure 18.1). In the dark, starch mobilization allows the constant production of sucrose and the uninterrupted flow of carbon to heterotrophic tissues (Winter and Huber 2000). The reactions that derive to sucrose synthesis include the action of sucrose-P synthase (SPSase, EC 2.4.1.14) and sucrose-P phosphatase (EC 3.1.3.24) (see Figure 18.1, and Equations 18.13 and 18.14 in Figure 18.3). Sucrose is then transported to sink tissues where its degradation can be catalyzed by two different classes of enzymes. Invertases (EC 3.2.1.26, Equation 18.15 in Figure 18.3) catalyze the irreversible hydrolysis of sucrose to Glc and Fru. On the other hand, a reversible cleavage of sucrose occurs by the action of sucrose synthase (SSase, EC 2.4.1.13, Equation 18.16 in Figure 18.3; see also Figure 18.2) (Iglesias and Podestá 2005, Winter and Huber 2000). The latter reaction conserves the energy of the glycosidic bond in UDP-glucose (UDPGlc), which can be used for cellulose synthesis or can enter glycolysis. In heterotrophic tissues, Glc6P or Glc1P are the preferred molecules carried

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through a specific translocator (GPT, see Figure 18.2) inside plastids, where it can be utilized for starch synthesis (Winter and Huber 2000). In addition to sucrose and starch, certain plants synthesize Gol (also known as sorbitol) or mannitol as important photosynthetic products (Loescher and Everard 2004). In these organisms, the analysis of [14C]CO2 assimilation reveals the presence of two major soluble compounds: the polyol and sucrose. In apple leaves, about 70% of the newly photosynthetically fixed carbon was found as Gol and sucrose (Grant and Rees 1981). In celery, almost 80% of the label in mature leaves was recovered as mannitol and sucrose, with similar quantities (on a molar basis) of each one (Loescher et al. 1992). Similar results were obtained in apricot, where Gol was found to be the compound with the highest label 30 min after the pulse (Bieleski and Redgwell 1977). Gol is a major photosynthetic product in many important fruit-bearing tree species of the Rosaceae family, such as apple, peach, pear, and loquat. In general, a few studies deal with sugar-alcohols metabolism, in particular if compared with the abundant bibliography concerning starch (Ballicora et al. 2003, 2004) and sucrose (Koch 2004, Winter and Huber 2000) pathways. Because in many plants sugar-alcohols are main photosynthetic products, their metabolism is expected to be tightly regulated, probably at different levels. It is worth noting that sugar-alcohols could have important roles in plant tolerance to certain types of abiotic stresses (Loescher and Everard 2004). Gol is synthesized in mature leaves trough the conversion of Glc6P into Gol6P by the action of a NADPH-dependent aldose-6-phosphate reductase (A6PRase, EC 1.1.1.200; see Equation 18.17 in Figure 18.3) (Figueroa and Iglesias 2010, Hirai 1981, Kanayama and Yamaki 1993, Zhou et al. 2003b) and the subsequent hydrolysis of the phosphate group catalyzed by a specific Gol6P phosphatase (EC 3.1.3.50, Equation 18.18 in Figure 18.3) (Zhou et al. 2003a). These enzymes can be found in the cytosol in soluble forms, without any association with vacuoles, chloroplasts, mitochondria, peroxysomes, or membranes (Loescher and Everard 2004). The NADPH necessary for synthesizing the sugar-alcohol may be derived from the reaction catalyzed by np-Ga3PDHase (see Figure 18.3, Equation 18.9), which has been found in celery leaves at levels capable of sustaining relevant production of mannitol by this plant (Gao and Loescher 2000, Rumpho et al. 1983). The sugar-alcohol produced in leaves can be transported to sink or developing tissues, such as fruits or immature leaves, where it can be converted to Fru by the action of a NAD-dependent Gol dehydrogenase (GolDHase, EC 1.1.1.14, Equation 18.19 in Figure 18.3) (Ohta et al. 2005, Oura et al. 2000, Yamaguchi et al. 1994).

18.4  Plants Carbohydrate Metabolism and Stress 18.4.1  Responses of Glycolytic Carbon Metabolism Enzymes to Stress 18.4.1.1  Hexose-P Metabolism Mertens et al. (1990) found that PPi-PFKase (but, not ATP-PFKase) is induced upon anoxia in rice seedlings. Fru2,6bisP also increases during anoxia in rice seedlings, helping to boost PPi-PFKase activity in a more acidic environment that could curtail this enzyme’s activity (Mertens 1990). In this tissue, sucrose degradation proceeds mainly through the uridylate requiring the sucrose synthase/nucleoside diphosphate kinase system, lowering the dependence on adenilates that usually have lower levels upon this condition (Ricard et al. 1991). Pi starvation increases PPi-PFKase levels in Brassica nigra and Brassica napus (Theodorou and Plaxton 1994, 1996). B. nigra seedlings respond to Pi starvation by increasing the extractable activity of PPi-PFKase, the ratio PPiPFKase:ATP-PFKase and by an increase in the amount of the α relative to the β subunit (Theodorou and Plaxton 1994). The same response in the α:β ratio has been observed in black mustard suspension cells under Pi deficit (Theodorou et al. 1992). In both cases, the sensitivity to Fru2,6bisP is increased. Thus, the response to Pi stress at the hexose-P pool level is clearly to circumvent the adenylate-dependent step catalyzed by ATP-PFKase. This is a result of the acquired ability of plants to use PPi as a phosphoryl donor, efficiently using the energy available in the anhydride bond

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(Stitt 1998). Assuming that PPi is a by-product of anabolism; no ATP is needed for the conversion of sucrose to hexose-P via the SSase pathway in heterotrophic tissues, whereas 2 ATPs are needed for the invertase pathway. Remarkably, although PPi can be hardly conceived as nothing more than a transient by-product of several cell reactions, which is readily hydrolyzed in animal cells, PPi levels in the cytosol of plant cells remain stable through a variety of conditions, including, but not limited to, severe Pi-deficiency or anoxia, which is consistent with the importance of PPi-dependent enzymes during stress (Plaxton and Podestá 2006, Stitt 1998). The importance of PPi in plant cell economy has been demonstrated by the creation of transgenic plants expressing a bacterial pyrophosphatase in the cytosol. These plants showed a threefold decreased PPi concentration and also severely impaired growth (Jellito et al. 1992). The link between Pi nutritional status and the regulation of ATP-PFKase and PPi-PFKase is also evident from the fact that ATP-PFKase is regulated by the Pi:PEP ratio (with Pi being an activator and PEP an inhibitor); while Pi is a potent inhibitor of PPi-PFKase in the glycolytic direction (Dennis and Blakeley 2000). This suggests that under Pi deficiency, the latter would be the predominant activity, thus allowing an adenylate-independent glycolytic pathway. Pi-PFKase has been implicated in the response to cold stress as well. An examination of the subunit composition of PPiPFKase in orange fruits showed a displacement of the α:β subunit ratio from 1.66 to 1 upon exposure to frost and almost a doubling of its activity, all this complemented by an increased sensitivity to Fru2,6bisP (Falcone Ferreyra et al. 2006). Cold stress leads to an initial fall in sucrose synthesis, accumulation of phosphorylated metabolites, and consequent Pi-limitation of photosynthesis (Stitt and Hurry 2002). Plants respond to the challenge by increasing sucrose synthesis. Within minutes of initiation the cold stress in Arabidopsis thaliana, SPSase is posttranslationally activated by phosphorylation (Stitt and Hurry 2002). On the longer term, SPSase and cytosolic Fru1,6bisP phosphatase expression are boosted, releasing the Pi necessary for sustained photosynthesis at the expense of phosphorylated metabolites (Stitt and Hurry 2002). The response also includes movement of Pi from the vacuole to the cytosol, to allow the replenishment of phosphorylated metabolites without depleting free Pi (Hurry et al. 2000). Thus, as expressed by Stitt and Hurry (2002): “changes in Pi modulate and may even act as a signal in the regulation of photosynthetic/metabolic acclimation to low temperatures, and lead to major changes in the ability of the different genotypes to develop frost tolerance.” 18.4.1.2  PEP Metabolism Levels of PEP carboxylase have been reported to vary in response to different abiotic stresses. Recently, a study in frost-damaged orange fruits showed increased levels of PEP carboxylase in stressed fruit, which correlate with a lower sensitivity to its natural feedback inhibitor malate (Falcone Ferreyra et al. 2006). Since the fermentative pathway is higher in this tissue, it has been proposed that PEP carboxylase could act, in combination with malate dehydrogenase, as an ancillary fermentative enzyme, helping in the provision of ATP when aerobic respiration is affected (Falcone Ferreyra et al. 2006). Vu et al. (1995) reported that cold-hardy citrus varieties responded with an increase in extractable PEP carboxylase activity upon acclimation, whereas a sensitive cultivar showed a decrease in foliar PEP carboxylase levels. Similar trends have been reported in other cultivars exposed to cold (Vu et al. 1995), underscoring an as yet little studied role of PEP carboxylase in plant primary metabolism. Abiotic stresses that affect water balance, including cold stress and anoxia, also provoke an induction of PEP carboxylase. Induction was root-specific, except for the cold treatment, which also induced the enzyme in shoots (González et al. 2003). In general, induction of PEP carboxylase has been linked to an increased need for synthesis of organic acids such as malate, in response to cytoplasmic alkalinization among other challenges. PEP carboxylase activity is also responsive to Pi levels. Several studies showed an increase in PEP carboxylase content upon nutritional deprivation of Pi. One of the first papers describing the bypass of adenylate-using enzymes upon Pi deprivation in B. nigra suspension cells reported that PEP carboxylase, np-Ga3PDHase, PPi-PFKase, and PEP phosphatase showed important increases

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in activity at the expense of the ATP-PFKase, PyrKase, and Ga3PDHase (Duff et al. 1989). B. napus efficiently uses rock Pi by excreting organic acids to the environment with the concurrent solubilization of the former. Pi deprivation in B. napus and the related hedge mustard caused an increase in PEP carboxylase levels and, probably as a result of this, also in malate exudation (Hoffland et al. 1992). Moraes and Plaxton (2000) reported the purification and properties of the Pi-deficient B. nigra PEP carboxylase. Pi starvation increased PEP carboxylase by 2.5-fold, while refeeding caused an immediate return to near control levels. The study of the purified enzyme showed that the increased activity under Pi shortage cannot be attributed to an increased phosphorylation state of the enzyme that would make it less susceptible to malate inhibition. It is possible that, at least in these cells, metabolite effects override the importance of phosphorylation in PEP carboxylase control (Moraes and Plaxton 2000). Several reports (Andaluz et al. 2009, Thimm et al. 2001) highlight the induction of PEP carboxylase in roots under iron deficiency. The induction of PEP carboxylase accompanies the apoplastic acidification caused by iron starvation (Thimm et al. 2001). PEP carboxylase induction in roots is paralleled by increased levels of several glycolytic enzymes, namely, Ga3PDHase, phosphoglyceromutase, enolase, and PyrKase. This, together with an enhanced mitochondrial electron transport complement, reveals a respiratory surge as a response to iron stress.

18.4.2  Soluble Carbohydrates and Their Role against Stress Many organisms accumulate low molecular weight compounds such as disaccharides (sucrose and trehalose), sugar-alcohols, quaternary amines, or amino acids. It has been proposed that these molecules allow organisms tolerate certain kinds of abiotic stresses (like salinity, cold or drought) through mass action. These compounds may be considered as compatible solutes, a term that was introduced in studies conducted with yeast accumulating nonreducing sugars (like trehalose) and sugar-alcohols (including glycerol) in response to osmotic stress (Brown and Simpson 1972). It is well known that different inorganic (K+, Na+, Cl−, and SO42−) and organic (reducing hexoses) molecules are critical to establish osmotic adjustments in aqueous systems. However, these compounds are different from compatible solutes, which may affect the properties of solutions by distinctive ways (Bohnert and Jensen 1996, Loescher and Everard 2004). Early work established that high concentrations of compatible solutes do not interfere with in vitro enzyme activities and that sometimes they can protect proteins from deleterious effects of salts or heat (Bohnert and Jensen 1996, Loescher and Everard 2004). It has been described that the concentration of these molecules should reach values as high as 500 mM to exert protection; interestingly, such levels may be achieved in cells (Moing et al. 1997, Nadwodnik and Lohaus 2008). Different explanations have been proposed to clarify the protective effect of compatible solutes on biological structures. One hypothesis is that they substitute water molecules in the hydration of proteins and membranes, thus allowing enzyme activity to occur even at extremely low water concentrations (Webb and Bhorjee 1968). Another alternative suggests that compatible solutes might be outside the hydration sphere of proteins, thus producing a particular rearrangement of the sphere and inducing the biological structure to adopt a preferential hydration (Timasheff 1993). Concerning plants, studies performed at the present time highly support the possibility to derivate cytosolic carbohydrates to different metabolic fates rendering compounds of the type of compatible solutes. Accumulation of these metabolites could help the plant to resist extreme conditions of low temperatures, high salt levels, and water deficit. The overall picture sustain that modifications in the balance of photosynthates partitioning can play critical roles for plant productivity and survival under physiological or stress conditions. 18.4.2.1  Osmotic Stress Adaptation Plants response to osmotic stress generally results in increased soluble sugars and decreased starch as a result of enhanced sugar synthesis. This rise in carbohydrates concentration has been related with higher activity levels of the synthesizing enzymes. For instance, SPSase activity dramatically increased in osmotically stressed spinach leaves and potato tubers. This activation is consequence of the phosphorylation of a single residue (serine-424 in spinach leaf SPSase), and is different from that involved

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in dark-light modulation (Winter and Huber 2000). This site is widely conserved among species and its phosphorylation in osmotically stressed leaves activates the enzyme, thus allowing sucrose synthesis to occur when it would otherwise be restricted (Toroser and Huber 1997). Accumulation of sucrose, cyclic, or acyclic sugar-alcohols, proline, and quaternary amines (like glycinebetaine) could potentially play a direct role in osmoregulation and could also provide quickly metabolizable carbohydrates for energy production when carbon is diverted from growth to other functions (Hare et al. 1998). 18.4.2.2  Low-Temperature Stress Carbohydrates such as sucrose, fructans, and sugar-alcohols can be important in tolerance and resistance to cold-induced damage (del Viso et al. 2009a,b, Loescher and Everard 2004, Pontis 1989, Tognetti et al. 1990). Cold damage results by desiccation due to water demand from the protoplast, as a consequence of the growing ice crystal (Loescher and Everard 2004). Even when results are not conclusive, many studies have related this kind of stress with the accumulation of compatible solutes. For instance, it was found that Gol concentration in apple shoot xylem increased with leaf senescence and low temperatures (Williams and Raese 1974). Similar changes have been described in plum trees, where the highest level of Gol in sap was found after exposing plants to temperatures below zero (Loescher et al. 1990). Further evidence in the same way was obtained by Hirai (1983), who reported a raise of Gol and A6PRase levels in loquat leaves during low-temperature seasons. In addition, accumulation of sucrose in photosynthetic and heterotrophic tissues has been linked with increased SPSase activity under nonfreezing temperatures, which was related to an increased level of SPSase protein in spinach leaves (Guy et al. 1992) and potato tubers (Hill et al. 1996). In spinach leaves, the rate of SPSase protein synthesis seems to be responsible for the raise in activity and the newly produced enzyme subunit appears to be identical to that found under normal conditions (Guy et al. 1992). On the other hand, cold-exposed potato tubers showed an increase in a particular subunit (1b) of SPSase, which correlates with a change in the kinetic properties of this enzyme. Thus, the altered kinetics of SPSase may play an important role in the regulation of sucrose synthesis in cold-stored tubers (Hill et al. 1996). 18.4.2.3  Drought Stress Results relating drought stress and carbohydrates accumulation are difficult to understand as a result of secondary effects, like growth inhibition and dehydration (Loescher and Everard 2004). However, it is important to draw attention to the evidences. For instance, Gol was the main soluble carbohydrate and its content was doubled in plants from the Prunus genus subjected to drought stress (Ranney et al. 1991). In apple trees, drought stress resulted in the preferential accumulation of Gol and Glc at the expense of sucrose and starch (Wang et al. 1995, 1996). When genetically transformed plants were used, it was demonstrated that the increased ability for synthesizing soluble sugar-alcohols promoted drought tolerance. In transformed tobacco plants synthesizing the nonreducing disaccharide trehalose, it was determined that their ability to survive after a drought period was enhanced. However, their phenotypes were altered and the growing rate was diminished up to 50% under normal growing conditions (Holmstrom et al. 1996). A similar work, where transformed tobacco plants were engineered to produce bacterial fructans, showed that these plants grew better than controls under polyethylenglycol-induced drought, conditions where the growing rates and the fresh and dry weights were also higher. On the other hand, compared with those plants producing trehalose, these transformed plants did not show differences with controls under normal conditions (Pilon-Smits et al. 1995). 18.4.2.4  High-Salinity-Induced Stress The correlation between salt stress and sugars accumulation is quite strong, and these works represent the most numerous reports linking one type of stress with polyols accumulation. A couple of studies, done with celery exposed to NaCl-induced stress (Everard et al. 1994) or macronutrients excess (Stoop and Pharr 1994) established that changes in mannitol metabolism were consequence of both carbon partitioning and utilization. For instance, in newly developed celery leaves exposed to 300 mM NaCl, the carbon flux into mannitol remained at similar rates to controls, although a 70%

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decrease in carbon assimilation was found. The maintenance of mannitol synthesis was at sucrose expenses, thus increasing fourfold the ratio between mannitol and sucrose labeled. This change was associated to an increase in the activity of mannose-6-P reductase (EC 1.1.1.224). However, this increase in activity was not related to a rise in the protein level of the enzyme, suggesting a possible posttranslational modification of the enzyme (Everard et al. 1994). On the other hand, a study conducted with Japanese persimmon (Diospyros kaki, a plant that normally does not produce sugaralcohols) transformed with the gene encoding for the A6PRase from apple leaves, found an increase in tolerance to NaCl-induced stress in those lines capable of accumulating Gol (Gao et al. 2001). 18.4.2.5  Hydroxyl-Radical Scavengers Reduced stomatal conductance can occur under salt and drought stress, thus increasing the production of free radicals. There is evidence that in fungi and other organisms sugar-alcohols (such as Gol and mannitol) and cyclic polyols (like mio-inositol) can act as free radical scavengers (Jennings and Burke 1990, Smirnoff and Cumbes 1989). Also, it was shown that this effect could be observed in stress caused by dehydration (Smirnoff 1993). However, the evidence of in vivo assays is limited. For instance, transformation of tobacco plants with a bacterial mannitol-1-P dehydrogenase (EC 1.1.1.17), directed to chloroplasts by the introduction of a transit peptide, rendered a line capable of accumulating mannitol in chloroplasts in concentrations up to 100 mM, with no alteration of the phenotype or the photosynthetic activity. The presence of mannitol in chloroplasts resulted in an increased tolerance to methylviologen, a compound that produces oxidative stress. It is important to emphasize that the presence of the polyol did not reduce the abundance of reactive oxygen species, but it conferred an additional protection to the already present in non-transformed plants (Shen et al. 1997).

18.5  Concluding Remarks As can be inferred from the numerous examples listed above, carbon metabolism and partitioning critically affect plant productivity under physiological and stress conditions. Thus, the understanding of key enzymes involved in different metabolic routes operating in plants is relevant to evaluate potential plant adaptation in different environments. Many factors can affect the expression pattern of genes and the activity of enzymes related to carbohydrates metabolism. In general, the modulation of enzyme activity by metabolites (allosterism) or posttranslational modifications (thiols oxidation and reduction or phosphorylation) have been relatively well established in sucrose and starch biosynthetic pathways. On the other hand, the enzymes involved in sugar-alcohols metabolism have been scarcely characterized. Thus, efforts should be made to develop accurate systems to express and purify these enzymes, and such a work is currently under way (Figueroa and Iglesias 2010, Ohta et al. 2005). Developing molecular tools to identify the functionality of different enzymes and to rationally modify plant metabolism is promissory to handle plant behavior in diverse habitats.

Acknowledgments The authors wish to thank the financial support of ANPCyT PICTO 2005 15–36129 and UNL CAI+D 2008 Redes y Orientados to AAI, ANPCyT PICT 2005 32459 to FEP, and CONICET PIP 2519 to FEP and AAI.

References Andaluz, S., J. Rodríguez-Celma, A. Abadía, J. Abadía, and A.-F. López-Millán. 2009. Time course induction of several key enzymes in Medicago truncatula roots in response to Fe deficiency. Plant Physiol. Biochem. 47:1082–1088. Bakrim, N., J. Brulfert, J. Vidal, and R. Chollet. 2001. Phosphoenolpyruvate carboxylase kinase is controlled by a similar signaling cascade in CAM and C(4) plants. Biochem. Biophys. Res. Commun. 286:1158–1162.

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Loescher, W. H., T. McCamant, and J. D. Keller. 1990. Carbohydrate reserves, translocation, and storage in woody plant roots. HortScience 25:274–281. Loescher, W. H., R. H. Tyson, J. D. Everard, R. J. Redgwell, and R. L. Bieleski. 1992. Mannitol synthesis in higher plants: Evidence for the role and characterization of a NADPH-dependent mannose 6-phosphate reductase. Plant Physiol. 98:1396–1402. Martín, M., W. C. Plaxton, and F. E. Podestá. 2007. Activity and concentration of non-proteolyzed phosphoenolpyruvate carboxykinase in the endosperm of germinating castor oil seeds: Effects of anoxia on its activity. Physiol. Plant. 130:484–494. Moing, A., F. Carbonne, B. Zipperlin, L. Svanella, and J.-P. Gaudillère. 1997. Phloem loading in peach: Symplastic or apoplastic? Physiol. Plant. 101:489–496. Moraes, T. F. and W. C. Plaxton. 2000. Purification and characterization of phosphoenolpyruvate carboxylase from Brassica napus (rapeseed) suspension cell cultures: Implications for phosphoenolpyruvate carboxylase regulation during phosphate starvation, and the integration of glycolysis with nitrogen assimilation. Eur. J. Biochem. 267:4465–4476. Murmu, J. and W. Plaxton. 2007. Phosphoenolpyruvate carboxylase protein kinase from developing castor oil seeds: Partial purification, characterization, and reversible control by photosynthate supply. Planta 226:1299–1310. Nadwodnik, J. and G. Lohaus. 2008. Subcellular concentrations of sugar alcohols and sugars in relation to phloem translocation in Plantago major, Plantago maritima, Prunus persica, and Apium graveolens. Planta 227:1079–1089. Nielsen, T. H. 1995. Fructose-1,6-bisphosphate is an allosteric activator of pyrophosphate: Fructose-6-phosphate 1-phosphotransferase. Plant Physiol. 108:69–620. Ohta, K., R. Moriguchi, K. Kanahama, S. Yamaki, and Y. Kanayama. 2005. Molecular evidence of sorbitol dehydrogenase in tomato, a non-Rosaceae plant. Phytochemistry 66:2822–2828. Oura, Y., K. Yamada, K. Shiratake, and S. Yamaki. 2000. Purification and characterization of a NAD+-dependent sorbitol dehydrogenase from Japanese pear fruit. Phytochemistry 54:567–572. Pilon-Smits, E., M. Ebskamp, M. J. Paul et al. 1995. Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant Physiol. 107:125–130. Plaxton, W. C. 1989. Molecular and immunological characterization of plastid and cytosolic pyruvate kinase isozymes from castor-oil-plant endosperm and leaf. Eur. J. Biochem. 181:443–451. Plaxton, W. C. 1996. The organization and regulation of plant glycolysis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47:185–214. Plaxton, W. C. and F. E. Podestá. 2006. The functional organization and control of plant respiration. Crit. Rev. Plant Sci. 25:159–198. Podestá, F. E. 2004. Glycolysis. In Encyclopedia of Plant and Crop Science, ed. T. Goodman, pp. 547–550. New York: Marcel Dekker. Podestá, F. E. and W. C. Plaxton. 1991. Kinetic and regulatory properties of cytosolic pyruvate kinase from germinating castor oil seeds. Biochem. J. 279(Pt 2):495–501. Podestá, F. E. and W. C. Plaxton. 1994a. Regulation of carbon metabolism in germinating Ricinus communis cotyledons. I. Developmental profiles for the activity, concentration, and molecular structure of pyrophosphate- and ATP-dependent phopshofructokinases, phosphoenolpyruvate carboxylase, and pyruvate kinase. Planta 194:374–380. Podestá, F. E. and W. C. Plaxton. 1994b. Regulation of carbon metabolism in germinating Ricinus communis cotyledons. II. Properties of phosphoenolpyruvate carboxylase and cytosolic pyruvate kinase associated with the regulation of glycolysis and nitrogen assimilation. Planta 194:406–417. Podestá, F. E. and W. C. Plaxton. 2003. Ligand binding to potato tuber pyrophosphate-dependent phosphofructokinase studied through intrinsic fluorescence quenching. Evidence of competitive binding among fructose-1,6-bisphosphate and fructose-2,6-bisphosphate. Arch. Biochem. Biophys. 414:101–107. Pontis, H. G. 1989. Fructan and cold stress. J. Plant Physiol. 134:148–150. Ranney, T. G., N. L. Bassuk, and T. H. Whitlow. 1991. Osmotic adjustment and solute constituents in leaves and roots of water-stressed cherry (Prunus) trees. J. Am. Soc. Hort. Sci. 116:684–688. Ricard, B., J. Rivoal, A. Spiteri, and A. Pradet. 1991. Anaerobic stress induces the transcription and translation of sucrose synthase in rice. Plant Physiol. 95:669–674. Rius, S. P., P. Casati, A. A. Iglesias, and D. F. Gomez-Casati. 2006. Characterization of an Arabidopsis thaliana mutant lacking a cytosolic non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase. Plant Mol. Biol. 61:945–957. Rumpho, M. E., G. E. Edwards, and W. H. Loescher. 1983. A pathway for photosynthetic carbon flow to mannitol in celery leaves: Activity and localization of key enzymes. Plant Physiol. 73:869–873.

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Synthesis by Plants 19 Protein under Stressful Conditions Pallavi Sharma and Rama Shanker Dubey Contents 19.1 Introduction...........................................................................................................................465 19.2 Stressed Environments and Protein Synthesis...................................................................... 470 19.2.1 Salinity..................................................................................................................... 470 19.2.1.1 Salt-Induced Protein Synthesis.................................................................. 470 19.2.1.2 Protein Level in Salt-Stressed Plants........................................................ 475 19.2.1.3 Enzyme Levels in Salt-Stressed Plants..................................................... 477 19.2.2 Drought.................................................................................................................... 479 19.2.2.1 Drought-Induced Protein Synthesis..........................................................480 19.2.2.2 Protein Level in Drought-Stressed Plants.................................................484 19.2.2.3 Enzyme Levels in Drought-Stressed Plants.............................................. 485 19.2.3 Heat Stress............................................................................................................... 488 19.2.3.1 Synthesis of Heat-Shock Proteins............................................................. 489 19.2.3.2 Types of Heat-Shock Proteins................................................................... 491 19.2.4 Chilling.................................................................................................................... 491 19.2.4.1 Cold Acclimation...................................................................................... 492 19.2.4.2 Abscisic Acid and CA............................................................................... 495 19.2.5 Anaerobic Stress...................................................................................................... 496 19.2.6 Pathogenesis............................................................................................................. 497 19.2.7 Wounding................................................................................................................. 499 19.2.8 Metal Toxicity..........................................................................................................500 19.2.8.1 Enzyme Levels in Metal-Stressed Plants.................................................. 502 19.2.9 Gaseous Pollutants................................................................................................... 503 19.2.10 UV Radiation...........................................................................................................504 19.3 Conclusions............................................................................................................................ 505 References....................................................................................................................................... 505

19.1  Introduction Environmental stresses present major challenges in our quest to achieve sustainable food production. The reactions of plants to environmental stresses are complex and involve a wide array of physiological and biochemical responses. Such responses are initiated by plants growing in stressed environments to overcome, avoid, or nullify the effects of stresses. Tolerance or sensitivity toward a particular stressful condition depends on the genetic and biochemical makeup of the species. Much attention has been focused during recent years to evolve crop species with adaptability built into their genetic and biochemical makeup to withstand various stressful environmental conditions. Plants are unable to express their full genetic potential for production when subjected to stressful environments (Zeigler, 1990; Gao et al., 2007). When exposed to stressed environments, plants initially recognize the stress stimulus, and thereafter a signal transduction cascade is 465

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invoked. Secondary messengers relay the signal, activating stress-responsive genes, the expression of which causes the accumulation or depletion of certain metabolites, alteration in the activity behaviors of many enzymes, overall changes in protein synthesis, and, of particular interest, synthesis of new sets of proteins that are specific to the particular type of stress (Jacobsen et al., 1986; Vierling, 1991; Kaur and Gupta, 2005; Sharma and Dubey, 2007; Kumar et al., 2008; Gupta et al., 2009). It has been shown that different environmental stresses induce the synthesis of novel proteins in plants, which possibly provide evolutionary value to the plants for enhanced survival in adverse environmental situations. The synthesis of such stress-induced proteins has been well documented under salinity stress (Ericson and Alfinito, 1984; Hurkman and Tanaka, 1987; Singh et al., 1987; Ben-Hayyim et al., 1989; Naot et al., 1995; Naqvi et al., 1995; Igarashi et al., 1997; Aarati et al., 2003; Mahmoodzadeh, 2009), osmotic stress or drought (Bewley and Larsen, 1982; Singh et al., 1987; Vance et al., 1990; Robertson and Chandler, 1994; Baker et al., 1995; Mantyla et al., 1995; Perezmolphebalch et al., 1996; Zhang et al., 1996; Jiang and Huang, 2002; Demirevska et al., 2008; Khurana et al., 2008), heat shock (Heikkila et al., 1984; Mansfield and Key, 1988; Vierling, 1991; Cordewener et al., 1995; Waters et al., 1996; Lee et al., 2007; Yildiz and Terzi, 2008), low-temperature treatment (Meza-Basso et al., 1986; Hahn and Walbot, 1989; Bruggemann et al., 1994; Griffith et al., 1997; Matsuba et al., 1997; John et al., 2009; Kikuchi and Masuda, 2009), anaerobiosis (Ricard et al., 1991; Christopher and Good, 1996; Sachs et al., 1996; Subbaiah and Sachs, 2003), infection with pathogens (Antoniw et al., 1980, Ohashi and Matsuoka, 1985; Abad et al., 1996; Herbers et al., 1996; Tornero et al., 1997; Almagro et al., 2009), wounding (Cabello et al., 1994; Jung et al., 1995; Schaller and Ryan, 1996; Jimenez et al., 2008; Dafoe et al., 2009), metal toxicity (Choi et al., 1995; Shah and Dubey, 1998a; Sharma and Dubey, 2007), gaseous pollutants (Kirtikara and Talbot, 1996), and ultraviolet (UV) radiation (Jung et al., 1995; Rao et al., 1996; Xu et al., 2008; Pan et al., 2009). Figure 19.1 shows the schematic diagram of synthesis of proteins under different stresses in plants. These proteins, which are stress specific, present newer avenues to improve stress tolerance of plants. The main idea underlying studies of stress-induced synthesis of proteins in plants is that the different sources of stresses, their duration, and severity lead to a differential expression of genetic Stress signal transduction

Stress sensor

Stress

Osmotic adjustments Membrane protection Chelation of metal ions Transcription factors Chaperons

Synthesis of “stress proteins” LEA Dehydrins HSPs CBFs Osmotin Phytochelatins Metallothioneins Anaerobic polypeptides Pathogenesis-related proteins

Traditional breeding

Altered gene expression

Genetic transformation

Stress-tolerant plants

FIGURE 19.1  Stress-induced protein synthesis in plants. Stresses cause important modifications in the gene expression in plants, which leads to the synthesis and accumulation of stress-related proteins. These proteins provide enhanced survival value to plants under adverse environmental situations and can be used to produce stress-tolerant plants by genetic transformations. For details, refer Section 19.2.

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information, resulting in changes in gene products, including mRNA and proteins. Such newly synthesized proteins are specific to the particular type of stress and possibly confer enhanced survival value to the plants (Ben-Hayyim et al., 1989). Stress-induced proteins identified from different organs of plants have been well characterized. Physicochemical parameters such as molecular weight and pI (isoelectric point) values of these proteins have been deduced (Ben-Hayyim et al., 1989; Robertson and Chandler, 1994; Sachs et al., 1996; Waters et al., 1996; Efeoglu and Terzioglu, 2007), and, in many cases, data regarding association characteristics and amino acid sequences have also been reported (Singh et al., 1987; Badur et al., 1994; Naot et al., 1995; Zhang et al., 1996). A sizeable volume of literature indicates quantitative and qualitative changes in proteins, when plants are stressed, mainly employing the methods of electrophoresis, the western analysis, enzyme kinetics studies, etc. Nowadays, high-throughput stress-induced protein identification and characterization systems using tools of proteomics are available. Improved protein extraction and purification protocols have been devised, and genomic sequence databases for peptide mass matches are available (Timperio et al., 2008). Although the stress proteins are synthesized in plants when they are subjected to stresses and can be revealed in tissues of plants adapted to stress, specific metabolic functions for most of these proteins have not been established as to how they confer adaptability toward stress (Ben-Hayyim et al., 1989; Artlip and Funkhouser, 1995; Khurana et al., 2008). Particularly, under anaerobic stress, the polypeptides that are synthesized have specific functions and belong to the enzymes of sugar phosphate metabolism (Ricard et al., 1991). Heat-shock proteins (HSPs), which are synthesized under heat stress, possibly assist in protein folding, protein–protein interactions, and the translocation of proteins across cellular compartments, and they have a possible role in protecting the organism from heat stress (Cordewener et al., 1995; Wang et al., 2004). Similarly, the pathogenesis-related (PR) proteins do act in the defense of the plants and have a putative role in pathogen resistance (Artlip and Funkhouser, 1995). Under salinity stress, it is suggested that the newly synthesized proteins, together with amino acids and soluble nitrogenous compounds, act as components of a salt-tolerance mechanism. These might function as compatible cytoplasmic solutes in osmotic adjustment in order to equalize the osmotic potential of the cytoplasm with the vacuoles in adverse conditions of salinity (Greenway and Munns, 1980; Dubey and Rani, 1989). Studies related to stress-induced synthesis of proteins have been performed using cultured plant cells (Ericson and Alfinito, 1984; Singh et al., 1987; Ben-Hayyim et al., 1989; Vance et al., 1990; Sobkowiak and Deckert, 2006), seedlings (Mansfield and Key, 1988; Hahn and Walbot, 1989; Han and Kermode, 1996; Igarashi et al., 1997; Efeoglu and Terzioglu, 2007), excised plant organs (Hurkman and Tanaka, 1987; Stuiver et al., 1988), and intact plants (Burke et al., 1985; Ohashi and Matsuoka, 1985; Kee and Nobel, 1986; Popova et al., 1995). Among these systems, cultured plant cells have proven to be superior to other systems as they show uniform response and are under better control of environmental parameters (Ben-Hayyim et al., 1989; Fadzilla et al., 1997). Cell cultures from tobacco, cowpea, potato, citrus, and many other plant species have been used to identify and characterize newly synthesized proteins under salinity, heat-shock, freezing, osmotic, and heavy metal stresses (Singh et al., 1987; Ben-Hayyim et al., 1989; Vierling, 1991; Naot et al., 1995; Fadzilla et al., 1997). During recent years, a wealth of literature has been available, dealing with environmental stress–induced proteome changes, based on whole tissue/organ analysis (Bae et al., 2003; Giacomelli et al., 2006; Goulas et al., 2006; Taylor et al., 2009). Taylor and coworkers (2009) analyzed data and reviewed a collection of both whole tissue and organellar proteomic studies that investigated the effects of environmental stress in the model plant Arabidopsis thaliana. They found 279 proteins that could change in abundance and could be assigned as protein components of the energy organelles (chloroplast, mitochondria, and peroxisomes). These could be placed into eight different functional categories, and nearly 80% of the specific protein isoforms detected were only reported to change in a single environmental stress. Besides, the identification of specific-stress-induced proteins, several investigators have tried to quantify the overall metabolic status of total and soluble proteins (including enzymes) of different metabolic pathways in stressed plant parts in order to evaluate the impact of stresses on various aspects

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of plant growth and metabolism (Dubey and Rani, 1987; Dubey and Rani, 1989; Gogorcena et al., 1995; Shah and Dubey, 1995a,b; Jha and Dubey, 2004a,b; Mishra and Dubey, 2006, 2008a,b; Sharma and Dubey, 2007; Maheshwari and Dubey, 2007, 2008, 2009). Environmental stresses generally are detrimental to plant growth, adversely affect the metabolism of plants, and cause an imbalance in the level of protein as a result of their effects on the synthesis and hydrolysis of proteins (Dubey and Rani, 1987; Elsamad and Shaddad, 1997; Moons et al., 1997; Shah and Dubey, 1998a,b; Mishra and Dubey, 2006; Maheshwari and Dubey, 2007, 2008). Abiotic stresses lead to an enhanced degradation of proteins. Amino acids derived from protein catabolism may be redistributed within the plant via the phloem and serve as a basis for protein synthesis in other plant parts (Feller et al., 2008). In saltand drought-stressed plant parts, the protein content decreases owing to the decreased rate of protein synthesis and the increased rate of proteolysis (Dubey and Rani, 1987, 1990; Perezmolphebalch et al., 1996; Sharma and Dubey, 2005a). In seeds germinating under salinity, moisture stress, or nickel toxicity, however, an increase in the protein level is observed. This increase can best be explained by the fact that in germinating seeds, stress causes decreased proteolysis in endosperms, resulting in a slower depletion of reserve proteins. This reflects an apparent increase in the endospermic protein level under stress, which is not a result of enhanced protein synthesis (Dubey, 1983a; Dubey and Rani, 1987, 1990; Maheshwari and Dubey, 2008). Salt tolerance is dependent on the genetic and biochemical characteristics of the species. Therefore, attempts have been made by certain groups of investigators to differentiate stress-tolerant and stress-sensitive genotypes of crops on the basis of profiles or levels of soluble proteins, specific enzymes in germinating seeds, and growing plant parts (Dubey and Rani, 1987, 1989; Perezmolphebalch et al., 1996; Elsamad and Shaddad, 1997; Mahmoodzadeh, 2009). The results of these attempts indicate that different levels of soluble proteins and many enzymes exist in the two sets of genotypes differing in stress tolerance. Studies conducted so far indicate that stressful conditions adversely affect the protein metabolism in plants and that in all different types of environmental stresses, such as salinity, drought, heat, chilling, anaerobiosis, pathogenesis, wounding, heavy metal toxicity, and gaseous Salinity

Water deficit

Chilling

Heat

Anaerobiosis Wounding

Abscisic acid

LEA 1: D-19 LEA 3: D-7 LEA 5: D-29 LEA 4: D-113 LE 25

Osmotin Stress-associated proteins (SAPs) HZ-Zip

LEA: 2 Dehydrins RAB protein D-11

Germin HVA 1

Δ1-pyrroline-5-carboxylase synthetase Δ1-pyrroline-5-carboxylase reductase

Betaine aldehyde dehydrogenase Ca2+-ATPase Superoxide dismutase Peroxidase Glutathione reductase RUBP carboxylase Nitrate reductase

Pathogenesis

Several novel proteins 21, 25, 75, 95, 160 kDa Antifreeze proteins LT 178

Heavy metal Gaseous pollutants

HSPs:

Cytosolic-60, 70, 81, 90 kDa Mitochondrial and other organellar-18, 20, 22, 24, 60, 70, 90 kDa

Low mol. Wt (17–30 kDa) LEA 5

Vegetative storage protein Chloroplastic CDSP-32, CDSP-34 Glycosylated cell wall protein α-amylase Sucrose synthase Δ1-pyrroline-5-carboxylase synthetase Proteases RUBP carboxylase PEP carboxylase

Cell wall proteins Phenylpropanoid pathway enzymes Peroxidase DHAP synthetase Glucanases Chitinases

Transit peptides Anaerobic polypeptides Glycolytic and fermentative enzymes Sucrose synthase ACC synthase Xyloglucan endo transglycosylase Superoxide dismutase

Phytochelatins Metal-binding protien complexes

PR proteins: 1, 2a, 2b, 3, 4, 5a, 5b Chitinases Glucanases Cell wall peroxidases

UV-radiation

Stress related proteins Cytosolic Cu, Zn-superoxide dismutase RUBP Carboxylase Glyceraldehyde 3-phosphate dehydrogenase

PR-3, PR-5 proteins OMT like proteins Peroxidases Flavonoid synthesizing enzymes

FIGURE 19.2  An overview of stress-induced protein-synthetic responses in plants. Different stresses induce the synthesis of various groups of proteins and cause either elevation (↑) or decline (↓) in the levels of enzymes. Some of the responses of salinity, drought, and chilling are common and are mediated via elevated levels of ABA. For details, refer Section 19.1.

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pollutants, new stress-specific proteins are synthesized. These proteins might play a role in signal transduction, antioxidative defense, antifreezing, heat shock, metal binding, anti-pathogenesis, or osmolyte synthesis (Qureshi et al., 2007; Timperio et al., 2008). Recent developments in sensitivity and accuracy for proteome analysis have provided new dimensions in identifying novel proteins and in assessing the changes in protein types and their expression levels under stresses (Qureshi et al., 2007). An overview of protein-synthetic responses in plants and alterations in the levels of key enzymes under various stresses is presented in Figure 19.2. These proteins that have been discussed in detail in Section 19.2 can be successfully used as attractive targets to produce stress-tolerant plants using biotechnological approaches (Table 19.1). The identification of novel stress-responsive proteins provides not only new insights into stress responses but also a good starting point for further dissection of their functions (Yan et al., 2006), and opens up new avenues for the production of stress-tolerant plants using traditional breeding or transgenic approaches. This chapter presents our current status of knowledge related to the effect of various environmental stresses on the overall aspects of protein synthesis in plants, and the possible role of stress-specific proteins in conferring an enhanced survival value to the plants against various environmental stress situations.

TABLE 19.1 Transgenic Stress-Tolerant Plants Produced by Using Stress-Related Proteins Stress Proteins Antifreeze protein LEA proteins

Dehydrins

HSPs

CBF/DREB

Gene

References

Freezing stress

Tobacco

BnLEA4–1 HVA1 OsLEA-3 LEA 4 Dhn5 Dhn24 RAB18 COR47 LTI29 LTI30 sHSP17.7 NtHSP70–1 AtHsp90.2 AtHsp90.5 AtHsp90.7 DREB1A OsDREB1B HvCBF4

Drought Salt

Arabidopsis Rice Tobacco

Salinity Osmotic stress Chilling

Arabidopsis Cucumber

Drought Salt Heat stress

Rice Tobacco Arabidopsis

Sato and Yokoya (2008) Cho and Choi (2009) Song et al. (2009)

Drought Freezing stress

Tobacco Paspalum notatum Flugge Rice Cotton Tobacco Strawberry Indian mustard Tobacco Arabidopsis

Oh et al. (2007) Gutha and Reddy (2008)

Osmotin gene

PC

AtPCS1 p5cs BrMT1 BjMT2 OgChitIVa PR-5

PR proteins

Plant

AFP

Osmotin

MT

Stress

High-salinity Salinity Drought Arsenic Cadmium Cadmium Copper B. cinerea A. alternata

Arabidopsis Tobacco

Worrall et al. (1998) Fan et al. (2002) Xu et al. (1996) Hu (2008) Dalal et al. (2009) Liu et al. (2009) Puhakainen et al. (2004) Yin et al. (2006) Brini et al. (2007)

James et al. (2008) Barthakur et al. (2001) Husaini and Abdin (2008) Parkhi et al. (2009) Pomponi et al. (2006) Gasic and Korban (2007) An et al. (2006) Kim et al. (2007) Velazhahan and Muthukrishnan (2003) Pak et al. (2009)

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19.2  Stressed Environments and Protein Synthesis The major type of stresses to which plants are exposed include salinity, drought, flood, heat, cold, anaerobiosis, infection by pathogens, metal toxicity, gaseous pollutants, and UV radiation. Plant metabolism and, more specifically, protein synthesis are adversely affected under these conditions. The effect of stress depends on the developmental stage of the plant, genotypes of the plant species, as well as the intensity and duration of the stress. The progress achieved so far in our understanding of the impact of different environmental stresses on protein synthesis is described in Sections 19.2.1 through 19.2.10.

19.2.1  Salinity Soil salinity is a major environmental stress that drastically affects crop productivity. Salinity poses a severe threat for the cultivation of crops in arid and semiarid regions of the world. Due to the continuous buildup of salinity in the soil, millions of hectares of usable land have now become unsuitable for cultivation. It is estimated that every year more than a million hectares of land is subjected to salinization. Soil salinity is thus threatening the civilization by persistently reducing the area for crop cultivation. Salinity not only causes great losses in crop yields but also has an impact on other economic, environmental, social, and political problems in the affected countries. The progress in developing salt-tolerant crop varieties has been very slow due to our incomplete knowledge of the mechanism of salt damage and the complex nature of salt tolerance. Even different varieties of a particular species may exhibit different tolerance behaviors. Salinity affects seed germination, plant growth, nutrient uptake, and metabolism owing to osmotic inhibition of water availability, ion imbalance, toxic effects of salt ions, and their effects on cellular gene expression machinery (Dubey and Pessarakli, 1995; Abdelkader et al., 2007). Different plant species have developed different mechanisms to cope up with salinity stress effects (Munns, 2002; Abdelkader et al., 2007). Salinity promotes the synthesis of salt stress–specific proteins (Hurkman and Tanaka, 1987; Singh et al., 1987; Ben-Hayyim et al., 1989; Artlip and Funkhouser, 1995; Mahmoodzadeh, 2009); causes either decreases (Popova et al., 1995) or increases (Dubey, 1983a; Elenany, 1997) in the level of total and/or soluble proteins, depending on the plant parts studied; and leads to increased activity/ synthesis of many enzymes (Dubey and Rani, 1987, 1990; Dubey et al., 1987; Mittal and Dubey, 1991, 1995; Igarashi et al., 1997). 19.2.1.1  Salt-Induced Protein Synthesis Plants growing in a saline environment show distinct changes in the pattern of synthesis and accumulation of proteins. Most of the experiments to study the salinity-induced synthesis of proteins have been conducted using plant cell cultures. Cell cultures rather than whole plant systems have proven to be more advantageous for such studies, because, in cell cultures, environmental parameters can be better controlled and the stress-tolerant cell lines generated can be readily selected and assayed for newly synthesized proteins. Several investigators have shown the synthesis of new proteins in cultured plant cells when subjected to salinity stress (Ericson and Alfinito, 1984; Singh et al., 1987; Ben-Hayyim et al., 1989; Artlip and Funkhouser, 1995; Elenany, 1997). The level of proteins differs in salt-tolerant and saltsensitive genotypes when they are subjected to salinity stress (Dubey and Rani, 1989; Elsamad and Shaddad, 1997; Mahmoodzadeh, 2009; Tada and Kashimura, 2009). Although it is well established that salt tolerance and sensitivity depend on the genetic and biochemical compositions of the species, it has been difficult so far to specify the exact genetic domain responsible for salt-adaptation expression leading to the synthesis of these proteins in salt-adapted plants. These specifically synthesized proteins under salt stress appear to have a role in providing tolerance or adaptation to the plants. However, the overall mechanism of how these proteins could provide adaptation is not clearly understood.

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To understand the mechanism of salt resistance in cultured tobacco cells, Ericson and Alfinito (1984) examined the protein patterns of NaCl-adapted as well as NaCl-nonadapted cell lines of tobacco (Nicotiana tabacum L.). Their results indicated that cells adapted to a medium containing NaCl showed two protein bands of 32 and 20 kDa in more abundance than unadapted cells. Further, in the salt-adapted cells, a unique protein of 26 kDa appeared that was specific for these cells and was not present in unadapetd cells or cells growing without NaCl. These investigators suggested that the three proteins synthesized in salt-adapted cells might be involved in a salt-adaptation process. Yildiz (2007) observed the synthesis of two new low-molecular-weight (LMW) proteins (28.9 and 30.0 kDa) and one intermediate-molecular-weight (IMW) protein (44.3 kDa) in response to the NaCl treatment in wheat cv. Ceyhan-99 (salt sensitive), whereas six LMW proteins (18.6, 19.4, 25.7, 25.9, 26.0, and 27.6 kDa) were newly synthesized in wheat cv. Fırat-93 (salt tolerant). The newly synthesized proteins were specific to each cultivar. Most of the newly synthesized proteins were acidic in nature with mol. wt. 32 subunits. Small Hsps are ATP-independent holdases. They bind nonnative proteins to form a soluble complex, preventing their aggregation. The N-terminal arm and/or C-terminal extension may mediate the binding to the substrate proteins. They may also be involved in the oligomer formation of small Hsps, and thus the dissociation of the small Hsp oligomers may be necessary to make the N-terminal arm and/or C-terminal extension available for interaction with nonnative proteins. The small Hsp/nonnative protein complex serves as a transient reservoir of substrates for subsequent refolding by ATP-dependent chaperone systems such as the Hsp70/DnaK chaperone system (see Ref. [35] and references therein). Small Hsps even coaggregate with nonnative proteins in order to mediate the resolubilization of the aggregates and subsequent refolding by ATP-dependent chaperone systems such as Hsp100/ClpB and the Hsp70/DnaK chaperone system (see Ref. [35] and references therein). One notable structural feature of chloroplast small Hsp (Hsp21) is that it contains a set of conserved methionines. Methionines M49, M52, M55, M59, M62, and M67 are located on one side of an amphipathic helix, which may fold back over two other conserved methionines (M97 and M101), to form a binding groove lined with methionines for the recognition of proteins with an overall hydrophobic character [43]. As small Hsps protect other proteins from aggregation by binding to their hydrophobic surfaces, keeping the conserved methionines in a reduced form is a prerequisite to maintain such binding. Evidence for the involvement of small Hsps in plant thermotolerance is found in literature, for example, enhanced thermotolerance of transformed tobacco plants with the introduction of the tomato mitochondrial small Hsp gene [44], increased thermotolerance by the overexpression of a small Hsp (sHsp17.7) in rice [45], and increased thermotolerance observed in Arabidopsis that constitutively expresses a cytosolic class I small Hsp from Rosa chinensis [46]. Carrot transgenic cells and regenerated plants, which constitutively expressed the carrot small Hsp (Hsp17.7) gene showed more thermotolerance than the vector controls [47]. In contrast, heat-inducible Hsp17.7 antisense lines were less thermotolerant. Small Hsp has not only the protein-protective activity, but also an ability to stabilize lipid membranes [48]. A mutant small Hsp with increased thylakoid association provided an elevated resistance against UV-B damage in the cyanobacterium Synechocystis sp. PCC 6803 [49]. In this cyanobacterium, it was shown that the wild type small Hsp (Hsp17) is equally distributed between the thylakoid and cytosolic fractions, whereas a mutant small Hsp17 mutated in position L9P appeared primarily in the soluble fraction, and another one mutated in position Q16R was found exclusively in the thylakoid membrane fraction. Compared with the wild type, the Q16R mutant had an enhanced lipid-mediated thylakoid membrane interaction, which affected directly the photosystem II complex and led to a greatly enhanced resistance to UV-induced photosystem II inactivation via facilitating photosystem II repair [49]. In this connection, it is interesting to note that immuno-cytochemical studies showed that cyanobacterial small Hsp has dynamic properties to change its localization between cytosol and thylakoid membranes during heat shock [50].

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20.5.3  Chaperonin (Hsp60)/GroEL Like small Hsps, chaperonins (Hsp60s)/GroELs are found throughout all three domains. They are ubiquitous in terms of cellular localization as well as the biological world, existing in cytosol, chloroplast, and mitochondrion. Chaperonins (Hsp60s)/GroELs (at least one of them when multiple homologues are present in a cell) are essential under all cellular conditions (for example, see Ref. [25]). The two subfamilies of this Hsp family divide along recognizable evolutional lines [51]. Type I is found in bacteria (GroEL) and endo-symbiotically related organelles, mitochondria (Hsp60), and chloroplasts (cpn60 or Rubisco binding protein), whereas type II resides in the archaebacterial/ eukaryotic cytosol (CCT/TriC). Type I and type II chaperonins are distantly related to each other. Both type I and type II chaperonins are megadalton-size double-ring assemblies that are composed of approximately 60 kDa subunits. The assemblies provide an encapsulated cavity where a nonnative polypeptide folds productively. What differentiates type I and type II chaperonins structurally is the dependency on a detachable “lid” structure for encapsulation. Type I chaperonins require a co-chaperonin (cpn10, cpn20, Hsp10, or GroES) to close the cavity, while type II chaperonins have a built-in protrusion structure to perform this function. Chaperonins are ATP-dependent foldases. They provide essential assistance to the folding/ refolding of newly translated/nonnative and newly translocated proteins. Despite the different encapsulation mechanisms, the ATP-directed chaperone cycles of the two subfamilies appear to be similar [51]. We will discuss about type I chaperonins, GroEL, and chloroplast chaperonin below. We will not discuss about type II chaperonins since very little is known about the type II chaperonins and cytosol protein folding in plants. 20.5.3.1  Type I Chaperonin Among the type I chaperonins, the E. coli GroEL is most extensively studied. The chaperonin forms a large oligomer that is composed of fourteen 60 kDa GroEL [52,53]. The subunits are arranged in a barrel-like complex that is made up of two stacked heptameric rings, which enclose a “cavity.” GroEL consists of three domains: an equatorial domain that forms the foundation of the assembly, a loosely structured apical domain that forms the ends of the ring, and a slender intermediate domain that connects the two [53,54]. The equatorial domain includes most of the connections between monomers of the same ring and between rings and contains the ATP/ADP/Mg+2-binding pocket. The intermediate domain closes on the binding pocket, providing essential residues for ATP hydrolysis. The apical domain binds GroES or a substrate polypeptide. GroES, also called co-chaperonin, forms a single heptamer ring and plays an important role in the GroEL’s chaperone action as described below. How does GroEL function as an ATP-dependent foldase? The principal mechanism is summarized as follows [51]. An “open” ring with or without ATP may accept a nonnative polypeptide. Binding of ATP to the equatorial domains of the ring renders the apical domains competent to bind GroES, whose association is accompanied by a further large movement of the apical domains. This drives the release of polypeptide substrate protein from the cavity wall into the now encapsulated hydrophilic, so-called cis cavity, where folding then commences before ATP hydrolysis in the cis ring weakens the affinity of GroEL for GroES and leads the entry of ATP into the sites of the opposite, so-called trans ring. The binding of ATP triggers release of the cis ligands (GroES, the substrate protein, and ADP). At the same time, ATP binding in the trans ring also enables binding of GroES to that ring, triggering a further round of protein folding. 20.5.3.2  Chloroplast Type I Chaperonin Chloroplast chaperonin (cpn60) was first discovered as the Rubisco binding protein [55]. There are two types of cpn60, α, and β, whose nuclear encoded precursors are synthesized outside the chloroplast and imported into it. The proteins are constitutively expressed and the level increases

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only slightly during heat shock. The two chaperonins are only 50% identical in terms of amino acid sequences, and the occurrence of highly divergent cpn60s in a higher plant plastid appears to be general [56–58]. In spinach, these subunits can form hetero-oligomers as well as homo-oligomers [59]. To find out the significance of these isoforms, the α and β subunits of cpn60 from pea were expressed individually in E. coli, and the purified cpn60s were subjected to in vitro reconstitution experiments [60]. In the presence of ATP, the β subunits formed homo-tetradecamers. In contrast, α subunits only assembled into α/β hetero-tetradecamers in the presence of β subunits. Nothing is known about the physiological significance for the presence of two types of chaperonins in chloroplasts. The following studies may provide a hint to solve the problem. In contrast with E. coli, cyanobacterial genomes generally contain two groEL homologues. The two GroELs are approximately 60% identical in terms of amino acid sequences. Although one of these GroELs is dispensable under normal growth conditions, it plays an important role(s) under stresses including heat and cold [61]. 20.5.3.3  Chloroplast Co-Chaperonin Chloroplast co-chaperonin was originally isolated as a protein from pea chloroplast lysate that formed a stable complex with GroEL in the presence of ATP [62]. Remarkably, it had twice the molecular mass of the GroES co-chaperonin. In vitro assay showed that it is a functional homolog of GroES. Genes encoding this “double” GroES-like co-chaperonin from spinach and Arabidopsis were cloned and the predicted amino acid sequences revealed that the co-chaperonin consists of two GroES-like sequences fused head-to-tail to form a single protein (see Ref. [63] and references therein). The two GroES-like sequences exhibit 40%–50% sequence identity. In vivo, the Arabidopsis homolog that was introduced and overexpressed in tobacco localized specifically to the chloroplast stroma [64]. Studies with purified proteins revealed a similar mechanism of protein folding in the case of the chloroplast chaperonin and co-chaperonin with that of GroEL and GroES [62,65,66]. Experiments showed that cpn60 functions equally well with bacterial, mitochondrial, or chloroplast co-chaperonin [60,66]. This means that the unique binary chloroplast protein is not obligatory for the cpn60mediated folding. In addition to the cpn20, Arabidopsis chloroplast has a cpn10 that has only one GroES-like sequence [67]. Chloroplast cpn60 forms a stable complex with a variety of proteins besides Rubisco [68–71]. These are the Rieske FeS protein [69], ferredoxin NADP+ reductase [70], and the multisubunit coupling factor CF1 core complex [71].

20.5.4  Hsp70/DnaK Hsp70 or DnaK (a prokaryotic homologue), is present in eukaryotic and eubacterial cells. Genes for Hsp70/DnaK homologues are only found in a subset of archaea [72]. Hsp70s that are highly conserved proteins [73] are present in cytoplasm, mitochondrion, chloroplast, endoplasmic reticulum, and nucleus [3,74–78]. Multiple members of the Hsp70 family can be present in one cellular compartment of higher plants. The Hsp70/DnaK family contains both heat-inducible and constitutively expressed members, the latter of which are sometimes called heat-shock cognate proteins (Hsc70). Many of higher plant Hsp70s show organ-specific expression pattern. Hsp70/DnaK consists of an N-terminal ATPase domain of 45 kDa and a C-terminal peptidebinding domain of ∼25 kDa [79,80]. The C-terminal domain is further subdivided into a β-sandwich subdomain (a substrate binding cavity) and an α-helical subdomain. The latter is a lid that closes the cavity. Structures of the individual domains have been available [79,80], and recently a two-domain crystal structure of bovine Hsc70 was reported [81]. Hsp70/DnaK interacts with extended hydrophobic peptide segments in an ATP-controlled fashion [79,80]. ATP binding to the N-terminal ATPase domain triggers the transition to the low affinity state of the C-terminal peptide-binding domain, while ATP hydrolysis (to the ADP-bound state) leads to the high affinity state. The ATP binding opens the lid described above, which is

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involved in substrate release. Substrate and/or Hsp40/DnaJ binding to Hsp70/DnaK stimulates ATP hydrolysis (see the following section). Thus, ATPase and substrate-binding domains mutually control each other allosterically. Hsp70/DnaK plays diverse roles in cells, including (re)folding of newly synthesized or unfolded polypeptides, assisting in the import of proteins into organelles, the dissociation of macromolecular complexes/aggregates, and targeting proteins to lysosomes or proteasomes for degradation [79,80,82,83]. Hsp70/DnaK can be an ATP-independent holdase by itself, but becomes ATPdependent foldase when it works with Hsp40/DnaJ and nucleotide exchange factor/GrpE. In a cell, Hsp70/DnaK works with these co-chaperones. Transgenic Arabidopsis whose level of Hsp70/Hsc70 was reduced by an Hsp70 antisense gene exhibited less thermotolerance than the wild type [84]. Mutational studies indicated that a chloroplast member plays a role for thermotolerance. Two plastid Hsc70 (cpHsc70-1 and cpHsc70-2) T-DNA insertion knockout mutants, Δcphsc70-1 and Δcphsc70-2, of Arabidopsis were isolated. Although no visible phenotype was observed in the Δcphsc70-2 mutant under normal growth conditions, the Δcphsc70-1 mutant plants exhibited variegated cotyledons, malformed leaves, growth retardation, and impaired root growth. After heat shock treatment of germinating seeds, root growth from Δcphsc70-1 seeds was further impaired, indicating that cpHsc70-1 is important for thermotolerance of germinating seeds [85]. Transgenic tobacco plants that constitutively expressed elevated levels of nucleus-localized Hsp70 (NtHSP70-1) showed higher levels of thermotolerance than antisense transgenic seedlings or transgenic seedlings carrying only the vector [78]. 20.5.4.1  The Hsp70/DnaK Chaperone System Hsp40/DnaJ (or J-domain proteins) is present in cytoplasm, mitochondrion, chloroplast, and endoplasmic reticulum [86–90]. The coexistence of Hsp70/DnaK and Hsp40/DnaJ in the same cellular compartment suggests that certain chaperone–co-chaperone interactions are permitted. Hsp40s/DnaJs are characterized by the presence of a J-domain, which is strictly essential for its co-chaperone functions [79,80]. Hsp40/DnaJ interacts with Hsp70/DnaK to enhance its ATPase activity, and thus controls its binding affinity to a substrate protein [79,80]. Like Hsp70/DnaK, Hsp40/DnaJ can bind a nonnative protein and protect it from aggregation. It is thought to recruit a nonnative protein and transfer it to the ATP-bound state of Hsp70/DnaK. At least one more key-player is involved in the Hsp70/DnaK chaperone system. It accelerates nucleotide (ADP/ATP) exchange in Hsp70/DnaK. In prokaryotes, GrpE performs this [79,80]. It has a molecular mass of 22 kDa and forms a stable dimer in solution. GrpE accelerates/stabilizes the open conformation of the nucleotide binding pocket of the N-terminal ATPase domain of DnaK, which facilitates the release of ADP and binding of ATP [79]. In eukaryotes, BAG proteins and others have been found to act as the nucleotide exchange factors for Hsp70 proteins. The chaperone cycle of the DnaK chaperone system is summarized as follows [80]. DnaJ binds a substrate, and then interacts with DnaK. The interaction induces a conformational change of DnaK, resulting in stimulation of ATP hydrolysis, and the closing of the substrate-binding cavity. Thus, the ADP-bound form of DnaK exhibits a high affinity for its substrate. GrpE helps release of ADP and binding of ATP from DnaK through its interaction with the DnaK ATPase domain. Upon ATP binding to the N-terminal ATPase domain of DnaK, the substrate-binding cavity becomes opened. The ATP-bound DnaK exhibits a low affinity for its substrate. Thus, the bound substrate is released from DnaK. The chaperone cycle is thought to facilitate protein folding.

20.5.5  Hsp90/HtpG Hsp90 or HtpG (a prokaryotic homologue), is present in eukaryotic and eubacterial cells. Archaea generally lack genes for Hsp90/HtpG [72]. Members of the Hsp90/HtpG family are present in chloroplast, mitochondrion, endoplasmic reticulum, and are predominantly localized in the cytoplasm [91]. The Hsp90/HtpG family contains both heat-inducible and constitutively expressed members [23,92].

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Hsp90 forms a constitutive dimer at physiological temperatures. Each monomer consists of three domains: N-terminal domain, middle-domain, and C-terminal domain. Hsp90 is a very weak ATPase, and its N-terminal domain possesses an ATP binding site [93]. The C-terminal domain is essential for Hsp90 dimerization [94,95]. Structural, biochemical, and mutational analysis of Hsp90 showed that conformational/domain rearrangements of Hsp90 are coupled to the ATPase reaction, which is thought to drive structural changes of a substrate protein and its release [96,97]. Nothing is known about the nature of the changes engendered in Hsp90 clients by association with Hsp90 and passage through the ATPase-coupled chaperone cycle [96]. In eukaryotes, Hsp90 collaborates with co-chaperones/cofactors such as Cdc37, Aha1, and p23/Sba1 to mediate the conformational regulation of a wide variety of substrate proteins including transcription factors and protein kinases under physiological conditions [96,97]. Like the other major classes of Hsps, Hsp90 can recognize and bind nonnative proteins, thereby preventing their nonspecific aggregation [98]. This general protective chaperone function may be especially important under stress conditions. In plant cells, Hsp90-based chaperone hetero-complexes have been identified. Hsp70, a p60/Sti1/Hop ortholog, and high-molecular-weight immunophilins were detected in these heterocomplexes [91,99–101]. As far as we know, there is no direct evidence that Hsp90 is involved in thermotolerance in higher plants. This may be due to the fact that Hsp90 is involved in numerous and diverse cellular functions as described below, and is essential in plants as it is essential for the viability in yeast [26]. The following results with the TU8 mutant suggest that Hsp90 plays a role in thermotolerance in plants [102]. The TU8 mutant of Arabidopsis that is deficient in glucosinolate metabolism and pathogen-induced auxin accumulation was found to be less thermotolerant than the wild-type plant. Among different Hsps only the expression level of cytoplasmic Hsp90 declined in the mutant at elevated temperatures. Transient expression of Hsp90 in mutant protoplasts increased their survival rate at higher temperatures to near equivalent that of wild-type protoplasts suggesting that the reduced level of Hsp90 in the mutant may be the primary cause for the reduction in thermotolerance. Although not a higher plant, htpG knockout mutants of the cyanobacterium Synechococcus elongatus PCC 7942 exhibited great loss of thermotolerance as compared with the wild type strain, indicating that Hsp90/HtpG plays a role in the thermal stress management [103]. Hsp90 has been assigned numerous and diverse functions including protein folding, signal transduction, protein transport across the endoplasmic reticulum and organeller membranes, and protein degradation in the eukaryotic cells [96,97]. One notable function of plant Hsp90 is that it is crucial for defense against pathogens. Hsp90 is involved in plant immunity signaling pathways [104]. The SGT1-Hsp90 pair, a chaperone complex is required for maintenance of immune sensors. Lastly, we should add that Hsp90 buffers genetic variation by keeping mutant proteins in wild-type conformations [105,106]. When this buffering is compromised, for example, by heat stress that diverts Hsp90s from its normal, specific target proteins to denatured proteins, the variations are exposed, resulting in production of an array of morphological phenotypes. The great effect of Hsp90’s effects on the buffering and release of genetic variation suggests it may have an impact on evolutionary processes.

20.5.6  Hsp100/ClpB Hsp100/ClpB is present in eukaryotic and eubacterial cells. Archaea generally lack genes encoding members of this family [72]. Hsp100/ClpB has been identified in chloroplast, mitochondrion, and cytosol of plants (Hsp101) [107–109]. Intriguingly it is not found in the cytosol of animal cells [110], while a homologue (Hsp104) is present in the yeast cytosol. Hsp100/ClpB belongs to the class 1 family of Clp/Hsp100 AAA+ (ATPases associated with various cellular activities) proteins [111,112]. Hsp100/ClpB forms large hexameric ring structures and contains two AAA+ modules. Unlike other Hsp100/Clp proteins, Hsp101, Hsp104, and ClpB are not involved in protein degradation. Rather, the members of the Hsp100/ClpB family function as a molecular

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chaperone. Hsp100/ClpB neither has foldase activity nor holdase activity. It neither promotes protein folding nor suppresses protein aggregation. It is an ATP dependent unfoldase/disaggregase that mediates the unfolding/dissolving of protein aggregates. The Hsp70/DnaK chaperone system can collaborate with Hsp100/ClpB to refold these nonnative proteins to their native structure. What is the mechanism for protein disaggregation? A probable mechanism may be as follows [83]. Hsp100/ClpB binds a substrate protein(s) that is subsequently translocated (mechanically pulled) through the central, narrow channel of the Hsp100/ClpB rings in response to ATP hydrolysis. The protein is unfolded during the translocation/the pulling process. This pulling action is associated with unfolding because the substrate protein is forced to enter a narrow channel that cannot otherwise be negotiated. The energy of ATP hydrolysis can be translated by the machine into the exertion of mechanical force needed for unfolding [113]. Upon release from Hsp100/ClpB, the substrate protein is taken care of by the Hsp70/DnaK system. In ClpB, its protein disaggregation activity is potentially associated with the unfolding that occurs during translocation down its central channel was supported by an experiment in which the distal surface of ClpB was engineered so that it interacted with the ClpP protease. This version of ClpB promoted the degradation of disaggregated substrates [114]. Hsp101 is required for acclimation to high temperatures. Successful complementation of yeast hsp104 mutants with higher plant hsp101 provided the first clue for their important role in imparting thermoprotection [115–118]. While hsp101 mutants such as hot-1 in Arabidopsis were found to be defective in the acquisition of thermotolerance against high temperature [119], the constitutive expression of hsp101 provided significant growth advantage to the transgenic seedlings at high temperatures [120]. When the expression of hsp101 was reduced by antisense or co-suppression, the modified plants had impaired acquired thermotolerance [120]. ClpB is also essential for thermotolerance in the cyanobacterium Synechococcus elongatus PCC 7942 [121]. These studies with model plants or organisms may prompt someone to construct an improved thermotolerant crop plant by overexpression of Hsp100. High temperature is detrimental to both the vegetative and reproductive stages of rice [122]. Thus development of improved elite rice varieties with enhanced tolerance to high temperature can help in this regard. A. thaliana Athsp101 cDNA was employed for overexpression in an elite indica rice cultivar to obtain transgenic plants. 40–45 days old transgenic plants were exposed to 45°C for 3 h and subsequently placed at 28°C for recovery. After 5 days, control plants totally collapsed whereas transgenic plants were green and healthy. Strong accumulation of AtHsp101 was observed in the transgenic lines at normal temperature as well as in response to high temperature stress. On the other hand, native OsHsp100 was significantly induced to almost similar extent in untransformed and transgenic plants at 47°C. Thus, it was speculated that heat-induced OsHsp100 alone was inadequate to impart higher level of protection against heat stress and the constitutive overexpression of AtHsp101 provided the improved thermotolerance capacity to the transgenic plants [123]. In addition to its essential role in acquired thermal tolerance, Hsp101 provides a substantial fitness benefit under normal growth conditions [124]. The T-DNA insertion mutants of chloroplast and mitochodrial homologues showed no evidence for heat stress phenotypes of seedling similar to those observed in hsp101 mutants [107]. However, the chloroplast homologue was shown to be essential for chloroplast development.

20.6  A Novel Hsp (Hsa32) Involved in Maintenance of Acquired Thermotolerance A heat-stress-associated 32-kD protein (Hsa32), which is highly conserved in land plants but absent in most other organisms was reported [125]. The gene responds to heat shock at the transcriptional level in moss, Arabidopsis, and rice. Disruption of Arabidopsis Hsa32 by T-DNA insertion resulted in a great loss of thermotolerance that was acquired at 37°C when recovery period at 22°C after

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the pre-heat-treatment was longer than 48 h, and severe heat shock was challenged at 44°C. This indicated that Hsa32 is essential for tolerance against a severe heat challenge after a long recovery following acclimation treatment, which is apparently due to a fast decay of acquired thermotolerance in the absence of Hsa32.

20.7  Engineering Plants Tolerant to Stresses Other Than Heat The roles of the Hsps in the thermotolerance were already discussed throughout the above sections. Hence, in this section, we will briefly introduce some publications that are related to the production of plants that are tolerant to stresses other than heat by introducing/inducing an Hsp gene(s). Temperature stress is not the only stress that leads to the elevated expression of Hsps. The cold induction of some Hsps (for example, see Ref. [126]) may be related to plant defense mechanisms against cold stress. Thus, it is reasonable to expect that the over-expression of Hsps can improve cold stress tolerance in plants. Low-temperature storage is one of the most important methods of reducing postharvest decay and maintaining the organoleptic and nutritional quality of fruits and vegetables. Exposing sensitive fruits to low temperatures induces chilling injury leading to significant changes in overall quality [127]. There is a report showing a simple way to obtain a cold-tolerant tomato fruit by preheat-treatment to induce Hsps. The Fortaleza tomato variety showed high sensitivity to cold storage (87% damage in untreated fruit after 21 days at 2°C). However, when they were heat treated at 38°C in a chamber for 24 and 48 h, the development of chilling-associated symptoms in a significant percentage of fruits (47% and 20% of damaged fruits, respectively) were prevented [128]. They detected increasing accumulation of a 17.6 kDa class I small Hsp in pericarp proteins of the tomato fruit by the heat treatment. Expression of some Hsps in different organisms has been shown to be affected by a number of chemicals: arsenite [6,14,46], heavy metals such as cadmium, cobalt, copper, nickel, and silver [6,14]. Cytosolic/nuclear Hsc70-1 overexpression in Arabidopsis specifically conferred gammaray hypersensitivity and tolerance to salt, cadmium, and arsenite [129]. Plants overexpressing Hsc70-1 accumulated less cadmium, thus providing a possible molecular explanation for their tolerance phenotype. DnaK1 from the halotolerant cyanobacterium Aphanothece halophytice was overexpressed in the cytosol of transgenic tobacco plants, and was found to improve their salt tolerance [130]. Interestingly, while evaluating researches aimed at developing transgenic crops and plants with enhanced tolerance to naturally occurring environmental conditions it was revealed that the response of plants to a combination of two different abiotic stresses is unique and cannot be directly extrapolated from the response of plants to each of the different stresses applied individually [12]. Plant acclimation to a particular abiotic stress may require a specific response that is tailored to the precise environmental conditions the plant encounters. To illustrate this point, transcriptome profiling studies of plants subjected to different abiotic stress conditions prompted a somewhat unique response and little overlap in transcript expression could be found between the responses of plants to abiotic stress conditions such as heat, drought, cold, salt, high light, or mechanical stress (reviewed in Ref. [12]). Drought and heat stress can be an excellent example of this type, which may occur in the field simultaneously. It was observed that a combination of drought and heat stress had a significantly greater detrimental effect on the growth and productivity of maize, barley, and sorghum as compared with each of the different stresses applied individually [12,131–134]. Transcriptome profiling studies of plants subjected to a combination of drought and heat stress suggest that the stress combination requires a unique acclimation response involving >770 transcripts that are not altered by drought or heat stress [135]. Among them, 11 transcripts encoding Hsps were specifically elevated during a combination of drought and heat stress. Therefore, to develop transgenic crops with enhanced tolerance to field conditions, (over) expression of several Hsps that is tailored to the environmental conditions in transgenic plants may be able to improve the way.

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20.8  Conclusions Hsps are a diverse group of proteins that share the property for the binding of substrate proteins that are in nonnative structural states. They play an essential role in the cellular protein homeostasis. They are involved in a broad array of cellular processes required for both normal cellular functions and survival under stress conditions. A volume of overwhelming evidence supports the assumption that the Hsps are some of the most important entities to provide heat tolerance to plants. The representative Hsps function as molecular chaperones. They must become even more indispensable at higher temperatures where the probabilities of denaturation, incorrect folding, and aggregation of cellular proteins are much higher. Genetic manipulation to introduce an Hsp(s) alone may not be sufficient in an attempt to provide a crop plant with traits such as heat tolerance, resistance to dehydration, and tolerance to salt and other stresses. Nevertheless, further understanding of Hsps and molecular chaperones is essential for the future development of such crop plant strains.

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92. Yabe N, Takahashi T, and Komeda Y. Analysis of tissue-specific expression of Arabidopsis thaliana HSP90-family gene HSP81. Plant Cell Physiol. 35:1207–1219, 1994. 93. Prodromou C, Roe SM, O’Brien R, Ladbury JE, Piper PW, and Pearl LH. Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone. Cell, 90:65–75, 1997. 94. Nemoto T, Ohara-Nemoto Y, Ota M, Takagi T, and Yokoyama K. Mechanism of dimer formation of the 90-kDa heat-shock protein. Eur. J. Biochem. 233:1–8, 1995. 95. Harris SF, Shiau AK, and Agard DA. The crystal structure of the carboxy-terminal dimerization domain of htpG, the Escherichia coli Hsp90, reveals a potential substrate binding site. Structure, 12:1087–1097, 2004. 96. Pearl LH and Prodromou C. Structure and mechanism of the Hsp90 molecular chaperone machinery. Annu. Rev. Biochem. 75:271–294, 2006. 97. Wandinger SK, Richter K, and Buchner J. The Hsp90 chaperone machinery. J. Biol. Chem. 283:18473– 18477, 2008. 98. Wiech H, Buchner J, Zimmermann R, and Jakob U. Hsp90 chaperones protein folding in vitro. Nature, 358:169–170, 1992. 99. Owens-Grillo JK, Stancato LF, Hoffmann K, Pratt WB, and Krishna P. Binding of immunophilins to the 90 kDa heat shock protein (hsp90) via a tetratricopeptide repeat domain is a conserved protein interaction in plants. Biochemistry 35:15249–15255, 1996. 100. Stancato LF, Hutchison KA, Krishna P, and Pratt WB. Animal and plant cell lysates share a conserved chaperone system that assembles the glucocorticoid receptor into a functional heterocomplex with Hsp90. Biochemistry 35:554–561, 1996. 101. Reddy RK, Kurek I, Silverstein AM, Chinkers M, Breiman A, and Krishna P. High molecular weight FK506-binding proteins are components of heat shock protein 90 heterocomplexes in wheat germ lysate. Plant Physiol. 118:1395–1401, 1998. 102. Ludwig-Muller J, Krishna P, and Forreiter C. A glucosinolate mutant of Arabidopsis is thermosensitive and defective in cytosolic Hsp90 expression after heat stress. Plant Physiol. 123:949–958, 2000. 103. Tanaka N and Nakamoto H. HtpG is essential for the thermal stress management in cyanobacteria. FEBS Lett. 458:117–123, 1999. 104. Shirasu K. The HSP90-SGT1 chaperone complex for NLR immune sensors. Annu. Rev. Plant Biol. 60:139–164, 2009. 105. Rutherford SL and Lindquist S. Hsp90 as a capacitor for morphological evolution. Nature 396:336–342, 1998. 106. Queitsch C, Sangster TA, and Lindquist S. Hsp90 as a capacitor of phenotypic variation. Nature 417:618–624, 2002. 107. Lee U, Rioflorido I, Hong SW, Larkindale J, Waters ER, and Vierling E. The Arabidopsis ClpB/Hsp100 family of proteins: Chaperones for stress and chloroplast development. Plant J. 49:115–127, 2007. 108. Keeler SJ, Boettger CM, Haynes JG, Kuches KA, Johnson MM, Thureen DL, Keeler CL Jr., and Kitto SL. Acquired thermotolerance and expression of the HSP100/ClpB genes of lima bean. Plant Physiol. 123: 1121–1132, 2000. 109. Katiyar-Agarwal S, Agarwal M, Gallie DR, and Grover A. Search for the cellular functions of plant Hsp100/Clp family proteins. Crit. Rev. Plant Sci. 20:277–295, 2001. 110. Mosser DD, Ho S, and Glover JR. Saccharomyces cerevisiae Hsp104 enhances the chaperone capacity of human cells and inhibits heat stress-induced proapoptotic signaling. Biochemistry 43:8107–8115, 2004. 111. Lee S, Sowa ME, Choi JM, and Tsai FT. The ClpB/Hsp104 molecular chaperone-a protein disaggregating machine. J. Struct. Biol. 146:99–105, 2004. 112. Neuwald AF, Aravind L, Spouge JL, and Koonin EV. AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res. 9:27–43, 1999. 113. Lee C, Schwartz MP, Prakash S, Iwakura M, and Matouschek A. ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal. Mol. Cell. 7:627–637, 2001. 114. Weibezahn J, Tessarz P, Schlieker C, Zahn R, Maglica Z, Lee S, Zentgraf H, Weber-Ban EU, Dougan DA, Tsai FT, Mogk A, and Bukau B. Thermotolerance requires refolding of aggregated proteins by substrate translocation through the central pore of ClpB. Cell, 119:653–665, 2004. 115. Lee YJ, Nagao RT, and Key JL. A soybean 101-KD heat shock protein complements yeast HSP 104 deletion mutant in acquiring thermotolerance. Plant Cell 6:1889–1897, 1994. 116. Schirmer EC, Lindquist S, and Vierling E. An Arabidopsis heat shock protein complements a thermotolerance defect in yeast. Plant Cell 6:1899–1909, 1994.

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117. Wells DR, Tanguay RL, Le H, and Gallie DR. HSP101 functions as a specific translational regulatory protein whose activity is regulated by nutrient status. Genes Dev. 12:3236–3251, 1998. 118. Young TE, Ling J, Geisler-Lee C, Robert LT, Caldwell C, and Gallie DR. Development and thermal regulation of the maize heat shock protein, HSP101. Plant Physiol. 127:777–791, 2001. 119. Hong SW and Vierling E. Mutants of Arabidopsis thaliana defective in the acquisition of tolerance to high temperature stress. Proc. Natl. Acad. Sci. USA, 97:4392–4397, 2000. 120. Queitsch C, Hong SW, Vierling E, and Lindquist S. Heat shock protein 101 plays a crucial role in thermotolerance in Arabidopsis. Plant Cell, 12:479–492, 2000. 121. Eriksson MJ and Clarke AK. The heat shock protein ClpB mediates the development of thermotolerance in the cyanobacterium Synechococcus sp. strain PCC 7942. J. Bacteriol. 178:4839–4846, 1996. 122. Pareek A, Singla SL, and Grover A. Protein alterations associated with salinity, desiccation, high and low temperature stresses and abscisic acid application in seedlings of Pusa 169, a high-yielding rice (Oryza sativa L.) cultivar. Curr. Sci. 75:1023–1035, 1998. 123. Katiyar-Agarwal S, Agarwal M, and Grover A. Heat-tolerant basmati rice engineered by over-expression of Hsp101. Plant Mol. Biol. 51:677–686, 2003. 124. Tonsor SJ, Scott C, Boumaza I, Liss TR, Brodsky JL, and Vierling E. Heat shock protein 101 effects in A. thaliana: Genetic variation, fitness and pleiotropy in controlled temperature conditions. Mol. Ecol. 17:1614–1626, 2008. 125. Charng YY, Liu HC, Liu NY, Hsu FC, and Ko SS. Arabidopsis Hsa32, a novel heat shock protein, is essential for acquired thermotolerance during long recovery after acclimation. Plant Physiol. 140:1297– 1305, 2006. 126. Neven LG, Haskell DW, Guy CL, Denslow N, Klein PA, Green LG, and Silverman A. Association of 70-Kilodalton heat-shock cognate proteins with acclimation to cold. Plant Physiol. 99:1362–1369, 1992. 127. Wang C. Approaches to reduce chilling injury of fruits and vegetables. Hort. Rev. 15:63–95, 1993. 128. Polenta GA, Calvete JJ, and Gonzalez CB. Isolation and characterization of the main small heat shock proteins induced in tomato pericarp by thermal treatment. FEBS J. 274:6447–6455, 2007. 129. Cazale AC, Clement M, Chiarenza S, Roncato MA, Pochon N, Creff A, Marin E, Leonhardt N, and Noel LD. Altered expression of cytosolic/nuclear HSC70–1 molecular chaperone affects development and abiotic stress tolerance in Arabidopsis thaliana. J. Exp. Bot. 60:2653–2664, 2009. 130. Sugino M, Hibino T, Tanaka Y, Nii N, and Takabe T. Overexpression of DnaK from a halotolerant cyanobacterium Aphanothece halophytice acquires resistance to salt stress in transgenic tobacco plants. Plant Sci. 146:81–88, 1999. 131. Heyne EG and Brunson AM. Genetic studies of heat and drought tolerance in maize. J. Amer. Soc. Agron. 32:803–814, 1940. 132. Craufurd PQ and Peacock JM. Effect of heat and drought stress on sorghum. Exp. Agric. 29:77–86, 1993. 133. Savin R and Nicolas ME. Effects of short periods of drought and high temperature on grain growth and starch accumulation of two malting barley cultivars. J. Plant Physiol. 23:201–210, 1996. 134. Jagtap V, Bhargava S, Streb P, and Feierabend J. Comparative effect of water, heat and light stresses on photosynthetic reactions in Sorghum bicolor (L.) Moench. J. Exp. Bot. 49, 1715–1721, 1998. 135. Rizhsky L, Liang H, Shuman J, Shulaev V, Davletova S, and Mittler R. When defense pathways collide. The response of Arabidopsis to a combination of drought and heat stress. Plant Physiol. 134:1683–1696, 2004.

of Low Temperatures 21 Effect on the Structure of Plant Cells: Structural, Biochemical, and Molecular Aspects L’udmila Slováková, Ildikó Matušíková, Ján Salaj, and Ján Hudák Contents 21.1 Introduction........................................................................................................................... 536 21.2 Chilling.................................................................................................................................. 536 21.2.1 Cell Membranes......................................................................................................... 536 21.2.2 Plastids and Mitochondria......................................................................................... 537 21.2.3 Endoplasmic Reticulum and Dictyosomes................................................................ 538 21.2.4 Nucleus...................................................................................................................... 539 21.2.5 Cytoskeleton.............................................................................................................. 539 21.3 Freezing.................................................................................................................................540 21.3.1 Cell Membranes......................................................................................................... 541 21.3.2 Endoplasmic Reticulum............................................................................................. 542 21.3.3 Vacuole...................................................................................................................... 543 21.3.4 Chloroplasts...............................................................................................................544 21.3.5 Nucleus......................................................................................................................546 21.3.6 Mitochondria.............................................................................................................546 21.3.7 Dictyosomes............................................................................................................... 547 21.3.8 Cytoskeleton.............................................................................................................. 547 21.4 Molecular Biology and Biochemistry of Response to Low Temperatures............................ 548 21.4.1 Cell Membranes......................................................................................................... 548 21.4.2 Endoplasmic Reticulum............................................................................................. 549 21.4.3 Vacuole...................................................................................................................... 549 21.4.4 Chloroplasts............................................................................................................... 550 21.4.5 Nucleus...................................................................................................................... 550 21.4.6 Mitochondria............................................................................................................. 553 21.4.7 Cytoskeleton.............................................................................................................. 553 21.4.8 Cell Wall.................................................................................................................... 553 21.5 Conclusion............................................................................................................................. 554 References....................................................................................................................................... 554

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21.1  Introduction Among the external factors that greatly affect cell development is temperature. A favorable temperature has a positive effect on structural and physiological processes of plant cells. When the temperature is increased or decreased, a harmful effect on the plant cells can be observed. Plant sensitivity to temperature depends on the plant’s origin and phylogeny. The effect of temperature on cell ontogeny has been extensively studied and there are reviews elsewhere concerning this topic [1,2]. In this chapter we have tried to submit the results regarding the effect of low temperatures on the structure of the plant cell. Mutual comparison of existing results and their generalization is not easy, because variable plant species in different ontogenetic phases have been used in the observation. The temperatures on the Earth’s surface are very different, changing during the seasons as well as during the day and nights. Despite these differences, the plants grow almost everywhere. However, to be able to survive the unfavorable temperatures, plants have to adapt to this temperature oscillation. Plants, on the basis of their sensitivity to temperature, have been divided into three groups [1]:

1. Chilling-sensitive plants: These plants are seriously injured by the temperatures above zero (usually below 15°C). 2. Chilling-resistant plants: These plants are able to tolerate low temperatures but are seriously injured when ice start to form in their tissues. 3. Frost resistant plants: These plants are able to tolerate exposure to very low temperatures (of −50°C to −100°C even when immersed in liquid nitrogen).

Most perennial plants growing in the temperate regions undergo a “hardening” process in the autumn of each year to prepare for overwintering. In most agricultural areas, unseasonal frost can occur throughout much of the growing season. During periods of active growth, most crop species do not tolerate freezing. Depending on the minimum temperature and the duration of the frost, plants may be partially damaged or killed, resulting in lower yield and quality of harvest or even complete crop failure. Most winter crops, however, have the ability to develop freezing tolerance when exposed to hardening conditions. Each plant is characterized by certain genetically fixed level of resistance to low temperatures, which reduces its metabolic activity at such low temperatures. This level of resistance (or survival capacity) can vary among individual plant species. Low temperatures act as a stress factor that has a strong impact on growth, reproduction, and distribution of plants. The ability of plants to survive and grow depends on different ecological and physiological mechanisms [1–4].

21.2  Chilling Chilling injury can be observed on many plants of tropical and subtropical origin when they are exposed to low, but nonfreezing temperatures, in their chilling range, which is usually from 25°C to 10°C [5]. For plants of temperate origin, chilling temperatures usually range from 15°C to 0°C. The chilling effect is manifested by physiological and cytological changes. Depending on the time and temperatures, the cytological changes can be either reversible or irreversible. However, chillingsensitive plants are also able to adapt to the chilling if they are hardened a certain amount time at temperatures slightly above their critical temperatures. Many light- and electron microscopic studies have shown different structural changes of the cells in chilling-sensitive plants after their exposure to a long period of chilling stress [6–8].

21.2.1  Cell Membranes The cellular membranes are those cell compartments, where the primary events of chilling stress occur [9]. An increase in the permeability of the plasmalemma and leakage of organic and inorganic

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substances is considered to be the first symptom of cell injury [10]. Light- and electron microscopical observations of tomato cotyledons growing at 5°C for 3 days have revealed the loss of cell turgor, vacuolization of cytoplasm, swelling, and disintegration of cell organelles [11]. More detailed ultrastructural time-course studies have shown injury of plasmalemma after 20–24 h. Disintegration of the plasmalemma can be observed after prolonged cold treatment or at lower temperature [12,13]. During plasmolysis of hardened and nonhardened cells of rape and alfalfa plants, the plasmalemma is pressed against the tonoplast and deleted into the vacuole as sac-like intrusions [14]. A similar sac-like invaginations of the tonoplast into the vacuole during hardening at 5°C can be seen in potato leaves [15]. Chilling of the roots of the tropical plant Episcia reptans results in tonoplast discontinuity within 1 h at 5°C and 3 h at 10°C [16]. Two types of crystalline deposits (cytoplasmic and tonoplast-associated) are seen in root cells after chilling stress. Since similar deposits have also been observed in epidermal, mesophyll, and vascular cells of E. reptans leaves [17] and on the tonoplast of potato cotyledons [13], and these deposits closely follow tonoplast disruption, it can be supposed that these deposits probably serve as an indication of cell injury in the plants with increased time of exposure. Although the injury of a majority of the membranes after a short period of chilling is usually reversible, injury of the tonoplast is irreversible [18] and may govern the ability of plants to survive rewarming [19]. Frequently, as a result of chilling stress or hardening at low, above-zero temperatures, lipid bodies accumulate in the cytoplasm or in close association with plasmalemma [20,21].

21.2.2  Plastids and Mitochondria Swelling of plastid membranes and mitochondria is a very common symptom of chilling temperatures. The harmful effect of these temperatures is mostly time dependent. Chloroplasts from the leaves of E. reptans chilled for 6 h at 5°C have an irregular and less organized membrane system and fewer plastoglobules. Increase in the exposure time results in both swelling of the chloroplast thylakoids and in a decrease in the size and number of starch grains [17]. After 4 h of exposure at 5°C, injured chloroplasts disintegrated thylakoids can be seen [11]. Full grana disintegration and an increase in the number and size of the plastoglobuli can be observed in hardened cucumber leaves after 11 days of chilling. Hardening of potato leaves for 10 days at 5°C causes dilation of the thylakoids and the disappearance of starch grains [15]. The chilling stress induces the reduction of starch grains and thylakoids in winter wheat and in maize [22]. When Ephedra cells are cultivated for 15 days at 2°C, the plastids together with mitochondria are organized into groups. Plastid grana are innumerous and plastids very often contain membrane-free stroma [23]. Contrary to this result, the long-term hardening of young seedlings of Norway spruce at 3°C increases the content of the starch grains in plastids and the thylakoids are not distinctly dilated. Such plastids possess numerous plastoglobules [20]. After the 1-day exposure of Ephedra cells to 2°C, the mitochondria have less dilated cristae and their matrix is transparent [23]. Swollen mitochondria with reduced cristae have been observed in chilled onion cells [24], in maize root cortex (Figure 21.1) [8], and in both root and leaves cells of E. reptans [16,17]. Owing to the mitochondrial swelling in chilled tissues, their volume is doubled in comparison with the mitochondria from the plants grown at a favorable temperature [17]. The first symptoms of visible injury of mitochondria have been recognized after exposure of tomato cotyledons at a temperature of 5°C for 4 h [13]. These mitochondria possess a reduced number of cristae and discontinuities in their envelope. Structural alterations of mitochondria have been seen in the microsporocytes and tapetum of Rhoeo discolor exposed to a temperature of 4°C–5°C for 4 days [25]. In contrast, no visible changes in the mitochondria have been detected in xylem ray cells of poplar trees at a temperature of 0°C for 14 days [26]. Recently, an electron microscopy study has shown that the mitochondria in the axis of soybean seeds imbibed at 10°C and 4°C were slightly diminished and lacked internal structure. In contrast,

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FIGURE 21.1  Mitochondria with dilated cristae from maize root cortex cells at 5°C (×24,000). (From Cˇiamporová, M. and Mistrík, I., Plant Cell under Unfavourable Conditions (in Slovak), Veda, Bratislava, 1991. With permission.)

the mitochondria in the axis imbibed at 22°C contained numerous well-developed cristae with easily distinguishable outer and inner boundary membranes [27].

21.2.3  Endoplasmic Reticulum and Dictyosomes The endoplasmic reticulum (ER) of plant cells seems to be very sensitive to cold. A strict correlation has been found between the temperature (between 30°C and 5°C) and the volume of the ER labyrinths. After exposure of plants to cold, an extensive dilation and vesiculation of smooth ER cisternae can be observed quite clearly, and the profiles of the rough ER almost completely disappear with the drop in temperature [28]. These dilated vesicular ER cisternae probably serve as accumulation sites of cryoprotective substances [26]. Prolonged exposure of Cornus stolonifera callus cells to 0°C for 12 h results in partial dilatation followed by microvesiculation of the rough ER and releasing of the ribosomes from the membranes. Vacuolization of smooth ER is visible after 24 h of chilling [18]. Dilation of rough ER without ribosomes has been observed in cooled microsporocytes [25]. The vesicles originating from the dilated rough ER without ribosomes have autolytic functions in chilled cells [11,12]. It might be suggested that the transformation of the rough ER into vacuolated smooth ER represents an early stage of chilling [18]. Full reversibility of the ultrastructural changes has been shown suggesting that the ER system is very dynamic; it is probably the most dynamic structure in plant cells [28]. Many studies have shown that the formation of parallel and concentric layering of ER cisternae can by induced by different types of stress and therefore it might have been suggested that these configurations are manifestation of an adaptive mechanism protecting the plant cells and of repairing processes within stress-damaged cells [29,30]. Dictyosomes are cell organelles that are metabolically very active, for example, in protein sorting, and membrane formation. They respond to chilling stress by swelling. The swollen dictyosome cisternae occur in tomato cotyledons after 4 h of chilling at 5°C [13] or after 24 h at 0°C in Cornus stolonifera cells [18]. During cold hardening of Arabidopsis thaliana cells contain more microvesicles that are either associated with dictyosome cisternae or located in their vicinity; the dictyosomes probably take part in structural and conformational modification of plasmalemma [31]. Longer exposure to chilling temperature causes disintegration of the dictyosomes [32,18].

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21.2.4  Nucleus Numerous studies of the effect of chilling temperatures from 0°C to 4°C on the functional and structural behavior of nuclei in pollen mother and tapetal cells of R. discolor have been done. A short treatment of nuclei with cold does not cause any important changes in morphology of the nuclei and in DNA synthesis [33]. However, a longer cold treatment considerably reduces both DNA and RNA synthesis [34]. Modification of the nuclear structure of plant cells has been observed by both light microscopy and electron microscopy. Lobed nuclei in the Ephedra cells have been observed after long-time (15 days) exposure to 2°C [23]. The nuclei of other plants respond to longer exposure to low temperatures by swelling and modification of the nuclear envelope [3,25] and chromatin coagulation [37]. Following cold stress at 5°C for 3 days, the nuclei in the root cells of maize contain rather dispersed chromatin, nuclear bodies often occur in the nucleoplasm, and the nucleolar organizer regions are pronounced (Figure 21.2) [35,36]. Fully nuclear disruption is observed in C. stolonifera cells and in very sensitive E. reptans cell after 2 days at 5°C [16,18]. In tomato cotyledons, irreversible injury of nuclei is seen after 20–24 h of chilling [13]. After exposure of wheat cells to the chilling temperatures, the fibrillar zone of the nucleolus is more abundant and granular zone becomes diffused [37,38]. A high amount of fibrillar components can result in the formation of nucleolus-like bodies in the cytoplasm [39] or in the nucleoplasm [8,22,36]. Besides the nucleus structure, a decrease in temperature from the optimum value to the minimum value (about 1°C) is accompanied by a progressive slowdown of the mitotic cycle as well as of the duration of mitosis. At the temperature of 3°C, for instance, the mitotic cycle in Vicia faba root cells may be 22 times longer than at 25°C [40].

21.2.5  Cytoskeleton According to the some investigators [6], the primary response to the chilling stress may be a breakdown of cytoskeleton, and alterations in membranes, that is, a physical phase transition of membranes from liquid-crystalline to solid gel state [9]. It has been suggested that chilling stress has a direct effect on the microtubules [41]. This major component of the cytoskeleton, have been found to depolymerase during cold treatment [42,43]. Disassembling of microtubules in response to low temperatures depends on the type of organisms and reflecting critical temperature. In chilling sensitive

FIGURE 21.2  Nucleus with a nucleolus, pronounced nucleolus organizer region, and nuclear bodies in the nucleoplasm from maize root cortex cells at 5°C (×18,000). (From Cˇiamporova, M. and Mistrík, I., Environ. Exp. Bot. 33, 11, 1993. With permission.)

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plants this disassembly start as early as at temperatures above 4°C, whereas in moderately resistant plants they can withstand 0°C; in cold-resistant species disassembly did not occur even at temperatures below −5°C the [41]. Depolymerization of cortical microtubules at chilling temperatures (0°C–4°C) has been repeatedly observed in several chilling-sensitive species of higher plants [42] and in various cell types, including the root cells of maize [45], the guard cells of onion [46], the suspension culture cells of maize [47], and protoplasts isolated from tobacco [48]. In experiments with cucumber cotyledons, it has been found that treatment with antimicrotubular drugs make the chilling worse, whereas treatment with abscisic acid (ABA) protects cotyledons from drug effect and chilling injury [49]. There is a connection between chilling of cytoskeleton and the inhibition of cytoplasmic streaming. The chilling temperatures can influence the equilibrium of Ca2+ and adenosine triphosphate (ATP), which is connected with F-actin activity [6,7,50]. Actin filaments have also been found to be involved in cold-induced conformational changes and the reorganization of the ER [51]. These results indicate that low temperatures (0°C–4°C) most likely influence either the interaction of the force-generating system, probably myosin, with actin filaments or the force-generating mechanism of the actomyosindriven intracellular movement, but they do not affect actin-filament integrity [51]. The effect of low, nonfreezing temperatures on the plants also is visible at anatomical and morphological levels. These aspects are connected mainly with adaptation reactions of less chilling-sensitive and cold-resistant plants (like winter cereals) to growth at low temperatures. Such anatomical and morphological changes like altered stomatal frequency [52], decreased epidermal cell size [53], increased mesophyll cell size, and suberinization [52,54] are associated with acclimation of plant to nonfreezing temperatures.

21.3  Freezing Generally, freezing in plants consists of the conversion of liquids in cells to a solid state, which is accompanied by loss of heat. Two types of freezing are recognized in plant cells and tissues: (a) vitrification—solidification of cellular content into noncrystalline (amorphous) state and (b) crystallization—arrangement of liquid molecules into orderly structures [55]. Vitrifiation of liquids in cells is a result of rapid freezing (at more than 3°C/min) of plant tissue to very low temperature. It is enhanced by hardening of plants at low temperatures. Although vitrification does not occur in nature, it is of great interest to researchers because it enables plants to survive temperatures close to absolute zero [56]. On the other hand, crystallization (or ice formation) is a very common phenomenon in nature. The crystallization of ice may occur either within or outside the cells, but the process depends on the speed of cooling. The formation of ice inside the cells may occur by both internal nucleation or by penetration of external ice crystals into the cells [57]. In both cases, this type of freezing, also called intracellular, is lethal because of the immediate disruption of the cells. The only exception of this rule may be in case of the cells that exhibit deep supercooling [58]. Plant cells can also survive intracellular ice formation when the ice crystals that form by freezing are very fine, cooling is extremely rapid, and these crystals melt before they reach a harmful size [59]. There are three types of intracellular ice formation in epidermal cells of onion plants at highspeed of cooling [60]: (a) Ice formation spreads from cell to cell through the plasmodesmata. Freezing from cell to cell is also observable on the Tradescantia staminal hair cells [61] and in mesophyll cells of Norway spruce during the winter frosts [62]. (b) Less frequently, ice can be formed in the cell walls adjacent to the intercellular spaces. Ice arises first in the plasmolyte between the cell wall and cytoplasm and then rapidly in the cytoplasm. (c) Intracellular ice originates spontaneously from centers of nucleation within the cytoplasm and later in surrounding plasmolyte. If the speed of cooling is slow enough (in nature, the cooling rate seldom exceeds 1°C/h), the liquids in the cells freeze extracellularly, causing cell dehydration of cytoplasmic solutes and a reduction in cell volume and surface area, all factors which can potentially damage the

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cells irreversibly [63]. Ice formation for most plant tissues begins on the surface of cell walls, in water transport elements, or on external surfaces. Although the cooling is slow and plasmalemma remains intact, ice formation will be confined outside of cells [64]. There are two major strategies allowing plants to survive freezing stress: freezing tolerance and freezing avoidance [2]. Tissues displaying freezing tolerance respond to freezing stress by the loss of cellular water to extracellular ice, resulting in collapse of the cell. As a consequence, an increased concentration of the cell sap and a lowered freezing point will occur. Cell wall structure, rigidity, and porosity play important roles in the capacity for extracellular and extra-organ freezing [65]. The control of ice formation and movement of water from the protoplast to extracellular spaces appears to be particularly important in nonconiferous plants [65]. For work which has been done on conifers, see [66]. In plants displaying second strategy—freezing avoidance, tissues exhibit deep supercooling, in which cellular water is isolated from the dehydrative and nucleating effects of extracellular ice [67]. The formation of ice in tissues and the appearance of frozen plant cells is well documented, mainly in studies employing light microscopy [56,61]. Descriptions of frozen cells at the electron microscopic level also have been done [68].

21.3.1  Cell Membranes As already mentioned [68], functionally intact cell membranes are an effective barrier to the propagation of ice; however, this barrier may vary depending on the temperature or cold hardening [69]. Although the mechanisms involved in plant cold acclimation and frost injury are extraordinarily complicated, the freezing and thawing of cellular water have been found to be basic elements of freezing injury in plant tissues [57]. It has been established that the cellular membranes are more susceptible to freezing damage than soluble enzymes. The plasma membrane seems to be the most susceptible and, therefore, it has been identified as the major site of lethal injury [70]. Leakage of ions from thawed tissues is a common phenomenon of freezing injury. The leakage is usually considered the consequence of the loss of membrane semipermeability or membrane rupture by freezing injury. However, observations on onion epidermal cells indicate that freezing injury is firstly due to a specific alteration in the membrane semipermeability to K+, and secondary effect is protoplasmic swelling [71]. There are numerous studies dealing with the physiological and biochemical changes occurring in membranes during the freezing and cold hardening processes [4,70,71,74] but observations regarding alterations in the cellular membranes are rather insufficient [59,68,72,73]. Isolated plant protoplasts are an excellent model system to study destabilization of the plasma membrane after freezing stress. The use of protoplasts has shown that destabilization manifests in various ways: by intracellular ice formation, by loss of osmotic responsiveness, or by expansioninduced lysis [69]. If cellular membranes are the site of freezing injury, then cellular alterations during cold acclimation that allow the cells to survive freezing also will appear in membranes [74]. Cold acclimation involves chemical and structural alterations of the plasma membrane to resist freeze dehydration, mechanical stress, molecular packing, and other events caused by extracellular freezing. Cytological changes associated with an abrupt increase in hardiness occur at 0°C or −3°C within 7–10 days. However, these changes may be indirect. Observations on Robinia pseudoacacia have revealed a seasonal transition in the plasmalemma from a physical state of relative smoothness and regularity in summer to a highly folded state in winter. It is considered that a highly folded membrane state would facilitate water flow and alleviate the stresses of contraction and expansion during freeze–thaw cycles [75]. However, the plasma membrane of cortical cells of mulberry twigs in winter is relatively smooth, and highly folded states have not been observed. Only after cold acclimation in October at 0°C for 20 days or −3°C for 7 days, when hardiness increased at temperatures ranging from −15°C to −70°C, was the plasma membrane highly folded and microvesicles with a double lipid layer membrane appeared

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in the peripheral cytoplasm. These microvesicles originate from the ER. A very similar ultrastructure has been observed in the cold-acclimated cells collected at the end of autumn. In April, at a decreased hardiness at −15°C, the plasma membrane is already smooth and regular. When these dehardened cells are rehardened at 0°C for 10–15 days, hardiness increased, the plasma membrane becomes folded, and microvesicles reappear near the periphery of the cytoplasm. From these results, it appears that a highly folded state of the plasma membrane and the formation of numerous microvesicles represent a transition associated with higher freezing tolerance rather than representing a special membrane structure characteristic for extremely hardy cells in the winter state [76]. Formation of osmiophilic regions associated with the plasmalemma has been also observed. Substantial regions of the plasmalemma bilayer are transformed into either amorphous, osmiophilic or densely packed regions or in multilayered structures with high surface curvatures [77]. Deep invaginations of the plasmalemma and formation of electron-dense deposits outside the plasmalemma in xylem parenchyma cells of peach and oak trees in a frozen state at −10°C occur [67]. We have observed similar changes of plasmalemma in the mesophyll cells of silver fir [106] and numerous electron-dense lipid bodies associated with the plasmalemma in mesophyll cells of Norway spruce during winter, when the frost resistance of these species is very high [62]. Augmentation of lipidic globules and their localization in the cytoplasm along the plasmalemma apparently results in the changes in lipidic part of membranes during the freezing treatment [78]. It has been found [79] that osmotic shrinkage of protoplasts isolated from Secale cereale results in an irreversible decrease of the surface area of the plasmalemma concurrent with the formation of endocytotic vesicles. This may lend support to the idea that the reduction of the plasmalemma surface area and the reduction of the volume of the protoplast through dehydration occur as initial responses to slow freezing [14,79]. Increasing of the intramembranous particles and plasmalemma invaginations has occurred in more frost-resistant Chloromonas cells, whereas in the frost-sensitive Chlamydomonas cells, they are absent [80]. A higher frequency of osmiophilic globules in acclimated (−25°C to −30°C) isolated protoplasts of S. cereale have been found than in nonacclimated (−3°C to −5°C) protoplasts. Osmiophilic regions observed under transmission electron microscopy corresponded to the extrusions of the surface of acclimated protoplasts observed under scanning electron microscopy (SEM) [69]. SEM observations on apple parenchyma cells have revealed similar copula-shaped protrusions on the surface membranes. The protrusions are associated with the fibrillar formations of exoplasm. It is clear that mechanical breaks of membrane may arise on the plasmalemma near protrusions under stress conditions of freezing. It can be supposed that plasmalemma instability zones are formed under freezing stress connected with protoplast compression under dehydration, whereas protrusions themselves consist of structural lipids of higher unsaturation. Intracellular processes leading to the membrane stabilization are evidently related to condensation of polyphenols, which make cell resistance under stress conditions at super-low temperatures essentially higher [81]. If plants are nonacclimated or the freezing stress is very severe, disruption of the plasmalemma and cell organelles and the collapse of the cell wall with the protoplast can occur [21,82]. In case of the moss, Physcomitrella patens, when frozen to −4°C, freezing injury-associated ultrastructural changes such as formation of nontreated aperticulate domains and fracture-jump lesions were frequently observed in the plasma membrane of protonema cells but not in that of ABA-treated cells [82].

21.3.2  Endoplasmic Reticulum If the plasmalemma is considerably damaged, its protective function against quick dehydration of cells or penetration of ice into cells can be replaced by parallel layering of the ER [84]. The ER is structurally and functionally highly dynamic part of the endomembrane system of plant cells. The response of the ER is immediate at the low temperatures, which is accompanied by shift in its structure and space organization in the cells.

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One of the specific features of wintering plants is the absence of rough ER in the cells. This type of ER, observed in the cortical cells of apple during the growing season, at freezing temperatures in winter becomes sparse and replaced by vesicular ER [85]. The cells enriched with numerous tubular and vesicular smooth ER cisternae have also been observed in ray parenchyma cells of poplar. These smooth ER cisternae are the most characteristic components of cells in the winter stage, and they are suspected to be the site of sugars accumulation [86]. In the cells that survive freezing temperatures by a deep supercooling mechanism, the presence of tubular ER is a feature of dehydration tolerance [87]. The ultrastructural study of such extremely cold hardy cells such as cortical parenchyma cells of mulberry collected in winter has shown that initiation of freezing at 5°C results in the formation of multiplex lamellae that completely cover the area in the vicinity of the plasmalemma. The multiplex lamellae are produced by fusion of preexisting vesicular ER via a reticular network. The completed multiplex lamellae are composed of a parallel array of sheet-like ER cisternae. The formation of multiplex lamellae on the initiation of freezing is largely dependent on seasonality in close association with the development of freezing tolerance [84,88]. Examples of stacked ER were also found in dormant buds in potato and in several other species, mainly trees, such as Betula [89], Sorbus, Quercus, Fraxinus [89,90], Rhododendron [91], and Salix [92]. The stacking of ER disappears in spring in connection with breaking of dormancy. In a study of dormant Tilia buds, using freeze-fractured material, no concentric layering of ER was observed, but an extensive network of ER close to the plasmalemma was found [93]. The groups of stacked ER cisternae have been observed in cells of wheat seedlings at −10°C, whereas at −30°C, ER has been present in the form of numerous vesicles and sacs [94]. The presence of numerous vesicles and cisternae of smooth ER close to the cell wall is considered to be a characteristic feature of frost-resistant cells [68,75,84]. The occurrence of the concentric type of rough ER in frozen cells is an adaptive mechanism protecting ribosomes against injury by low temperature [32].

21.3.3  Vacuole The vacuolization of the cytoplasm is a very important phenomenon, and it is often described as a structural reaction of cells to freezing. Reversible splitting of the large central vacuole into many smaller ones has been observed in many plants; namely, woody species. At the beginning of cold acclimation of peach stem tissue, the cells have their typical architecture—a large central vacuole and a thin band of peripheral cytoplasm—but with the continuing cold acclimation, distribution of the cytoplasm gradually becomes more homogenous, that is, the nucleus is located centrally and many small vacuoles appear in the cells [67]. Splitting of the central vacuole has been recorded in the mesophyll cells of Pinus cembra and Picea excelsa after the first autumn frosts [44,83], in the phloem cells of Metasequoia glyptostroboides [32], and in the mesophyll cells of Norway spruce and silver fir during winter period [62]. A dense and extensive cytoplasm containing numerous small vacuoles is characteristic for winter-hardy cells. Autophagic activity of the vacuoles after severe cold injury has been observed in many plant cells, which results in the digestion of cytoplasmic structures and reorganization of distinct cytoplasmic organelles. The release of protein-toxic vacuolar substances resulted in frost injury of spruce needles due to loss of cell compartmentation and concomitant flooding of the cell interior [32]. Functional stability of the tonoplast, therefore, can play an important role in the frost resistance of spruce needles. Seasonal changes in the vacuole from winter to spring in mulberry cortical cells consist of an engulfment of the tonoplast, fusion and inflation of small vacuoles, and coalescence into larger vacuoles [75]. Similar findings have been seen in the mesophyll cells of both Norway spruce and silver fir [62,106] and in the leaves of evergreen species Aucuba japonica and Prunus laurocerasus [21]. Decay of the central vacuole to small vacuoles is an adapting mechanism of the plants to low

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temperatures in autumn and in winter, although this process of adaptation is reversible. With the increasing temperatures in spring, the central vacuole is differentiated again by the fusion of small vacuoles [95]. On the other hand, although the splitting of the central vacuole is also observed in other woody trees (e.g., Sambucus and Betula) at temperature −30°C to −50°C, this phenomenon can be considered the consequence of a decrease in frost resistance [84]. A decrease of frost resistance in the cells of Robinia pseudoacacia also results in degradation of cell membranes, including the tonoplast [96]. Vesiculation of the tonoplast into the vacuole may represent a similar mechanism (like vesiculation of the plasmalemma) for the reduction of the surface area of the tonoplast and the volume of the vacuole. Observations using osmotically manipulated isolated cells of Brassica napus and S. cereale support this assumption [14].

21.3.4  Chloroplasts The response of chloroplasts to low-temperature stress depends on the temperature and hardening capacity of particular species. Numerous data from some extremely hardy conifers and from a few moderately frost-resistant herbaceous plants indicate variable changes in the chloroplast membranes in different species [97]. For instance, coniferous species tolerate temperatures at around −40°C (and lower), whereas moderately frost-resistant plants such as winter annual herbs and grasses are already killed at −10°C to −15°C. It is often assumed that the chloroplasts are the cell organelles most sensitive to low temperatures [32]. Observations of three grass species have been shown that the transition from 25°C to lowtemperature conditions (10°C, 0°C, −5°C) causes the swelling of chloroplasts in mesophyll cells at 0°C. Dilatation of thylakoids has occurred at −5°C. Similar structural changes of chloroplasts and the disappearance of starch grains were observed in the mesophyll cells of Sorgum and Paspalum [98], and in winter wheat [95]. Chloroplasts of resistant wheat varieties [99] and winter rape [100] change their shape during the hardening, even as retaining their individuality, but during winter they are clumped together. After rewarming during the spring, the structural recovery of the chloroplasts can be observed [100]. According to the position of chloroplasts in the cells during freezing stress, plants can be divided into two groups: (a) the chloroplasts retain their integrity but migrate from a summer position near the cell wall to a crowded position in the cell center and (b) the chloroplasts agglutinate, lose their integrity, and merge with each other to become a continuous mass from which the chloroplasts separate again when spring approaches [56]. Observations on broadleaf evergreen woody species as A. japonica [101], P. laurocerasus [102], Skimmia japonica [103] and Mahonia aquifolium (unpublished observations) have revealed remarkable changes in the chloroplast structure during the year. In summer, chloroplasts are oval shaped, they are placed along the cell walls, and their inner architecture is the same as in other higher plants [104] and contains starch grains. In autumn, the originally lens-shaped chloroplasts of Aucuba and Skimmia become globular and move gradually from the cell wall to the center of the cell. The chloroplasts of Prunus and Mahonia, which are more frost resistant than Aucuba and Skimmia, are still positioned at the cell wall. At this season of year, no starch grains have been observed in the chloroplasts of the plants studied [105]. In winter, the chloroplasts of Prunus are still distributed along the cell wall, whereas the chloroplasts of Skimmia and Aucuba create irregular formations in a different part of the cell. The welldeveloped membrane system with the signs of slight dilatation can be observed. The membrane system of the chloroplasts is often located in one part of chloroplast, leaving only membrane-free stroma in the other part (Figure 21.3). In the chloroplast stroma, small groups of plastoglobuli are present and no starch grains can be visible. Aggregation of the chloroplasts around the nucleus is a

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FIGURE 21.3  Chloroplast of Aucuba japonica in winter with membrane-free stroma and slight thylakoid dilation (×22,000). (From Hudak, J. and Salaj, J. Photobiochem. Photobiophys., 12, 173, 1986, With permission.)

phenomenon connected with the winter metabolism of the cell and is a characteristic feature of the most frost resistant species [83]. A characteristic feature of the chloroplasts of broad leaf species in the spring is the presence of a large number of starch grains resulting in irregular plastid shape. These plastids represent an atypical stage within the plastid ontogenesis chloroamyloplasts [104]. Similarly large content of starch is also observed in the cells of Abies alba and Picea abies [20,106]. Reports have confirmed that the plasticity of the chloroplast membrane system enable the cells to overcome unfavorable conditions during the winter. Mistletoe is well-known semiparasitic plant in which its leaves exhibit a high level of frost resistance. We have not found any striking differences in the ultrastructure of Viscum chloroplasts in the winter and summer. The thylakoid system of chloroplasts is composed of numerous grana and stroma lamellae. Both the stroma lamellae and marginal thylakoids of grana show signs of slight dilatation. The membrane system remains well differentiated even at a temperature as low as −7°C. Small plastoglobuli and a low amount of starch grains are regularly distributed in stroma. The chloroplasts are regularly sheathed by membranes of the ER during the summer, but in the winter, these membranes are fragmented into vesicles of variable shape and size (Figure 21.4) [107].

FIGURE 21.4  Mistletoe chloroplast with an extensive fragmentation of endoplasmic reticulum into vesicles at −7°C (×19,000). (From Hudak, J. and Lux, A., Photosynthetica 20, 223, 1986. With permission.)

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The chloroplasts of conifers [108,109] respond to cold acclimation and freezing by extensive changes in their architecture and localization in the cells. Generally, the chloroplasts of conifers respond to low temperatures mainly by a reduction of starch content, with the increase in the number of osmiophilic globules and of membrane-free stroma, and by swelling of the chloroplasts and their aggregation in one part of the cell [20,62,110]. Similar studies have been done on white spruce [111], balsam fir [112], and Scots pine [110]. The significant presence of numerous starch grains, incorporated into thylakoid system, has been recorded in all investigated evergreen species in the spring, when the chloroplasts are again less frost resistant. This, so called “spring starch” serves as a source of energy for the growth processes in this period [101,102,108]. On the other hand, the disappearance of the starch from the chloroplasts during cold acclimation, even in ABA-treated plants [82] is a typical reaction of both broadleaf and coniferous evergreen species [108,111], as well as of the cells cultivated in vitro with decreasing of temperature [20,113]. The hydrolysis of starch is one of the basic physiological mechanisms for the increase in the frost resistance of plants [114].

21.3.5  Nucleus Although the nucleus, because of its regulation of cell metabolism is considered to be the cell organelle most resistant to the nonlethal effect of low temperature [115], there are not many studies concerning its structural response to the freezing stress and to the process of developing frost resistance. The nuclei in the cells of black locust bark become denser during cold acclimation in the autumn [116]. The nuclei of the acclimated cells in the shoot apex of the rhododendron are ovoid and contain relatively large nucleoli and little heterochromatin, or they are irregularly shaped with small protruding lobes or nucleoplasmic extensions [91]. In the cortical cells of apple [85] at the stage of cold acclimation, each nucleus contains relatively lower amounts of heterochromatin and is located in the central part of the cell. The cooling of tobacco cells to −10°C induces formation of numerous small vacuoles in the nucleus [117]. Similar vacuolization of nuclei also have been observed in the cells of the wheat leaves at −4°C after 8 days of freezing, whereas in the nuclei exposed to −12°C, vacuoles already occur after 1 day [118]. The second step of wheat hardening at −16°C results in folding of the nuclear membrane and condensation of the chromatin [95]. Heterochromatin condensation seems to be a common reaction of the nuclei to a freezing temperature both in perennial grasses [72,118] and in woody plants [62,119]. In the mesophyll cells of spruce, in addition to nuclei with condensed heterochromatin, large nucleoli and changed nuclear membrane also occur during winter at −10°C to −15°C [62]. During winter, the nuclei move to the central position of the cells and are surrounded by the aggregations of the swollen chloroplasts. The movement of nuclei toward a central position in the cortical cells of apple during cold acclimation also has been observed.

21.3.6  Mitochondria The mitochondria are the primary site of intermediary metabolism in the cell and are therefore an excellent means to the study of plant response to the changes in the environment [120]. The alterations of the mitochondrial membranes directly influence the process of cell respiration. Decreasing the temperature in the environment is accompanied by a decrease in the number of mitochondrial cristae and the matrix becomes electron transparent [76,84]. Swollen mitochondria and a reduction in the number of the cristae or their atypical orientation in the cell have been found in the dormant buds of Salix in early winter [92] and in the shoot apex cells of Rhododendron [91]. The reaction of herbaceous plants to freezing stress is similar to that seen in woody species. The mesophyll cells of winter rape in October at the temperature of −6°C possess mitochondria with reduced number of cristae and low electron density of the matrix. In December, when the cells are

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highly injured (about 80%), the mitochondria are hardly visible because of their changed structure in the strongly vacuolated cytoplasm. However, after 48 h recovery of the conditions, the swollen mitochondria are able to rebuild their membrane system [100]. Well-developed mitochondria are present in the rye mesophyll cells at 5°C, but in the cells of both cold-acclimated and cold-nonacclimated rye plants slowly frozen to −12°C, the mitochondrial cristae are strongly disorganized [121]. No significant differences in the respiration of the mitochondria in the extracellularly frozen cells of both acclimated and nonacclimated rye seedlings have been detected [122]. It can be concluded that mitochondria in situ retain normal function even after the cells have been killed by extracellular freezing. However, reports have shown that the mitochondria of rye leaf cells frozen in situ are much more susceptible to frost injury than the chloroplasts [121].

21.3.7  Dictyosomes Abundant dictyomes, usually composed of four to seven cisternae with numerous vesicles originating from their ends, are a common feature in the cells of different species not only during growth season but also during cold acclimation [85,91]. The presence of dictyosomes in poplar cortical cells in September is common. The number of dictyosomes decreases with fall in temperature in October and their level continues to decrease until the next spring [123]. In mulberry parenchyma cells, the dictyosomes secrete numerous vesicles and some of them are located beneath the plasmalemma during slow freezing at −5°C [88]. It may be possible that these secretory vesicles might participate in the formation of the multiplex lamellae that are very often found during slow freezing in mulberry cells. In spite of the frozen state of tissues, dictyosomes in the cells of woody plants can be occasionally identified in their original form. There is evidence [85] that in mid November, when the cortical cells of apple survive freezing at a temperature of −20°C, the dictyosomes are still active and they produce vesicles. In late January, when the cells are hardy to a temperature of −30°C, dictyosomes can be observed, but they are not active. Similar alterations in the dictyosome ultrastructure and their localization in the cell have been observed in the cortical parenchyma cells from mulberry twigs [76], in the xylem ray parenchyma cells of P. persica [67], and in bark tissue of R. pseudoacacia [75].

21.3.8  Cytoskeleton The altered stability of the cytoskeletal elements at low temperatures has been recognized in different plant species. However, the lack of consensus regarding positive correlation between cold acclimation and the cold stability of microtubules still prevails [124,125]. Cold-induced depolymerization of microtubules at temperatures below 0°C has been observed; for example, in the cells of onion [43], cotton [126], spinach [124], garlic and winter wheat [127], and rye [125,128]. The effect of cold acclimation on cortical microtubule stability during freezing has been studied in cold-acclimated and cold-nonacclimated rye leaves [128]. The experiments have shown that unchanged microtubule arrays are still present in cold-acclimated leaf cells after −4°C temperature treatment. In contrast in the leaf cells of nonacclimated plants and in the root cells of both coldacclimated and cold-nonacclimated plants the microtubules are shorter and less abundant. After a −10°C temperature treatment, the cortical microtubules are almost totally depolymerized in both types of root cells and in the leaf cells of nonacclimated plants, whereas cold-acclimated leaf cells constantly have abundant cortical microtubule arrays. Semiquantitative analyses of the cortical microtubules of protoplasts have confirmed the findings with intact leaf cells. These experiments have shown that the cortical microtubules of nonacclimated leaf cells are cold labile and cold acclimation induces cold-stable microtubules in leaf cells as well as in protoplasts (Figure 21.5) isolated from cold-acclimated leaves [128]. Hence, the cells need to have enough long cortical microtubules to keep their plasmalemma intact and responsive to the osmotic changes caused by subzero

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FIGURE 21.5  Responses of microtubules of isolated leaf protoplasts to freezing visualized by indirect immunofluorescence with anti-α-tubulin (1:100). Upper line (from left to the right) nonacclimated protoplasts: control protoplast, after freezing to −4°C and after freezing to −10°C. Lower line (from left to the right) cold acclimated protoplasts: control protoplast, after −4°C treatment and after −10°C treatment (×800). (From Niki,  T. et al. Plant Cell Physiol. 19, 139, 1978. With permission.)

temperatures. Under these stressful conditions, the microtubules may serve as a necessary support for the plasmalemma [128]. The crucial role of microtubules and/or microfilaments in the movement and reconstruction of the ER on the freezing has been reported in the cortical parenchyma cells of mulberry [88]. A contraction of the ER tubule (functional state) to a central rod (nonfunctional state) in the plasmodesmata during cold treatment is caused by changes in the actin–myosin filaments [129]. The partial disruption of actin filaments can accompany or promote freezing tolerance of carrot cell suspensions during preservation at extremely low temperatures [130].

21.4  Molecular Biology  and Biochemistry of Response to Low Temperatures 21.4.1  Cell Membranes Complex aspects of compositional alterations in the plasma membrane during cold acclimation have been reported. The concept of existence of microdomains in the plasma membrane has been introduced [131]. Microdomains were isolated from plasma membrane of A. thaliana as detergentresistant membrane fraction (DRM). After cold acclimation, the proportion of free sterols, P-type H+-ATPases, aquaporins, and endocytosis-related proteins increased in the DRM, and, conversely, tubulins, actins, and V-type H+-ATPase subunits decreased [132]. This suggests that plasma membrane microdomains function as platforms of membrane transport, membrane trafficking, and cytoskeleton interaction [133]. There are still a limited number of known genes that are ultimately contributing to the increased stability of plasma membranes during low temperature stress. A low molecular weight plasma membrane protein from banana, encoded by MpRC1, was shown to directly modulate the physical properties of membrane upon stress, before an overall and more permanent response is initiated [134]. Closely related cold-induced genes/proteins were also identified in other plant species such as the WPI6 from wheat [135], BLST101 from barley [136], RCI2 from Arabidopsis [137], and OsLTi6 from rice [138]. In response to low temperatures, the plant membrane lipids have the tendency to change from gel to liquid-crystalline phase which is caused by increased lipid desaturation [139]. Modifications

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of lipid unsaturation, controlled by key desaturases [140–142], lipases and invertases [143], account for a fraction of acquired freezing tolerance. Positive correlation between the unsaturated fatty acids and the electron transport activities, repairing PSII from damage from chilling, and integrity of the membranes has been reported [144]. However, interactions with other protective proteins such as dehydrins and chaperonins are necessary to fulfill a higher level of freezing tolerance [140]. In Arabidopsis [145] and rice [146], most of known genes for plasma membrane intrinsic proteins (the group of aquaporins) were repressed, while expression of the AtPIP2;5 [145] and the abundance of both PIP1 and PIP2 proteins from maize [147] were induced by cold. Cold stress has also been shown to affect the post-translational modifications of proteins. Glycosylation and phosphorylation profiles of calreticulin, a key protein that regulates quality control of other proteins, are changed in rice leaf sheets by low temperatures [148].

21.4.2  Endoplasmic Reticulum The ER is considered to be the locus of lipid biosynthesis and is intimately concerned with the turnover of the membrane components. There is a fairly good correlation between the degree of unsaturation of the lipid phosphatidylglycerol (PG) and the known chill-sensitivity of many species, though it is not possible to generalize since such correlation was also absent in some species. It has been suggested that the expression of the stearoyl-acyl carrier protein (ACP) desaturase gene in potato [140] and rapeseed [149] plants contributes to increase of cold tolerance in plants due to the increased desaturation of the fatty acids and thus a better membrane control of damage at the membrane level. In potato, the membrane lipids upon stress change their composition showing increase (by 5%–10%) in unsaturated fatty acids. The cold-inducible WAP27 was found as an accumulate localized specifically in vesicularform ER and also localized in dehydration-induced multiplex lamellae (MLP)-form ER [150]. It is hypothesized that in the ER it can stabilize the membrane in a manner similar to that of COR15am. Conversion of the ER to MPL and accumulation of WAP27 in the ER during winter have the specific effects of inhibiting or minimizing plasma membrane destabilization due to the close approach of membranes and consequently confer extremely high freezing tolerance to cortical parenchyma cells of mulberry tree [151]. Other cold-inducible proteins identified in the ER are the WAP27B that shows homology to LEA3 proteins [151] and WAP20 exhibiting homology to ER-localized small heat shock proteins (smHSPs) [150]. Both WAP27 and WAP20 are localized inside the ER vesicle and possibly play a significant role in the acquisition of freezing tolerance in cortical parenchyma cells of mulberry trees [151]. The ER structure reveals relative stability even in the absence (disorganization) of cytoskeletal structures during cold stress (see later) that manifests a certain organizational independence of this membranous organelle [152]. In addition, ER membranes might provide cytoskeletal monomers with the information important for their spatial organization during cold and also during subsequent recovery of actin filaments and microtubules at optimal temperatures.

21.4.3  Vacuole In plant cells with acquired tolerance to freezing, the segmentation of the central vacuole is commonly known. This phenomenon was reported in winter rye [53], winter oilseed rape [153], as well as in moss cells Physcomitrella patens [82]. Restriction of vacuole volume associated with the appearance of numerous membrane-bound vesicles may be advantageous to frost-hardened cells. Interaction between water molecules and the polar head groups of membrane lipids might result in lowering of water potential in these compartments, which is an important factor for avoidance of excessive dehydration during formation of extracellular ice [153]. In A. thaliana, the reduction in vacuole volume and increase in cytoplasmic space in leaf cells induced cold acclimation. This is a

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possible mechanism for accumulation of sucrose in a cytoplasm which contributes to protection of membranes from damages caused by dehydration and freezing [154]. Cryoprotectants like fructans are synthesized in vacuoles and they are probably transported to the apoplast by postsynthesis mechanisms which are probably induced by cold [155]. These findings indicate that segmentation of vacuoles might contribute to enhancement of freezing tolerance. The vacuole also plays the role in calcium influx elicited by cold shock where the immediate rise in cytosolic free calcium concentration occurred. In this case, the vacuole served as an intracellular source of calcium [156]. The aquaporin GhTIP1;1 in cotton, that displays water channel activity and facilitates water transport through the vacuolar membrane, has been suggested to play a role in response to cold stress [157].

21.4.4  Chloroplasts The photosynthetic activity declines in chilling-sensitive plants exposed to low temperatures as a consequence of decrease in the efficiencies of the both photosystems (PSI and PSII), the ATP synthase, and the stromal enzymes of the C3 carbon reduction cycle [158]. Although chilling can initiate the formation of reactive oxygen species (ROS) in membranes [159,160], peroxidation of lipids and finally leakage of cell content and loss of water [161], the chloroplast does not appear to be the primary target for oxidative damage at low temperatures [162]. In thylakoids, ROS are eliminated by photoprotective mechanisms [163], nonenzymatic antioxidants like ascorbic acid and α-tocopherol [164,165], or by enzymes for example, superoxide dismutases (SOD) or peroxidases [166–168]. In the outer chloroplast membrane, a steroid binding protein lipocalin has been identified in wheat and Arabidopsis [169] that possibly contributes to increase of membrane fluidity at low temperature [170]. Further, enhanced expression of lipid desaturases like fad7 [171] and desC [172] has resulted in increased number of thylakoids per granum in transgenic tobacco plants. The cold regulated products of the COR15a gene of A. thaliana [173,174] and the cor14b from barley [175] in stroma probably protect the thylakoid membranes against freeze-induced damage. However, unlike the other cryoprotective proteins [176], COR15am appears to function by decreasing the tendency of membranes to form the lamella-to-hexagonal II phase, which leads to membrane damage during freezing [177]. Similarly, the COR15b gene of A. thaliana, the cold regulated BN115, BN26, and BN19 genes of Brassica napus [178] and the Cbcor15b from Capsella [179], all apparent homologs of COR15a, encode for polypeptides that affect freezing tolerance. Freezing tolerance is achieved by sequential stages of cold acclimation that is initiated independently by low/freezing temperatures and by short day length [180]. Phytochromes are responsible for detection of photoperiod. Modifications of the expression level of the phytochrome phyA gene from Arabidopsis have been shown to prevent cold acclimation or change in sensitivity to day length [181] by regulating levels of ABA and dehydrin (LEA group 2) [182]. However, these genes are regulated by a different mechanism when induced by extracellular freezing [180]. Plastid factors, like changes in plastoquinone redox state, affect nuclear genome activity [183] and elicit stress response mechanisms [184].

21.4.5  Nucleus The ability of plants to cold acclimate is a quantitative trait involving the action of many genes with small additive effects [185]. It has been proposed that perception occurs through low-temperature induced changes in membrane fluidity (rigidification), protein and nucleic acid conformation, specific metabolite- and/or redox status [186]. Activation of calcium channels or secondary signals such as ABA or ROS leads to cold-induced Ca2+ increase in the cytosol and calcium signal amplification,

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and possibly phospholipid signaling are triggered [187]. It is proposed that in cabbage the gene csp5 encoding a cold shock protein is a final link of signal-transmitting chain [188]. Metabolic profiling revealed that cold acclimation increases ∼75% of the 434 metabolites detected in Arabidopsis [189]. In addition to their role as osmoprotectants, certain metabolites induced during cold acclimation might act as regulators of gene expression, for example, proline [190] or signal transducers for example, different soluble sugars [191]. Transcriptome remodeling induced by cold represents up to ∼20% of the genome [192,193]. In cold-acclimated winter wheat at slowly freezing conditions the number of upregulated genes reached a ratio of 9:1 with those of the downregulated [194]. In these analyses, cold regulated (COR), dehydration responsive, and ice re-crystallization inhibition (IRI) genes were upregulated and photosynthesis and respiration-related genes were repressed upon cold stress. Many COR genes have in their promoters one or several copies of the dehydration responsive element/C-repeat cis-element (DRE/ CRT) that is recognized by transcription factors known as CRT-binding factors (CBFs) or DREbinding factors (DREBs) [195,196]. The CBFs are expressed early and transiently by cold stress and have been identified in many plant species (for review see [197]). Their expression levels are sensitively regulated positively by, for example, cold-activated protein kinases, mitogen activated protein (MAP) kinase cascade [198], ABA, circadian clock [199], by ubiquitination-mediated proteosomal protein degradation of the ICE1 (Inducer of CBF Expression 1) [200] as well as by calmodulin binding transcription activator (CAMTA) [201]. On the other hand, CBFs are negatively regulated by the HOS1 (high expression of osmotically responsive gene 1) that targets ICE1 for proteosomal degradation [202–204]. The HOS1 gene is constitutively expressed but gets drastically downregulated within 10 min of cold stress. Posttranscriptional RNA processing and nucleocytoplasmic transport have been shown to play an important role in cold acclimation. Different RNA chaperones, RNA helicases, zinc-finger-containing glycine rich RNA binding proteins are upregulated by cold stress and contribute to freezing tolerance [205]. CBFs activate a core set of robustly COR “downstream” plant genes including genes known as COR, KIN (cold-induced), LTI (low-temperature-induced), or RD (responsive to dehydration) genes. Products of these genes include different dehydration-responsive proteins, enzymes required for biosynthesis of osmoprotectants and antifreeze proteins (AFPs). The AFPs (products of, e.g., CHT9 and CHT46 from winter rye, sthp-64 from Solanum dulcamara, PsAFP from Populus suaveolens) belong to effective cryoprotectants accumulated only during cold acclimation [206]. They possess multiple, hydrophilic ice-binding domains and appear to function as inhibitors of ice recrystallization and ice nucleation. Accumulation of the downstream gene products acts to minimize the injury caused by dehydration, or would limit ice crystal growth. For example, many COR or LEA proteins including dehydrins function as membrane stabilizers and thus enhance cold tolerance in plants [185]. Overall, cold acclimation results in protection and stabilization of the integrity of membranes, activating antioxidative mechanisms, accumulation of soluble sugars and other cryoprotectants, and activation of repair mechanisms (e.g., inducing chaperones) (Figure 21.6). Although the CBF cold regulatory pathway is commonly thought to play a key role in coldresponsive mechanisms, CBFs regulate only ∼12% of the cold-responsive genes [207]. There have been several CBF-independent regulons identified that are critical for cold acclimation. For example, mutations in the HOS9, HOS10 [208] or ESK1 [209] genes result in stronger or shifted induction of several CBF-target genes and lead to altered freezing tolerance. However, they exert no effects on the expression of CBFs. For a more detailed view on the network of COR genes in cold acclimation we refer the reader to reviews reported by [186,197,210]. Interestingly, the data indicate that the genetic and molecular basis of subzero acclimation is probably different from that of cold acclimation [211]. Proteomic analyses identified changes in nuclear proteome in response to cold stress. From all identified proteins 54 were up- or down-regulated in Arabidopsis [212] and sixty was upregulated in rice [213]. More recently, increased evidence points toward the existence of plant nuclear proteins

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Channel transport

Co

Sig

pm Metabolites

?

nal

per cep t

ion

Ca2+

HOS1

ROS tio da xi ro Pe

Phosphorylation cascade

cp

ld

n

ICE1

HOS1

PSI PSII

CRT/DREB

ROS detoxification COR, LEA

HOS9 HOS10

Membrane composition CBF

RD KIN LTI COR Nucl.

cox

ABA

mt ROS detoxification FA desaturation uncoupling

LEA, HSP FA desaturation ER Gol

FIGURE 21.6  Molecular interactions in the cell under low temperatures. After signal perception, the composition of the membrane (pm) is altered and calcium (Ca2+), ABA or reactive oxygen species (ROS) mediate signal amplification. In the nucleus (nucl.) the transcription of many genes is altered, largely repressed. Many genes containing in their promoters DRE/CTR elements are regulated by CTR-binding factors (SBFs), DRE-binding factors (DREBs), ICE (inducer of CBF expression) activator or by HOS (high expression of osmotically active genes) negative regulators. CBFs affect different cold regulated—(COR), cold induced— (KIN), low-temperature induced—(LTI) or responsive-to-dehydration (RD) genes that leads to production of specific protective proteins to avoid chilling damages. These include among others different heat shock proteins (HSPs), late embryogenesis abundant proteins (LEAs), and ROS detoxification proteins. In mitochondria (mt) the level of fatty acid (FA) saturation is altered and activity of cytochrome c oxidase (cox) is negatively affected. In chloroplast (cp), the activity of the photosystems (PSI and PSII) is repressed. Water status is carefully controlled, e.g., by LEAs or channel transport.

that are also targeted to other organelles—either mitochondria or plastids [214]. Most of such dualtargeted proteins identified so far are transcription factors that play a role in the genome structure maintenance by binding to telomeric DNA. Though the knowledge on their distribution patterns and distribution mechanisms is still rather limited, proteins with dual locations are excellent candidates for signal transduction and coordination of gene expression between the nucleus and the organelles involved in (cold) stress perception like chloroplasts and mitochondria [214].

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21.4.6  Mitochondria Mitochondria have been found to be the main cellular compartment affected by chilling treatment. Some of the most notable effects of cold on mitochondria are reversible uncoupling [215], decrease in protein import [216], and accumulation of stress related proteins. These include, for example, heat shock proteins such as the HSP22 [217], late embryogenesis abundant (LEA) proteins [218], peroxidases [219], and catalases [220–222]. Under low-temperature condition the involvement of alternative oxidase (AOX) [223] has been suggested to play role in Arabidopsis shoot acclimation [224], and cold acclimation and freezing tolerance in wheat [225]. Plant mitochondria possess alternative enzymes enabling oxidizing of both external NAD(P)H and internal NAD(P)H independently of complex I [226], providing additional flexibility to the mitochondrial metabolism during cold stress [227,228]. Mitochondria from the cold-tolerant population of maize genotypes revealed a higher percentage of 18-carbon unsaturated fatty acids when compared to the sensitive ones that has been associated with the physiological need to maintain membrane fluidity at low temperatures [230]. The level of 18:1 and 18:3 unsaturated fatty acids can control the activity of cytochrome c oxidase (COX) [229] that decreases in the inner mitochondrial membrane at chilling temperatures and causes enhanced production of H2O2 and subsequent lipid peroxidation in the membranes [219]. Efficient scavenging of H2O2 by antioxidant enzymes such as superoxide dismutase, catalase, and peroxidases [219,220,222,231] are necessary to prevent the irreversible damage of mitochondrial membrane components [231]. In addition, the functional state of mitochondria affects expression of nuclear genes [232] leading to further responses to low temperatures.

21.4.7  Cytoskeleton Inside the cells, the cytoskeleton can serve as a template where components transducing extracellular signals interact. Recently, the novel protein HOS1, a putative E3 ubiquitin ligase controlling degradation, was shown to reside in the cytoplasm at normal growth temperatures but accumulate in the nucleus in response to low temperatures [203,204]. The HOS1 is then likely to contribute to the communication between the cytoplasm and the nucleus under cold conditions. In addition, cold acclimation induced receptor kinase from wheat [233] and a large number of regulatory proteins [234] have been found to interact with microtubules supporting the view that microtubules play a central role in cold signaling and cold acclimation [235]. Despite the fact that cold stress conditions strongly inhibit protein synthesis in vivo, the levels of actin and tubulin in cytosolic and sedimentable protein fractions do not change under low temperatures (neither degradation of particular mRNAs occur) [152]. Cold acclimation caused the aggregation of microtubules, which is demonstrated by increased fluorescence and stability of microfilaments in root cells of Triticum aestivum [236]. Further, appearance of cold-stable microtubule was accompanied by reduced abundance of type TUA ½ α-tubulin isotypes [41] as well as they were fine and transverse strands [237]. Cold-resistance of microtubules is explained by amino acid substitution in sequences of tubulin molecules [238]. Microtubules are not only the target of cold stress, but they also seem to participate in cold sensing itself, triggering a chain of events that results in increased cold hardiness [239].

21.4.8  Cell Wall The cell wall acts as a barrier against propagation of extracellular ice. This is documented by experiments with plasma membrane disrupted cells by repeated freezing and thawing. In chilling-sensitive plant cells intracellular freezing occurred, whereas in chilling-resistant ones, the response to freezing was only by extracellular freezing [240]. Different pore size of the cell walls

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of chilling-sensitive and of chilling-resistant plants is believed to reflect on long-term cold acclimation due to the evolutional adaptation of plants to cold climates [241]. The process of cold acclimation also includes increased cell tension accompanied by ability of cell walls to undergo deformation. It was demonstrated that in suspension-cultured cells of grape and apple the cold acclimation was the result of increase in the cell-wall strength and a decrease in the cell wall pores [242]. Mechanical properties of cell wall are influenced by temperature dependent modifications in pectin content and their methyl esterification degree which increased its resistance to freezing of oil-seed rape plants [243] and chicory root [244]. The mechanism of deep supercooling in xylem ray parenchyma cells in hardwood species was proposed to allow adaptation to freezing [245].

21.5  Conclusion From the presented results it is obvious that low-temperature stress considerably affects the structure of plant cells. The structural response of the cells is variable and is determined by external (strength and duration of stress) and internal (plant species, ontogenetic phase of the plant, type of the tissue, and genetically determined level of resistance) factors. Therefore, it is difficult to decide which cell compartment plays the primary or the most important role in cell responses to both chilling and freezing stresses.

References

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213. Cui, S., F. Huang, J. Wang, X. Ma, Y. Cheng, and J. Liu. 2005. A proteomic analysis of cold stress responses in rice seedlings. Proteomics 5:3162–3172. 214. Krause, K. and K. Krupinska. 2009. Nuclear regulators with a second home in organelles. Trends Plant Sci 14:194–199. 215. Popov, V. N., O. V. Markova, E. N. Mokhova, and V. P. Skulachev. 2002. Effects of cold exposure in vivo and uncouplers and recouplers in vitro on potato tuber mitochondria. Biochim Biophys Acta Bioenerg 1553:232–237. 216. Taylor, N. L., C. Rudhe, J. M. Hulett, T. Lithgow, E. Glaser, D. A. Daya, A. H. Millar, and J. Whelan. 2003. Environmental stresses inhibit and stimulate different protein import pathways in plant mitochondria. FEBS Lett 547:125–130. 217. Stupnikova, I., A. Benamar, D. Tolleter, J. Grelet, G. Borovskii, A. J. Dorne, and D. Macherel. 2006. Pea seed mitochondria are endowed with a remarkable tolerance to extreme physiological temperatures. Plant Physiol 140:326–335. 218. Borovskii, G. B., I. V. Stupnikova, A. I. Antipina, C. A. Downs, and V. K. Voinikov. 2000. Accumulation of dehydrin-like-proteins in the mitochondria of cold-treated plants. J Plant Physiol 156:797–800. 219. Prasad, T. K., M. D. Anderson, and C. R. Stewart. 1994. Acclimation, hydrogen-peroxide, and abscisic-acid protect mitochondria against irreversible chilling injury in maize seedlings. Plant Physiol 105:619–627. 220. Prasad, T. K., M. D. Anderson, B. A. Martin, and C. R. Stewart. 1994. Evidence for chilling-induced oxidative stress in maize seedlings and a regulatory role for hydrogen-peroxide. Plant Cell 6:65–74. 221. Anderson, M. D., T. K. Prasad, and C. R. Stewart. 1995. Changes in isozymes profiles of catalase, peroxidase, and glulathione reductase during acclimation to chilling in mesocotyls of maize seedling. Plant Physiol 109:1247–57. 222. Prasad, T. K. 1997. Role of catalase in inducing chilling tolerance in pre-emergent maize seedlings. Plant Physiol 114:1369–1376. 223. Takumi, S., M. Tomioka, K. Eto, N. Naydenov, and C. Nakamura. 2002. Characterization of two nonhomoeologous nuclear genes encoding mitochondrial alternative oxidase in common wheat. Genes Genet Syst 77:81–82. 224. Fiorani, F., A. L. Umbach, and J. N. Siedow. 2005. The alternative oxidase of plant mitochondria is involved in the acclimation of shoot growth at low temperature. A study of Arabidopsis AOX1a transgenic plants. Plant Physiol 139:1795–1805. 225. Mizuno, N., A. Sugie, F. Kobayashi, and S. Takumi. 2008. Mitochondrial alternative pathway is associated with development of freezing tolerance in common wheat. J Plant Physiol 165:462–467. 226. Douce, R. and M. Neuburger. 1989. The uniqueness of plant-mitochondria. Annu Rev Plant Physiol Plant Mol Biol 40:371–414. 227. Vanlerberghe, G. C. and L. McIntosh. 1997. Alternative oxidase: From gene to function. Annu Rev Plant Physiol Plant Mol Biol 48:703–734. 228. Rasmusson, A. G., K. L. Soole, and T. E. Elthon. 2004. Alternative NAD(P)H dehydrogenases of plant mitochondria. Annu Rev Plant Biol 55:23–39. 229. De Santis, A., P. Landi, and G. Genchi. 1999. Changes of mitochondrial properties in maize seedlings associated with selection for germination at low temperature. Fatty acid composition, cytochrome c oxidase, and adenine nucleotide translocase activities. Plant Physiol 119:743–754. 230. Nishida, I. and N. Murata. 1996. Chilling sensitivity in plants and cyanobacteria: The crucial contribution of membrane lipids. Annu Rev Plant Physiol Plant Mol Biol 47:541–568. 231. Prasad, T. K., M. D. Anderson, and C. R. Stewart. 1995. Localization and characterization of peroxidases in the mitochondria of chilling-acclimated maize seedlings. Plant Physiol 108:1597–1605. 232. Lee, B. H., H. Lee, H. Lee, L. Xiong, and J. K. Zhu. 2002. A mitochondrial complex I defect impairs cold-regulated nuclear gene expression. Plant Cell 14:1235–1251. 233. Tardif, G., N. A. Kane, H. Adam, L. Labrie, G. Major, P. Gulick, F. Sarhan, and J. F. Laliberte. 2007. Interaction network of proteins associated with abiotic stress response and development in wheat. Plant Mol Biol 63:703–718. 234. Chuong, S. D. X., A. G. Good, G. J. Taylor, M. C. Freeman, G. B. G. Moorhead, and D. G. Muench. 2004. Large-scale identification of tubulin-binding proteins provides insight on subcellular trafficking, metabolic channeling, and signaling in plant cells. Mol Cell Proteomics 3:970–983. 235. Christov, N. K., R. Imai, and Y. Blume. 2008. Differential expression of two winter wheat alpha-tubulin genes during cold acclimation. Cell Biol Int 32:574–578.

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236. Khokholova, L. P., O. V. Olinevich, and M. Raudaskoski. 2003. Reorganisation of microtubule and actin cytoskeleton in root cells of Triticum aestivum L. during low temperature and abscisic acid treatment. Cell Biol Int 27:211–212. 237. Wang, Q. Y. and P. Nick. 2001. Cold acclimation can induce microtubular cold stability in a manner distinct from abscisic acid. Plant Cell Physiol 42:999–1005. 238. Nyporko, A. Yu., O. N. Demchuk, and Ya. B. Blume. 2003. Cold adaptation of plant microtubules: Structural interpretation of primary sequence changes in a highly conserved region of α-tubulin. Cell Biol Int 27:241–243. 239. Nick, P. 2008. Microtubules as sensor for abiotic stimuli. In Plant Microtubules, Plant Cell Monograph 11, P. Nick (ed.). Berlin/Heidelberg, Germany: Springer-Verlag, pp. 157–203. 240. Yamada, T., K. Kuroda, Y. Jitsuyama, D. Takezawa, K. Arakawa, and S. Fujikawa. 2002. Roles of the plasma membrane and the cell wall in the responses of plant cells to freezing. Planta 215:770–778. 241. Fujikawa, S., Y. Jitsuyama, and K. Kuroda. 1999. Determination of the role of cold acclimationinduced diverse changes in plant cells from the viewpoint of avoidance of freezing injury. J Plant Res 112:237–244. 242. Rajashekar, C. B. and A. Lafta. 1996. Cell-wall and cell tension in response to cold acclimation and exogenous abscisic acid in leaves and cell cultures. Plant Physiol 111:605–612. 243. Solecka, D., J. Zebrovski, and A. Kacperska. 2008. Are pectins involved in cold acclimation and deacclimation of winter oil-seed rape plants? Ann Bot 101:521–530. 244. Thonar, C., F. Liners, and P. Van Cutsem. 2006. Polymorphism and modulation of cell wall esterase enzyme activities in the chicory root during the growth season. J Exp Bot 57:81–89. 245. Fujikawa, S. and K. Kuroda. 2000. Cryo-scanning electron microscopic study on freezing behavior of xylem ray parenchyma cells in hardwood species. Micron 31:669–686.

of UV-B Radiation 22 Effects on Plants: Molecular Mechanisms Involved in UV-B Responses Brian R. Jordan Contents 22.1 Introduction........................................................................................................................... 565 22.2 UV-B Perception.................................................................................................................... 568 22.3 Signal Transduction Pathways............................................................................................... 569 22.4 Gene Activation and Expression............................................................................................ 571 22.5 Grape Development: A Real-Life Example of UV-B Responses.......................................... 572 22.6 Concluding Comments.......................................................................................................... 573 References....................................................................................................................................... 574

22.1  Introduction Ultraviolet (UV) radiation is divided into UV-A (320–380 nm), UV-B (280–320 nm), and UV-C (below 280 nm). UV-C is essentially a lethal radiation and is excluded from reaching the earth’s surface by the ozone layer. UV-B, however, is only partially excluded (90%) but showed stunted growth. Ash, alder, maple, and R. pseudoacacia contained normal concentrations of Cd, Cu, Pb, and Zn in their foliage. Consequently, these species reduce the risk of metal dispersal and are therefore suitable species for phytostabilization under the given conditions. White poplar accumulated high concentrations of Cd (8.0 mg kg−1) and Zn (465 mg kg−1) in its leaves and might therefore cause a risk of Cd and Zn input into the ecosystem because of autumn litter fall. This species is thus unsuitable for phytostabilization. Celik et  al. [161] evaluated the leaves of R. pseudoacacia L. as biomonitors of heavy metal contamination in Denizli city, Turkey. Concentrations of Fe, Zn, Pb, Cu, Mn, and Cd were determined in washed and unwashed leaves and soils collected from a wide range of sites with different degrees of metal pollution (industry, urban roadside, and suburban) and from a rural (control) site. All the above-mentioned elements were found to be at high levels in samples collected at industrial sites, except for lead and copper that were found at high levels in samples collected from urban roadsides that associated with the road traffic. The strong correlation between the degree of contamination and concentrations in all plant leaves assessed display that the leaves of R. pseudoacacia reflect the environmental changes accurately, and that they seem as an effective biomonitor (bioindicator) of environmental quality in areas subjected to industrial and traffic pollutions.

24.6  Metal Toxicity–Induced Alterations in Crops From a global perspective, it can be stated that crops are predominantly used as food and fodder. Only in advanced and highly developed countries are these plants also used as technical plants (e.g., for alternative source of energy or environment protection). Therefore, the ethical aspect is emphasized if the crops (e.g., maize, cereals, potatoes, rapeseed, and sunflower) could be used exclusively for alimentary purposes or also as an alternative energy source. Moreover, the effect of the toxic substances including heavy metals on physiological and production characteristics of the crops is in general extraordinarily important. Considering all the above-mentioned aspects of the crops, this would require a separate chapter in this book. However, we want to present a brief choice related to the effect of metals on cytology, anatomy, physiology, and production of these important group of plants. Toxic metals are known as mitotic inhibitors that finally cause the reduction of root growth. Jiang et al. [162] observed the toxic effect of CuSO 4 (10 −4 to 10 −1 mol dm−3) on chromosomal morphology.

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Doncheva [163] found that copper interrupts the progression of nuclei at the crucial G1/S transition point of the cell cycle, when it prevents their entry into mitosis. The decreased root growth could be due to the effect of copper on the root–meristem cell proliferation. In Hordeum vulgare and Setaria italica plants Yadav and Srivastava [164] confirmed the inhibitory effect of Cd2+ ions on the mitotic and active mitotic indices whereby Cd2+ ions induced various types of mitotic anomalies. Ivanov [165] investigated the inhibition of maize root growth by metal ions and found that the inhibitory efficiency decreased in the following order: Cu ≈ Tl > Ag > Cd > Hg > Co > Zn > Pb (with concentrations calculated as g dm−3) and Tl3+ > Cu2+ ≫ Ag+ > Hg2+ ≈ Cd2+ > Zn2+ ≈ Pb2+ ≈ Co2+ (for molar concentrations). Metal affinity for SH-groups of biological compounds was closely correlated (r = 0.9) with the molar concentration that inhibited primary root growth by 50%. A similar sequence of the efficiency of phytotoxic effects of metals reflected in root growth inhibition (Cu2+ > Hg2+ > Cd2+ > Zn2+) was also observed in tests with Brassica napus plants [166]. Sagardoy et al. [167] ascertained that the leaves of hydroponically cultivated sugar beet (Beta vulgaris L.) plants treated with 50 and 100 μM zinc sulfate developed symptoms of Fe deficiency, including decreases in Fe, chlorophyll, and carotenoid concentrations; increases in carotenoid/chlorophyll and ­chlorophyll a/b ratios; and deepoxidation of violaxanthin cycle pigments. Sunakar and Sumita [168] published that although lead ion (Pb2+) induces thylakoid-membrane lipid peroxidation, it prevents the loss of pigments (chlorophylls and carotenoids) and loss of proteins. The cation-induced retardation in pigment and protein loss is explained in terms of metal ion binding to lipid protein complex through carboxylic acid group of lipids and sulfhydryl group of protein. Chu et al. [169] using pot-culture experiments examined the individual and combined effects of Cu and Cd pollutants on Trifolium repens L. seedlings and observed that the contents of their leaf pigments decreased. Chlorophyll a was more sensitive than chlorophyll b to Cu and Cd pollutants, and chlorophyll b was more sensitive than carotenoid. It was also shown that the active oxygen metabolism of T. repens seedlings was destroyed by high amounts of Cu and Cd. Souza et al. [170] using scanning electron microscopy for the investigation of 8 day old maize plants exposed to Cd and Zn observed changes in the leaf surface, particularly in the guard cells of the stomata. The ultrastructural analyses of the parenchyma mesophyll cells showed extensive chloroplast disorganization, mainly affecting the thylakoid membranes and grana. Hydroponically cultivated seedlings of the soybean (Glycine max (L.) Merr.) exposed to PbCl2 (0, 10, 20, and 40 mg dm−3) were characterized by Pb-induced changes in the leaf epidermis structure involving a reduction in the cell size, more abundant wax coating, and an increase in the number of stomata and trichomes per unit leaf area with simultaneous reduction in the size of the guard cells [171]. Burst stroma of the thylakoid system and the cracked chloroplast envelopes were also observed. The importance of the increase in the number of stomata and trichomes for plants under the metal stress was examined. Kastori et al. [114] found that excess concentrations of lead (Pb), cadmium (Cd), copper (Cu), and zinc (Zn) significantly decreased transpiration and relative water content in hydroponically cultivated young sunflower (Helianthus annuus L.) plants. On the other hand, the number of stomata per unit leaf area increased while the size of the stomata decreased. Thus, excess concentrations of the heavy metals significantly affected plant water status, causing water deficit and subsequent changes in the plants. Cd exerted the most intensive effect on the plants, less intensive effects were found for Cu and Zn, at least harmful by Pb. Llamas et al. [172] investigated Cd2+ effects on transmembrane electrical potential difference, respiration, and membrane permeability of rice (Oryza sativa L.) roots and found that upon addition of 0.1 or 1 mmol dm−3 Cd2+ to the experimental solution, root cell membranes depolarized in few minutes, reaching very low Em values. This effect was transient and the initial membrane potential recovered totally within 6–8 h. Only the highest concentration used had an inhibitory effect on root respiration. Significant respiratory inhibition appeared after 2 h of exposure to Cd2+ and lasted for at least 4 h. In turn, membrane permeability increased in the presence of Cd2+ for at least 8 h, inducing K+ efflux from the roots. According to Burzynski and Buczek [173], application of 10 μmol dm−3 Cd, Cu, and Ni, and 50 μmol dm−3 Pb did not change the intensity of root respiration of cucumber

Plant Responses to Toxic Metal Stress

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seedlings. Moreover, the authors found that the respiration of cucumber roots during one hour of tissue incubation with heavy metals in concentrations inhibiting NO3− absorption was similar as in the control and thus the decrease in nitrate uptake by seedlings exposed to Cd, Cu, Ni, or Pb was not due to the influence of these metals on the respiratory processes. Metal toxicity–induced alterations in crop photosynthesis (different organization levels) were published by many authors, see, e.g., [174–178] for details. It should be stressed that the final consequence of the above-mentioned negative effects of toxic metals on crops is manifested in an undesirable decrease of crop yield, what can seriously affect the whole human population [179–181].

24.7  Response of Medicinal Plants to Metal Presence It was recognized that out of the 350,000 vascular plant species identified so far, about 35,000 (the estimates vary) species have at one time or the other been used by some people or cultures for medicinal purpose [182]. According to research carried out by the World Wildlife Fund (WWF), up to 90% of species of medicinal and aromatic plants (MAPs) traded in Europe are still harvested from the wild, and a rapid growth in the market is now resulting in the over-exploitation of wild stocks of some species [183]. Totally, from about 2,000 MAPs traded in Europe, 1,200–1,300 are native to the continent with only 130–140 species predominantly derived from cultivated stock. Wild harvesting of MAPs in Europe is still prominent in many former Eastern Bloc countries including Slovakia where the climate, soil, and low levels of pollution in these countries are some of the best in middle Europe for the cultivation of medicinal plants. Details concerning the abovementioned topic can be found in the project of Interactive European Network for Industrial Crops and their Applications, IENICA INFORRM Project [184]. Medicinal plants could be regarded as potential plant factories for new natural drugs. Hence, it could be stressed that it is necessary to check and monitor the herbs for the content of harmful substances including toxic metals (see [185] for details). Additionally, medicinal plants have a great potential for their exploitation in modern phytotechnologies, such as phytoremediation and phytofortification [186]. At present, it can be stated that the anthropogenic activity and its effects on the environment showed that medicinal plants have also responded to the changing environmental conditions. Some medicinal plants produce specific secondary metabolites that can detoxify some of the toxic metals. Hypericum perforatum and Matricaria recutita belong to cadmium hyperaccumulators because they accumulate in their shoots over 100 μg g−1 d.m. This property can be used in the future in phytoremediation technologies. Moreover, the rest of the biomass of medicinal plants after drug isolation can be utilized as organic fertilizers and pesticide preparations. On the other hand, in the last years, the practical use of alternative medicine in healing processes showed a continually increasing tendency. Several species of medicinal plants can be used as supplementary nutrition due to their ability to accumulate some essential nutrition elements (e.g., Se, Zn, and Fe) in the edible parts of these plants. Such fortification of plants with essential nutrients (phytofortification) in an easily assimilated form can help to feed the rapidly increasing world population and improve human health through a balanced mineral nutrition. In general, data related to toxic metal contents (e.g., Cd) in pharmaceutically utilized parts of the medicinal plants are also considered from the aspect of “food safety”(see [186] for details). In our experiments, we focused on three medicinal species: Hypericum perfolatum L., Matricaria recutiia L., and Salvia officinalis L. that are in general the most frequent medicinal plants used in phytotherapy. Some of the found and important results are presented in the following text. Hypericum perforatum is a plant that has been used as a medicinal herb since ancient time. Major constituents of this plant extracts include several classes of compounds exemplified by flavonols, flavonol glycosides, biflavones, naphthodianthrones, phloroglucinols, tannins, coumarins, essential oils, xanthophylls, and others [187]. The content of naphthodianthrone derivatives hypericin and pseudohypericin is approximately 0.05%–0.15%, that of flavonoid quercetin and biflavone

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biapigenin is 0.3% and 0.26%, respectively. From the other secondary metabolites, the highest content belongs to phlorogluclinole derivatives hyperforin and adhyperforin (up to 4%) [188]. These compounds are very important for the medicinal plants to preserve them against environmental stress. Thus, we studied the tolerance of H. perforatum to the toxic effect of copper and cadmium with respect to metal accumulation in individual plant organs [189]. The 6 week (Cu experiment) or 5 month old plants (Cd experiment) were exposed in hydroponics for 7 days to the following metal concentrations: 15, 30, 60, 90, and 120 μmol dm−3 CuCl2 · 2H2O and 12 μmol dm−3 Cd(NO3)2 · 4H2O. Thereafter, the length and dry mass of shoots and roots were measured and the metal content in the plant organs determined. It was confirmed that as the most sensitive parameter to Cu treatment was found to be the root dry mass. The length of shoot as well as shoot dry mass was not significantly affected. Lower values of root dry mass could be explained with a significant reduction of lateral roots and root hairs by Cu treatment. The roots of H. perforatum accumulated markedly higher concentrations of Cu than the shoots. The metal accumulated in both plant organs showed an increase with increasing metal concentration. Bioaccumulation factors (BAF), that is, quotients obtained by dividing the concentration of the metal in dry mass of individual plant tissues (root and shoot, respectively) by its concentration in the external exposure medium, were also calculated (Table 24.3). Taking into account the actual dry mass of individual plant organs (root and shoot), the Cu concentration in the shoot was within 20% of the investigated concentration range of total metal content uptake by the whole plant (Table 24.3, Pshoot). With respect to relatively high Cd content in the shoot dry mss (1,087 μg g−1), H. perforatum could be classified as a Cd hyperaccumulator. In this chapter, the possible contribution of the formation of metal complexes with the secondary metabolites of H. perforatum to the plant metal tolerance has been discussed first. Later, we stated [186] that for Table 24.3 Metal Content in Dry Mass of H. perforatum Plant Organs (μg g−1 d.m.) Metal Cu

Cd

Plant Organ Root Shoot Root Shoot Root Shoot Root Shoot Root Shoot Root Shoot Root Shoot Root Shoot

Ext. Metal Conc. (μmol dm−3) 0 0 15 15 30 30 60 60 90 90 120 120 0 0 12 12

Metal Content in Dry Mass (μg g−1) 56.6 13.3 1616 27.0 3698 62.1 5874 116.5 7856 141.8 6767 154.3 α-bisabolole oxide A, that is, these cultivars belong to the chemical type A of M. recutita (see [205] for details). Traditionally, in Eastern Slovakia, large regions are used for commercial chamomile cultivation. As chamomile species are long-term cultivated in field conditions, it is important to know how many of the Cd is taken up from the soil, transported, and accumulated in individual parts of plants. Therefore, the Cd content in the pharmaceutically important plant part—anthodium—was also estimated [205]. The values of Cd concentrations in the soil (determined in 2 M HNO3 leachate) and in chamomile anthodium as well as corresponding BAF related to some localities in Slovakia in which field cultivation of chamomile occurs (for the period 1999–2001) are shown in Table 24.6. The presented BAF are quotients obtained by dividing the concentration of Cd in the dry mass of anthodium by its concentration in the soil. The Cd fluctuation in chamomile anthodium (1995–2002) related to plants cultivated in different localities of Eastern Slovakia is presented in Figure 24.9. We also evaluated the relationship between the Cd content in chamomile anthodium dry mass and mean hydrothermic coefficient of Seljaninov (HC) as an integrated index of hydrothermic parameters. This coefficient could be calculated according to the following formula:

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Table 24.6 Cadmium Concentrations in Soil (Determined in 2 M HNO3 Leachate) and in Chamomile Anthodium Dry Mass as well as Corresponding BAF Related to Some Localities in Slovakia in Which Field Cultivation of Chamomile Occurs Locality

Cd in Soil (mg kg−1)

Streda nad Bodrogom Košice Michalovce Nová Lubovňa

0.111 ± 0.042 0.334 ± 0.050 0.335 ± 0.054 0.222 ± 0.025

Cd in Anthodia (mg kg−1)

BAF

0.168 ± 0.078 0.078 ± 0.022 0.179 ± 0.112 0.150 ± 0.021

1.514 0.234 0.534 0.676

Source: Šalamon, I. et al., Acta Hort. (ISHS), 749, 217, 2007. With permission. Note: The data are related to the period 1999–2001.

0.6 0.5

mg Cd kg–1

0.4 0.3 0.2 0.1 0.0 2002 2001 2000 1999 1998 1997 1996 1995

Figure 24.9  Cadmium content in anthodium of chamomile plants cultivated and collected in different localities of Eastern Slovakia in the period 1995–2002. (From Šalamon, I. et al., Acta Hort. (ISHS), 749, 217, 2007. With permission.)

HC = ∑R/(0.1 × TSm), where ∑R is the total amount of precipitations and TSm is the thermal sum of the mean daily temperatures exceeding 10°C in the investigated period. The dependence of Cd content in the chamomile anthodium dry mass on the mean hydrothermic coefficient for the period April–June evaluated from the data measured in the meteorological stations Streda nad Bodrogom and Michalovce in the period 1995–2002 is presented in Figure 24.10. The correlation between the accumulated Cd and HC value supported enormous significance of the actual climatic relations on the metal uptake and accumulation. Marquard and Schneider [193] were the first to confirm that chamomile plants had the potential to accumulate high levels of cadmium from the soil. In our paper [206], two tetraploid cultivars of Matricaria recutita L. (cv. Goral and cv. Lutea) were investigated in response to Cd application. Treated plants were cultivated in Hoagland solution with the following Cd concentrations: 3, 6, 12, 24, and 60 μmol dm−3 Cd(NO3)2. The plants growing in Hoagland solution without Cd served as control. Primary root length, root increment (root length after treatment − root length before treatment) was calculated [(root increment of Cd-treated plants/root increment of control plants) × 100] in %, root and shoot dry mass, as well as Cd content in plant organs were determined after 7 days of treatment. In other experiments, the older plants were grown in the greenhouse conditions in the soil for 7 weeks after germination. Plants used for photosynthetic and respiration measurements as well

619

Plant Responses to Toxic Metal Stress 0.6 2001

0.5

mg Cd kg–1

0.4 0.3 2002

0.2 0.1 0.0 0.75

2000

1999

1.00 1.25 Mean HC for the period April–June

1.50

Figure 24.10  Dependence of Cd content in chamomile anthodium dry mass on the mean HC for the period April–June evaluated from the data measured in the meteorological stations Streda nad Bodrogom and Michalovce. (From Šalamon, I. et al., Acta Hort. (ISHS), 749, 217, 2007. With permission.)

as for the analysis of assimilation pigment concentration were grown under greenhouse conditions for 9 weeks after germination. Then their roots were washed, transferred to hydroponic Hoagland solutions (control), and Hoagland solution with 12 μM Cd(NO3)2 and placed in the growth chamber for 10 days. The concentration gradient that was used in our experiments reflects the Cd content in the soils from non-contaminated to highly contaminated sites [207]. For the estimation on Cd toxicity of roots, we used primary root length and root increment, which are considered a reliable parameter for heavy metal tolerance [208]. At the beginning of Cd-treatment, nonsignificant variance in root length was observed. Significant inhibition of root growth was observed in both the chamomile cultivars after Cd-treatment (Table 24.7). We did not find any differences in the Cd accumulation in the roots of different cultivars, but cv Lutea accumulated a slightly higher amount of Cd in the shoot. No differences between the cultivars were recorded after Cd-treatment in growth parameters (Table 24.7). In the root test, we observed fragility, browning, and twisting of roots. In the shoots, leaf roll, chlorosis, and leaf growth inhibition occurred. During the root test chamomile plants cv. Goral formed the anthodia in all the concentrations except that of the control, despite the fact that the plants were only 3 weeks old. According to our observation, the plants started blossoming when they are 8–12 weeks old; however, Cd treatment resulted in reduced size of flowers. In the Cd hyperaccumulator Arabidopsis halleri, 4–5 weeks earlier blossoming under Cd administration was recorded [209]. Cd concentration 12 μmol dm−3 in the hydroponic solution (used in our other experiment, Table 24.8) represents strongly contaminated soil [207], however, the Cd effect on the plant was stronger in comparison to the soil, because Cd is not bound to the soil particles and so all the ions are available for plant uptake. cv. Lutea seemed to be more sensitive to Cd treatment, e.g., it exhibited greater leaf chlorosis. The measurements confirmed a higher inhibition of photosynthesis in cv. Lutea, although they accumulated less Cd than cv. Goral. Similar decrease of shoot dry weight in both cultivars was also detected (Table 24.8). Decrease of net photosynthetic rate could be due to structural and functional disorders in many different levels. Shoot and root respiration rates were not changed significantly in both chamomile cultivars (Table 24.8). We confirmed that chamomile belongs to the group of Cd accumulator species. If we take into account the high content of Cd in chamomile shoot (over 300 μg g−1 at 12 μmol dm−3 Cd in solution), only a small extant of damages occurred in Cd treated plants. Therefore, this medicinal plant species exhibited high tolerance to Cd treatment. This was also confirmed by Masarovičová et al. [199] through the effect of cadmium and zinc separately (10 μmol dm−3 for Cd and 50 μmol dm−3 for Zn), the combined application of these ions on physiological processes (photosynthetic rate, dark respiration rates of leaves and roots, and

620

Table 24.7 Concentration of Cd in Solution in Relation to Cd Accumulation, and Cd Accumulation in Relation to Growth Parameter (Root Length, Shoot, and Root Weight) Species and Cultivar

Cd in Solution (μM)

Shoot Cd Accumulation (μg g−1 d.w.)a

Root Weight (mg d.w.)

Shoot Weight (mg d.w.)

Primary Root Length (cm)

6.0 32.1 41.8 50.2 66.2 117.3

3.92 ± 0.29 3.01 ± 0.28 3.16 ± 0.41 2.87 ± 0.53 2.08 ± 0.13 1.53 ± 0.12

14.21 ± 1.55 10.62 ± 1.15 9.52 ± 1.21 9.76 ± 1.12 8.89 ± 1.05 5.99 ± 0.59

8.73 ± 0.44 7.83 ± 0.36 8.01 ± 0.55 7.60 ± 0.55 7.14 ± 0.54 5.60 ± 0.21

y = 370.8x − 42.4 R2 = 0.95 p < 0.001

y = 56.4x + 0.6 R2 = 0.91 p = 0.002

y = −0.003x + 3.7 R2 = 0.85 p = 0.009

y = −0.067x + 13.2 R2 = 0.84 p = 0.01

y = −0.004x + 8.7 R2 = 0.93 p = 0.002

10.6 139.3 226.0 313.0 381.2 702.0

5.5 51.8 68.0 75.4 91.2 140.5

6.29 ± 0.76 4.81 ± 0.62 3.73 ± 0.57 2.51 ± 0.17 2.92 ± 0.53 3.62 ± 0.78

13.29 ± 1.33 10.37± 1.31 8.55 ± 1.09 8.36 ± 0.99 8.42 ± 1.23 9.24 ± 0.80

11.93 ± 0.35 10.62 ± 0.42 9.37 ± 0.36 7.80 ± 0.41 7.40 ± 0.31 7.37 ± 0.30

Regression equation

y = 359.6x − 33.9 R2 = 0.95 p < 0.001

y = 68.2x + 9.6 R2 = 0.95 p < 0.001

y = −0.004x + 5.1 R2 = 0.40 p = 0.18

y = −0.031x + 11.9 R2 = 0.52 p = 0.08

y = −0.007x + 11.1 R2 = 0.74 p = 0.03

ANCOVA

Slope p = 0.867 y-inter P = 0.962

Slope p = 0.962 y-inter P = 0.025

Slope p = 0.853 y-inter p = 0.033

Slope p = 0.127 y-inter p = 0.332

Slope p = 0.251 y-inter p = 0.009

Regression equation

Matricaria recutita cv. Lutea

Control 3 6 12 24 60

Source: Pavlovič, A., et al., Bull. Environ. Contam. Toxicol., 77, 763, 2006. With permission. Note: The regression lines for cv. Goral and for cv. Lutea had similar slopes (p > 0.01), mean ± SE, n = 20. a Cd concentration in solution was log transformed.

Root Increment (%) 100 78 91 71 65 39

100 77 72 42 42 38

Handbook of Plant and Crop Stress

12.2 103.8 236.0 323.0 412.5 695.0

Matricaria recutita cv. Goral

Control 3 6 12 24 60

Root Cd Accumulation (μg g−1 d.w.)a

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Table 24.8 Values of Net Photosynthetic rate (PN) and Dark Respiration Rate (RD), Dry Weight, Root Length, and Cd Accumulation in the Plants Used for Photosynthetic Measurement (Mean ± SE) Parameter

Variant

PN (nmol CO2 g d.w. s ) n=4 RD shoot (nmol CO2 g−1 d.w. s−1) n=4 RD root (nmol CO2 g−1 d.w. s−1) n=4 Shoot biomass (mg) n = 15 Root biomass (mg) n = 15 Root length (cm) n = 15 −1

−1

Control Cd 12 μM Control Cd 12 μM Control

Shoot Cd content (μg g−1 d.w.)

Cd 12 μM Control Cd 12 μM Control Cd 12 μM Control Cd 12 μM Control

Root Cd content (μg g−1 d.w.)

Cd 12 μM Control Cd 12 μM

cv. Goral

cv. Lutea

148.48 ± 4.77 110.12 ± 8.27* 34.22 ± 1.45 36.75 ± 3.03 132.36 ± 21.43 181.87 ± 22.42 116.92 ± 11.61 85.00 ± 11.81 25.86 ± 3.08 15.44 ± 2.41* 14.14 ± 1.07 10.50 ± 0.84* 15.9 360.5 19.9 1081.0

177.39 ± 8.63♠ 124.85 ± 10.91** 38.62 ± 1.59 40.61 ± 3.81 44.19 ± 5.12♠ 55.83 ± 9.60♠♠ 136.92 ± 8.36 101.18 ± 15.88 24.56 ± 2.77 12.33 ± 1.83** 13.97 ± 0.81 9.65 ± 1.11** 4.61 248.3 44.8 895.0

Source: Pavlovič, A. et  al., Bull. Environ. Contam. Toxicol., 77, 763, 2006. With permission. Note: Comparisons were done between the control variant and Cd treatment (Cd 12 μM) at p = 0.05 (*) and p = 0.01 (**), and between cultivars at p = 0.05 (♠) and p = 0.01 (♠♠). Student’s t-test was used.

chlorophyll concentration), and production parameters (shoot and root biomass, shoot: root ratio, and lengths of shoots and roots) of young plants of Hypericum perforatum and Chamomilla recutita that were investigated. As the applied metal concentrations did not significantly affect the studied parameters (except for the root respiration rate), we can conclude that both investigated medicinal plants could be used in phytoextraction and the subsequent remediation of soils that are contaminated with cadmium and zinc. Jakovljevic et al. [210] investigated the influence of the different doses of sodium selenate (0, 100, and 500 g Se per ha) applied by foliar spraying on the yield and quality of chamomile (Chamomilla recutita (L.) Rausch.—M. recutita L.). The applied doses of Se did not influence the formation of dry chamomile flowers’ yield and the content of essential oil. However, the applied Se caused a significant increase in the content of bisabolol oxide A and B, followed by the decrease of the chamazulene content in the chamomile essential oil. Significant increase of Se content in the chamomile flowers (12.9–53.6 ppm) has also been observed. In our experiments with hydroponically cultivated chamomile plants, cv. Lutea [211] and cv Goral [212] treated with CdSeO4, CdSeO3, and Cd(NCSe)2(nia) we investigated Cd accumulation in the roots and shoots of plants (Table 24.9). The highest applied CdSeO3 concentration (60 μmol dm−3) caused a higher Cd content in the roots than the amount observed by employing CdSeO4, whereas in the case of treatment with 12 and 24 μmol dm−3 solutions of Cd(II) selenite and selenate adverse effects were observed. This could be correlated with the damage of the root cell membrane system due to the high concentration of cadmium and selenite ions. Immobilization of Cd ions in root tissue was manifested by a large amount of bioaccumulated Cd in this plant organ. The highest Cd content in the shoots was observed after the application

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Table 24.9 The Amount of Accumulated Metal in Dry Mass of Roots and Shoots of M. recutita (cv. Goral) Plants w (Cd) (g kg−1)

c Compound Control CdSeO3

CdSeO4

Cd(NCSe)2(nia)2

Cd(NCS)2(nia)2

μmol dm 0 12 24 60 12 24 60 12 24 60 12 24 60

−3

BAF

Root

Shoot

Root

Shoot

TF

Fraction of Cd in Shoot (%)

0.02 0.75 1.10 4.93 1.17 1.66 1.85 0.85 1.23 5.12 0.79 1.21 2.10

0.01 0.07 0.07 0.19 0.21 0.36 0.48 0.07 0.09 0.17 0.14 0.23 0.41

— 555 408 731 863 613 280 633 457 759 584 449 311

— 54.4 27.2 28.7 155 133 70.7 48.6 35.0 25.1 90.5 84.0 60.3

1.62 0.20 0.25 0.16 0.60 0.68 0.93 0.27 0.26 0.12 0.60 0.70 0.63

61.9 16.6 19.8 13.2 37.6 40.5 48.3 21.0 20.5 10.5 37.4 41.2 38.5

Source: Král’ová, K. et al., Chem. Pap., 61, 171, 2007. With permission. Note: Corresponding values of bioaccumulation and translocation factors and fraction of accumulated Cd allocated in shoots related to the total amount of Cd accumulated by the plant.

of CdSeO4. The change of S to Se in the complex Cd(NCX)2(nia)2 led to a increase of Cd content in the shoots. In general, the content of Cd accumulated in plant organs after the application of Cd(NCSe)2(nia)2 was comparable with that observed after the application of CdSeO3. The values of the translocation factor for Cd estimated for the experiments with CdSeO4 and Cd(NCS)2(nia)2 were more than two times higher than those found for the CdSeO3 and Cd(NCSe)2(nia)2. The highest fraction of Cd accumulated in the shoots was observed for CdSeO4, while the lowest fraction observed was for Cd(NCSe)2(nia)2. The obtained results correspond with those obtained for the chamomile cultivar Goral [212] that was found to be more tolerant to the cadmium exposure compared to the cultivar Lutea [211]. The treatment with CdSeO4 and Cd(NCS)2(nia)2 caused approximately 40% of the total amount of Cd that was accumulated by the plant in its shoots. On the other hand, approximately 80% (or more) from the total amount of the metal accumulated by the plant remain in the roots after the treatment with CdSeO3 and Cd(NCSe)2(nia)2. These data correlate well with the results of Shanker et al. [213]. The observed fact could be explained by taking into account the fact that the less mobile selenite after being reduced to the selenide tends to form Cd–Se complex, which appears to be unavailable for the plants. On the other hand, the more mobile anion selenate is available for Cd–Se formation only after following a more complicated redox process involving Se(VI) in SeO 42−, Se(IV) in SeO32−, and Se(0) species. According to Whanger [214], the presumed protective effect of Se against cadmium and mercury toxicity is through the diversion in their binding from low-molecular-mass proteins to higher-molecular-mass ones. The experiments with chamomile cv. Goral also showed that the BAF values related to Se accumulation in plant organs were significantly influenced by the oxidation state of Se—the application of selenate resulted in an intensive translocation of Se into the shoots and for this compound the BAF values determined for shoots were approximately 2.5 times higher than those determined for roots. The corresponding BAF values for Cd(NCSe)2(nia)2 were similar to those of CdSeO3. At application of CdSeO4, 90% of the uptaken Se and 50% of the uptaken Cd was situated in the shoots [212].

Plant Responses to Toxic Metal Stress

623

The presence of chelators can alter the mobility and transport of Zn, Cd, and Ni in soils because of the formation of water-soluble chelates, thus increasing the potential for the metal pollution of natural waters. However, chelators could also increase the bioavailability and uptake of toxic metals. Chelated metals are taken up via the apoplastic pathway. Disruption of the Casparian band is required to achieve the high-shoot concentrations that are needed for phytoextraction. Therefore, adding chelators to a soil increases not only the total dissolved metal concentration but also changes the primary route of the plant metal-uptake from the symplastic to the apoplastic pathway and depending on metal, plant species, and chelant concentration, significant increases in the metal uptake are likely [215]. The addition of an organic chelator (citric acid) enhanced the zinc and cadmium accumulation, mostly at the root level [216]. Our experiments with a set of Cu(II) chelates confirmed that the application of copper in the form of chelates led to more effective Cu translocation into the shoots in comparison to CuSO4 · 5H2O treatment [217]. Artificial chelator ethylenediaminetetraacetic acid (EDTA) promoted Cu translocation into the shoots of hydroponically cultivated chamomile plants very effectively which was reflected in the fact that at the treatment with higher metal concentrations (24 and 60 μmol dm−3) even 45% and 59% Cu accumulated by plants was allocated in the shoots (in the absence of a chelator this portion reached only 5.2% and 4.8%, respectively). Very efficient translocation of copper into the shoots observed at the presence of EDTA could be connected with the largest value of Cu-EDTA stability constant (log K1 = 18.8). Whereas the chelate formation between the EDTA and Zn or Cu resulted in a significantly decreased metal uptake into the chamomile roots, the decrease of Cd uptake due to chelate formation was very low [218]. Salvia officinalis L. is in general also one of the most important medicinal and aromatic plants with a wide spectrum of application in phytotherapy, cosmetics, and food industry. The genus Salvia includes more than 400 species. S. officinalis as a perennial plant originates from the Mediterranean region. Concerning the analysis of sage essential oil, the major compounds are thujone, cineole, camphor, and caryophyllene. These secondary metabolites are biologically active compounds present in Herba salviae with applications in phytotherapy. In the food industry, this aromatic plant species is recommended as a spice or as an additive substance (cf. [219,220]). From all the above-mentioned aspects, it is important to have information of toxic metal effects on growth and metal accumulation into the different plant organs of this species. Since Marquard and Schneider [193] characterized S. officinalis as the excluder of cadmium, we studied the effect of large external concentration ranges of cadmium (30–480 μmol dm−3 Cd(NO3)2) on the production characteristics (length of roots and shoots as well as dry mass of roots and shoots) of this species. We tested two cultivars: cv. Krajova (Slovakian provenance) and cv. Primorska (Yugoslavian provenance). Two months old plants were cultivated hydroponically for 7 days under controlled conditions in a Hoagland solution, without and in the presence of Cd(NO3)2 [221]. The plants were exposed in hydroponia for 7 days in controlled conditions: control variant in Hoagland solution and metal treated variants in Hoagland solution with 30, 60, 120, 240, 360, and 480 μmol dm−3 Cd(NO3)2. Then the length and dry mass of the shoots and roots were estimated. Metal content in aboveground and underground parts of the studied species were determined using FAAS. Differences were found in the phenology and production parameters between the two tested cultivars of different provenance. cv. Krajova was already sensitive to the concentration of 60 μmol dm−3 of Cd(NO3)2 when the oldest leaves dried. At a concentration of 120 μmol dm−3 Cd(NO3)2, all the older leaves were dried and the younger leaves wilted. At 240 μmol dm−3 of Cd(NO3)2, brown spots were observed on the leaves, and at the applied highest metal concentrations of 360 and 480 μmol dm−3 Cd(NO3)2, all the leaves were dried and on the apical side of the leaves depigmentation was observed. Cultivar Primorska seems to be more tolerant to metal treatment. Visual changes occurred up to 120 μmol dm−3 Cd(NO3)2 when only some of older leaves of the plant dried up. At the concentration of 240 μmol dm−3 Cd(NO3)2, the leaves were dried but they were green colored. This fact confirms the disturbance of the water regime and indicates strong water stress. At the highest tested Cd concentrations (360 and 480 μmol dm−3 Cd(NO3)2) all the leaves dried and the damage of leaf pigmentation was observed as the brown colored spots. In spite of the high concentration of Cd (30–480 μmol dm−3), the length of the roots

624

Handbook of Plant and Crop Stress 25,000 Cd in roots (mg kg–1 d.m.)

1 20,000

2

15,000 10,000 5,000 0

0

100

200 300 c (μmol dm–3)

400

500

Figure 24.11  Dependence of Cd content in roots of Salvia officinalis plants on the concentration of Cd(NO3)2 in the hydroponics (1, cv. Primorska; 2, cv. Krajova).

in both cultivars was almost not influenced. Also for the shoots of both cultivars, only a slight reduction of length was found. On the other hand, the dry mass of the shoots decreased at all applied Cd concentrations more expressively than the dry mass of the roots. The negative effect of the high Cd concentrations on the shoot dry mass was manifested mainly in cv. Krajova. The greatest portion of Cd into the roots was uptaken by both the cultivars (Figure 24.11). cv. Primorska accumulated two times more Cd than cv. Krajova in the shoot app. (Figure 24.12). However, the differences were found in the translocation of the cadmium from the roots into the shoots. cv. Krajova did not allocate the Cd from the roots into the shoots already at 240 μmol dm−3 Cd(NO3)2, which confirms the existence of some barriers in the roots. Bioaccumulation factor (BAF) for root depending on Cd concentration ranged from 565 to 357 and for shoot it was 47 to 22. Percent of Cd in the shoot was 27.8–29.0. On the other hand, for cv. Primorska, at increasing applied Cd concentrations, the Cd translocation from the roots into the shoots increased in the whole applied Cd concentration range. BAF for the root ranged from 817 to 419 and for the shoot from 30 to 81. The percent of Cd in the shoot was 9.4–41.0. On the basis of the results and definition of the Cd hyperaccumulators [222], it could be concluded that both studied cultivars belong to the category of hyperaccumulators of cadmium because they accumulated more than 100 μg g−1 of Cd in the shoots. However, cv. Primorska is more effective mainly for the translocation of cadmium into the aboveground part of plants. Our 5000 Cd in shoots (mg kg–1 d.m.)

1 4000 3000 2000 2

1000 0

0

100

200 300 c (μmol dm–3)

400

500

Figure 24.12  Dependence of Cd content in shoots of Salvia officinalis plants on the concentration of Cd(NO3)2 in the hydroponics (1, cv. Primorska; 2, cv. Krajova).

Plant Responses to Toxic Metal Stress

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findings for both the studied cultivars of Salvia officinalis, cv. Primorska and cv. Krajova, did not support the results of Marquard and Schneider [193], which characterized this medicinal plant species as an excluder of Cd.

Acknowledgments The authors wish to thank the Journal of Plant Physiology (Elsevier Publisher) for providing us with the original data and figures published in this journal (Ref. [21]). We are also grateful to SanofiAventis Pharma Slovakia for financial support.

References





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164. Yadav, P. and A.K. Srivastava. 1998. Cadmium induced mitotic anomalies in Hordeum vulgare and Setaria italica. J. Environ. Biol. 19:25–32. 165. Ivanov, V.B., E.I. Bystrova, and I.V. Seregin. 2003. Comparative impacts of heavy metals on root growth as related to their specificity and selectivity. Russ. J. Plant Physiol. 50:398–406. 166. Masarovičová, E., M. Peško, and K. Král’ová. 2009. Negative effect of abiotic factors on rapeseed growth. In 29th International Symposium on Industrial Toxicology 09, Svit, Slovak Republic, June 16–18, 2009, ed. V. Koprda and F. Čacho, pp. 107–111. Bratislava, Slovak Republic: Slovak Technical University Press. 167. Sagardoy, R., F. Morales, A.F. López-Millán, A. Abadía, and J. Abadía. 2009. Effects of zinc toxicity on sugar beet (Beta vulgaris L.) plants grown in hydroponics. Plant Biol. 11:339–350. 168. Sunakar, P. and P. Sumita. 2009. Impact of lead ion on the stability of lipid-protein organization of photosynthetic organell. Res. J. Biotech. 4:57–62. 169. Chu, L., D.Y. Liu, Y.B. Wang, J.H. Ding, and L.L. Wang. 2006. Separate and combined effects of Cu and Cd on seedling growth and active oxygen metabolism system of Trifolium repens L. Front. Biosci. 11:2861–U68 170. Souza, J.F., H. Dolder, and A.L. Cortellazzo. 2005. Effect of excess cadmium and zinc ions on roots and shoots of maize seedlings. J. Plant Nutr. 28:1923–1931. 171. Weryszko-Chmielewska, E. and M. Chwil. 2005. Lead-induced histological and ultrastructural changes in the leaves of soybean (Glycine max (L.) Merr.). Soil Sci. Plant Nutr. 51:203–212. 172. Llamas, A., C.I. Ullrich, and A. Sanz. 2000. Cd2+ effects on transmembrane electrical potential difference, respiration and membrane permeability of rice (Oryza sativa L) roots. Plant Soil 219:21–28. 173. Burzynski, M. and J. Buczek. 1994. The influence of Cd, Pb, Cu and Ni on NO3− uptake by cucumber seedlings. 1. Nitrate uptake and respiration of cucumber seedlings roots treated with Cd, Pb, Cu and Ni. Acta Physiol. Plant. 16:291–296. 174. Seregin, I.V. and V.B. Ivanov. 2001. Physiological aspects of cadmium and lead toxic effect on higher plants. Russ. J. Plant Physiol. 48:523–544. 175. Bertrand, M. and J.C. Guary. 2002. How plant adopt their physiology to an excess of metals. In Handbook of Plant and Crop Physiology, 2nd edn, ed. M. Pessarakli, pp. 751–761. New York: Marcel Dekker Inc. 176. Mishra, S. and R.S. Dubey. 2005. Heavy metal toxicity induced alterations in photosynthetic ­metabolism in plants. In Handbook of Photosynthesis, 2nd edn, ed. M. Pessarakli, pp. 845–863. Boca Raton, FL: Taylor & Francis Group. 177. Shukla, I., J. Singh, P. Joshi, and P. Kakkar. 2003. Effect of bioaccumulation of cadmium on biomass productivity, essential trace elements, chlorophyll biosynthesis, and macromolecules of wheat seedlings. Biol. Trace Element Res. 92:257–273. 178. Garcia, W.J., Blessin, CH.W., Inglett, G.E., Kwolek, W.F., Carliste, J.N., Hughes, L.N., and Meister, J.F. 1981. Metal accumulation and crop yield for a variety of edible-crops grown diverse soil media amended with sewage sludge. Environ. Sci. Technol. 15:793–804. 179. Wanga, M., Zoua, J., Duana, X., Jianga, W. 2007. Cadmium accumulation and its effects on metal uptake in maize (Zea mays L.). Bioresource Technol. 98:82–88. 180. Shanker, A.K., C. Cervantes, H. Loza-Tavera, and S. Avudainayagam. 2005. Chromium toxicity in plants. Environ. Int. 31:739–753. 181. Xu, J.K., L.X. Yang, Z.Q. Wang, G.C. Dong, J.Y. Huang, and Y.L. Wang. 2005. Effects of soil copper concentration on growth, development and yield formation of rice (Oryza sativa). Rice Sci. 12:125–132. 182. WWF-UK, August 2002. Fact Sheet 1: Towards Sustainable Herbal Medicine. 183. WWF-UK, August 2002. Fact Sheet 2: Cultivation versus Wild Harvesting of Medicinal Plants: Is Cultivation The Sole Solution? 184. Kozlowski, R., P. Braniecki, and M. Mackiewicz-Talarczyk. 2004. Report from the State of Poland. Forming Part of the IENICA-INFORRM Project. Poznan. 185. Král’ová, K. and E. Masarovičová. 2006. Plants for the future. Ecol. Chem. Eng. 13:1179–1207. 186. Masarovičová, E. and K. Král’ová. 2007. Medicinal plants—Past, nowadays, future. Acta Hort. (ISHS) 749:19–27. 187. Nahrstedt, A. and V. Butterweck. 1997. Biologically active and other chemical constituents of the herb of Hypericum perforatum L. Pharmacopsychiatry 30S:129–134. 188. Bilia, A.R., S. Gallori, and F.F. Vincier. 2002. St. John’s wort and depression. Efficacy safety and tolerability—An update. Life Sci. 70:3077–3096. 189. Král’ová, K. and E. Masarovičová. 2004: Could complexes of heavy metals with secondary metabolites induce enhanced metal tolerance of Hypericum perforatum? In Macro and Trace Elements. Mengen- und Spurenelemente. 22. Workshop, Jena, Germany, September 24–25, 2004, ed. M. Anke et al., pp. 411–416. Jena, Germany: Friedrich Schiller Universität.

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190. Falk, H. and W. Schmitzberger. 1992. On the nature of soluble hypericin in Hypericum species. Monatsh. Chem. 123:731–739. 191. Falk, H. and E. Mayr. 1997. Concerning bay salt and peri chelate formation of hydroxyphenanthroperylene quinones (fringelites). Monatsh. Chem. 128:353–360. 192. Palivan, C.G., G. Gescheidt, and L. Weiner. 2001. The formation of copper complexes with hypericin, in solutions: An EPR Study. J. Inorg. Biochem. 86:369–369. 193. Marquard, R. and M. Schneider. 1998. Zur Cadmiumproblematik im Arzneipflanzenbau. In Fachtagung Arznei- und Gewűrzpflanzen, Giessen, Germany, October 1–2, 1998, ed. R. Marquard and E. Schubert, pp. 9–15. 194. Masarovičová, E., K. Král’ová, F. Šeršeň, A. Bumbálová, and A. Lux. 1999. Effect of toxic metals on medicinal plants. In Mengen- und Spurelemente. 19. Arbeitstagung, Jena, February 3–4, 1999, ed. M. Anke et al., pp. 189–196. Leipzig, Germany: Verlag Harald Schubert. 195. Král’ová, K., E. Masarovičová, and A. Bumbálová. 2000. Toxic effect of cadmium on Hyperiucum perforatum plants and green alga Chlorella vulgaris. Chem. Inz. Ekol. 7:1200–1205. 196. Murch, S.J., K. Haq, H.P.V. Rupasinghe, and P.K. Saxena. 2003. Nickel contamination affects growth and secondary metabolite composition of St. John’s wort (Hypericum perforatum L.). Environ. Exp. Bot. 49:251–257. 197. Pandey, S., K. Gupta, and A.K. Mukherjee. 2007. Impact of cadmium and lead on Catharanthus roseus— A phytoremediation study. J. Environ. Biol. 28:655–662. 198. Grejtovský, A. and R. Prič. 2000. The effect of high cadmium concentration in soil on growth, uptake of nutrient and some heavy metals on Chamomilla recutita (L.) Rauschert. J. Appl. Bot., Angew. Bot. 74:169–174. 199. Masarovičová, E., K. Král’ová, and V. Streško. 2003. Effect of metal ions on some medicinal plants. Chem. Inz. Ekol. 10:275–279. 200. Eliáš, P. 1994. Research of flora and vegetation of settlements (towns, villages, castle ruins) in Slovakia (in Slovak). Zpravodaj Čes. Bot. Spol. 29:45–75. 201. Karmazín, M., J. Hubík, and J. Dušek. 1984. Catalog of Medicaments of Plant Origin (in Czech), 5th edn. Praha, Czech Republic: VHJ Spofa. 202. Magiatis, P., A. Michaelakis, A.L. Skaltsounis, and S.A. Haroutounian. 2001. Volatile secondary metabolite pattern of callus cultures of Chamomilla recutita. Nat. Product Lett. 15:125–130. 203. Repčák, M., A. Eliášová, and A. Ruscančinová. 1998. Production of herniarin by diploid and tetraploid Chamomilla recutita. Pharmazie 53:278–279. 204. Schlicher, H. 1973. Neuere Erkenntnisse bei der Qualitätsbeurteilung von Kamillenbluten bzw, Kamillenöl. Teil 2: Qualitative Beurteilung des Ätherischen Öles in Flores Chamomillae. Aufteilung der Handelskamillen in vier, bzw. fünf chemischen Typen. Planta Medica 28:133–144. 205. Šalamon, I., K. Král’ová, and E. Masarovičová. 2007. Accumulation of cadmium in chamomile plants cultivated in Eastern Slovakia regions. Acta Hort. (ISHS) 749:217–222. 206. Pavlovič, A., E. Masarovičová, K. Král’ová, and J. Kubová. 2006. Response of chamomile plants (Matricaria recutita L.) to cadmium treatment. Bull. Environ. Contam. Toxicol. 77:763–771. 207. Linkeš, V., J. Kobza, M. Švec et  al. 1997. Soil Monitoring in Slovakia. Actual State of the Soils in 1992–1996 (in Slovak). Bratislava, Slovak Republic: Research Institute of the Soil Fertility. 208. Murphy, A. and L. Taiz. 1995. A new vertical mesh transfer technique for metal tolerance studies in Arabidopsis ecotypic variation and copper-sensitive mutants. Plant Physiol. 108:29–38. 209. Küpper, H., E. Lombi, F. Zhao, and S.P. McGrath. 2000. Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri. Planta 212:75–84. 210. Jakovljevic, M., S. Antic-Mladenovic, M. Ristic, S. Maksimovic, and S. Blagojevic. 2000. Influence of selenium on the yield and quality of chamomile (Chamomilla recutita (L.) Rausch.). Rostlinná VýrobaPlant Production 46:123–126. 211. Král’ová, K., E. Masarovičová, I. Ondrejkovičová, and M. Bujdoš. 2007. Effect of selenium oxidation state on cadmium translocation in chamomile plants. Chem. Pap. 61:171–175. 212. Lešíková, J., K. Král’ová, E. Masarovičová, J. Kubová, and I. Ondrejkovičová. 2007. Effect of different cadmium compounds on chamomile plants. Acta Hort. (ISHS) 749:223–229. 213. Shanker, K., S. Mishra, S. Srivastava, R. Srivastava, S. Dass, S. Prakash, and M.M. Srivastava. 1996. Effect of selenite and selenate on plant uptake of cadmium by maize (Zea mays). Bull. Environ. Contam. Toxicol. 56:419–424. 214. Whanger, P.D. 1992. Selenium in the treatment of heavy-metal poisoning and chemical carcinogenesis. J. Trace Elem. Electrolytes Health Dis. 6:209–221. 215. Nowack, B., R. Schulin, and B.H. Robinson. 2006. A critical assessment of chelant- enhanced metal phytoextraction. Environ. Sci. Technol. 40:5225–5232.

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216. Mugnai, S., E. Azzarello, C. Pandolfi, and S. Mancuso. 2006. Zinc and cadmium accumulation in Hyssopus officinalis L. and Satureja montana L. Acta Hort. (ISHS) 723:361–366. 217. Král’ová, K., E. Masarovičová, J. Kubová, and O. Švajlenová. 2007. Response of Matricaria recutita plants to some copper(II) chelates. Acta Hort. (ISHS) 749:237–243. 218. Král’ová, K. and E. Masarovičová. 2008. EDTA-assisted phytoextraction of copper, cadmium and zinc using chamomile plants. Ecol. Chem. Engin. 15:213–220. 219. Langer, R., C. Mechtler, and J. Jurenitsch. 1996. Composition of essential oils of commercial samples of Salvia officinalis L. and S. fruticosa Mill.: A comparison of oils obtained by extraction and steam distillation. Phytochem. Anal. 7:289–293. 220. Perry, N.B., R.E. Anderson, N.J. Brennan, M.H, Douglas, A.J. Heaney, J.A. McGimpsey, and B.M. Smallfield. 1999. Essential oils from dalmatian sage (Salvia officinalis L.): Variations among individuals, plant parts, seasons, and sites. J. Agr. Food Chem. 47:2048–2054. 221. Masarovičová, E., K. Král’ová, and V. Streško. 2004. Comparative study of uptake, accumulation and some effects of cadmium in two cultivars of Salvia officinalis L. Chem. Inz. Ekol. 11:209–214. 222. Baker, A.J.M. 1995. Metal hyperaccumulation by plants: Our present knowledge of the ecophysiological phenomenon. In Will Plants Have a Role in Bioremediation? ed. D. Randall, I. Raskin, A.J.M. Baker, D. Blevins, and R. Smith, pp. 7–8. Columbia, MO: University of Missouri.

Metal Pollution: 25 Heavy Damage and Defense Strategies in Plants Flavia Navari-Izzo and Nicoletta Rascio Contents 25.1 Introduction........................................................................................................................... 635 25.2 Heavy Metal Toxicity in Plants............................................................................................. 636 25.2.1 Reactive Oxygen Species Production........................................................................ 636 25.3 Metal Tolerance..................................................................................................................... 638 25.3.1 Mechanisms of Metal Tolerance in Plants................................................................. 638 25.3.1.1 Cellular Antioxidant Defense against Oxidative Stress Induced by Metals...............................................................................638 25.3.1.2 Metal Compartmentation............................................................................ 643 25.4 Chelation of the Metals by Ligands.......................................................................................644 25.4.1 Organic Acids and Amino Acids...............................................................................644 25.4.2 Root Exudates............................................................................................................ 645 25.4.3 Phytochelatins............................................................................................................646 25.4.4 Metallothioneins........................................................................................................649 25.5 Mycorrhizas and Heavy Metal Tolerance.............................................................................. 650 25.6 Heavy Metal Hyperaccumulation.......................................................................................... 651 25.6.1 Hyperaccumulator Plants........................................................................................... 651 25.6.2 Enhanced Heavy Metal Uptake................................................................................. 654 25.6.3 Improved Root to Shoot Heavy Metal Translocation................................................ 655 25.6.4 Heavy Metal Detoxification in Leaves...................................................................... 657 25.7 Conclusions............................................................................................................................ 659 References.......................................................................................................................................660

25.1  Introduction In the literature the term “heavy metal” is used with a very broad and misleading meaning but in a strict sense it includes a group of metals with density higher than 5.0 g cm−3 and an atomic weight above 20. Actually, there is a strong tendency to classify metals according to their propensity to interact with biological ligands, which largely defines their toxicity. This chapter speaks of heavy metals as metals and metalloids that are toxic to plants. Phytotoxic amounts of heavy metals are occasionally found in soils under natural conditions but more frequently they are mobilized and released by technological and agricultural activities and tend to persist indefinitely, circulating and eventually accumulating throughout the food chain, thus posing a serious threat to animals, humans, and the environment. In Europe, the polluted agricultural lands likely encompass several million hectares (Flathman and Lanza, 1998). Some heavy metals (Mn, Fe; Cu, Zn, Mo, and Ni) are essential nutrients necessary for the normal growth of plants but 635

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when present at supra-optimal concentrations they can easily become phytotoxic. Of major concern with respect to plant exposure as well as accumulation in the human food chain are the metalloid arsenic (As), and the metals cadmium (Cd), mercury (Hg), and lead (Pb) (McLaughlin et al., 1999). The similarity to essential elements makes these nonessential elements potentially toxic to plants. In the environment, plants cannot move and can encounter elevated levels of both essential and nonessential metals much more than other organisms. Toxicity symptoms may derive from interactions at cellular/molecular level, such as blocking functional groups of biologically important molecules, displacing and/or substituting essential elements, inactivating enzymes, and disrupting cell and organelle membrane integrity. The high amount of metals in the tissue of hyperaccumulator plants suggests the existence of defense mechanisms to avoid the harmful effects caused by metals. These mechanisms are quite complex and their importance may vary in accordance with the metal, its concentration, the species, and even the plant organs and stages of development in the same plant, etc. This chapter focuses on responses of plants to toxic metals and the metalloid As exposure, and on mechanisms of tolerance that help plants to maintain growth avoiding interferences with the normal cellular metabolism.

25.2  Heavy Metal Toxicity in Plants At low metal concentrations the plant cell can resort to a number of avoidance mechanisms such as metal exclusion, translocation, and complexation in the cytoplasm (Vansgroveld and Clijsters, 1994). At high concentrations, when primary barriers are broken down, avoidance is insufficient, free metal concentration increases, and both redox and nonredox metals can stimulate production of reactive oxygen species (ROS) imposing oxidative stress (Aust et al., 1998; Navari-Izzo et al., 1998, 1999; Quartacci et al., 2001). Although O2 itself is not a harmful molecule, it can potentially be reduced to form toxic ROS. In the plant system, including algae, ROS are always formed by the inevitable leakage of electrons into molecular oxygen from the electron transport activities of chloroplasts, mitochondria, and plasma membranes. A growing body of evidence indicates that various toxic metals act as catalyst in the oxidative deterioration of biological macromolecules, and therefore the toxicities associated with these metals may be due, at least in part, to oxidative damage to the tissues. Actually, metals such as Cr, Cu, Fe, Hg, Ni, Pb, and V exhibit the ability to increase the normal production of ROS, resulting in lipid peroxidation (Quartacci et al., 2001), DNA damage, depletion of sulfhydryl groups, and altered calcium homeostasis (Stohs and Bagchi, 1995). There is significant evidence that exposure to the metalloid As also enhances the production of ROS, leading to membrane damage through the peroxidation of membrane lipids (Hartley-Whitaker et al., 2001a).

25.2.1  Reactive Oxygen Species Production Redox metals, performing one-electron oxireduction reactions, can easily catalyze the formation of free radical through a Fenton-type reaction (e): (a) Me( n −1) + + O2 → Me( n −1) +  O2 → Me n + + O2• − In aqueous solutions at neutral pH, O2• − can generate H2O2 (b) 2O2•− + 2H + → H 2O2 + O2 which can decompose to produce the Haber-Weiss reaction (c): (c) H 2O2 + O2• − → O2 + • OH + − OH (d) Me n + + O2• − → Me( n −1) + + O2 (e) H2O2 + Me(n−1)+ → Men+ + •OH + −OH Cycling of redox active metals, such as Cu and Fe, at/or near the binding sites of cell membrane constituents may also lead to a site-specific production of hydroxyl radicals (•OH) via Haber–Weiss

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reaction (c) (Chevion, 1988). In PSII isolated from wheat grown in excess Cu, the production of harmful oxygen species, such as hydroxyl radicals arising from superoxide has been demonstrated (Navari-Izzo et al., 1998, 1999). However, the kind of free radicals involved in DNA-strand cleavage is still a controversial problem. In fact, some authors suggested that singlet oxygen rather than hydroxyl radical may play a role in the induction of DNA strand breaks (Li and Trush, 1993) while others reported the involvement of both forms (Yamamoto and Kawanishi, 1989). As Hg2+ cannot replace Cu+ or Fe2+ in the Fenton reaction a different mechanism should cause an accumulation of ROS (Cho and Park, 2000; Patra et al., 2004; Han et al., 2007). The Hg2+ ions bind avidly to –SH groups and likely inhibit the activities of antioxidative enzymes especially of glutathione reductase (GR), and also raise a transient depletion of reduced glutathione (GSH) (Lomonte et al., 2010). Since glutathione is of pivotal importance for the redox status of the cells (Foyer et al., 1997) it may be guessed that a short-term depletion of GSH would have as consequence a natural accumulation of ROS. Mercury also can displace Cu2+ ions from metallothioneins in vitro, which might potentiate oxidative damage if it occurs in vivo. The question rises whether nonredox metals, which are incapable of univalent oxireduction reactions, are capable of producing ROS. In animal tissues, it has been demonstrated that Cd induces changes in the antioxidant status by either increasing superoxide radical production and lipid peroxidation or by decreasing enzymatic and nonenzymatic antioxidant levels (Stohs and Bagchi, 1995). Evidences have been reported that Cd leads to oxidative stress acquired in tolerant and sensitive clones of Holcus lanatus (Hendry et al., 1992) and in germinating seedlings of Phaseolus vulgaris (Somashekaraiah et al., 1992). In contrast with other heavy metals such as Cu or Fe, Cd does not directly produce ROS via Fenton and/or Haber–Weiss reactions but the production of ROS in plants may result from the activity of redox enzymes bound or associated to the plasma membrane of the cells. The induction of lipoxygenase (LOX, EC 1.13.11.12) in the presence of nonredox metals might explain its effect on the cellular redox status possibly related •− to the ability of this enzyme to produce superoxide anion ( O2 ) by oxidizing NADPH (Roy et al., 1994; Quartacci et al., 2001). LOX mediates polyunsaturated fatty acid oxidation and produces free radicals from fatty acids which in turn cause the destruction of the membranes. The iron-containing LOX is known to initiate lipid peroxidation (Thompson et al., 1987), and a high level of Zn promotes free radical generation and hence peroxidative degradation of polyunsaturated fatty acids (Weckx and Clijsters, 1997). The increase in free radical production could be due to interference of Zn with the normal functioning of electron transport chains in mitochondria and chloroplasts. In fact, heavy metals, including Zn, have been reported to suppress electron transport chain associated with these organelles (Weckx and Clijsters, 1997). Excess of metal ions blocks the electron flow in PSII (Pagliano et al., 2006) which leads to the formation of excess energy which in its turn causes production of ROS. Therefore, damage to biomembranes by lipid peroxidation might not be limited to redox metal only. Recently, it became evident that ROS play a dual role in plants both as toxic compounds and as key regulators of many biological processes. The identification of ROS-generating enzymes such as plant homolog of respiratory burst NADPH oxidase (Rboh) demonstrates that plant cells can initiate and most likely amplify ROS production for signaling (Miller et al., 2008). An oxidative burst can be observed in plants as an early response to pathogen attack (Frahry and Schopfer, 2001). O2•−, released in the apoplast by NAD(P)H oxidase, dismutates to H2O2 spontaneously and this reaction is enhanced by superoxide dismutase (SOD, EC 1.15.1.1) that has been also shown to be present in this compartment (Ogawa et al., 1996). The release of O2•− and H2O2 in the apoplastic space is believed to contribute to several disease resistance strategies in plants. ROS, and, in particular H2O2, have been considered as signal molecules in the environmental stress response since they induce the expression of a variety of defense-related genes (Foyer et al., 1997). However, very little is known in the case of excess of metals. In wheat seedlings, excess Cu induced a biphasic increase of O2•− in the apoplast. A very high SOD activity in the apoplast dismutated the superoxide anion giving rise to an increase in H2O2. The highest value of H2O2 was detected at 15 min of Cu exposure when peroxidase (POD, EC 1.11.1.9) activity reached the lowest value (Sgherri et al., 2007). Furthermore, in

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Brassica napus, a link between lipid signals and redox antioxidative signals was found as an early response to a treatment with excess Cu (Russo et al., 2008).

25.3  Metal Tolerance In order to minimize the negative effects of nonessential toxic metal and to maintain the concentration of essential metals within physiological limits, plants, as have other organisms, have evolved a range of homeostatic mechanisms to control the uptake, accumulation, and transport of the metals to possess a basic metal tolerance (Clemens, 2001). Only certain plants species and genotypes posses a naturally selected hyper tolerance toward particular metals different from the basal tolerance common to all plant species and varieties. Metal hypertolerance or tolerance in plants is the ability of certain plants to survive and reproduce in metal-rich soils with concentrations of metals toxic to other plants (MacNair and Baker, 1994), which is different from the basic normal tolerance held by all plants (Clemens, 2006). Basic strategies include exclusion, efflux of toxic metal ions, immobilization, compartmentalization and metal chelation, reduction of metal transport, and expression of other general stress response mechanisms. In addition, metallothioneins (MTs) and phytochelatins (PCS) are molecular components which play a significant role in tolerance. In metal tolerance test root growth is the most widely used parameter and the quantification of tolerance index (TI) in plants has been based on the use of relative measurements of the rates of root growth of plants tested in control and in metal-exposed plants (Wilkins, 1978; Baker, 1987). On this basis, a tolerance index (TI) can be calculated: TI (%) =

root growth in solution with metal root growth in solution without metal

However, there are some limitations for the use of TI (%) (MacNair, 1981, 1983; Verkleij and Prast, 1989; Schat and Ten Bookum, 1992). An important generalization is that due to the distinct chemical properties of the various metals, the tolerance machineries are essentially metal-specific. Higher plants employ two basic strategies to tolerate heavy metals: (1) avoidance or exclusion, which restricts the uptake and/or root to shoot transport; and (2) accumulation and sequestration, which allow plants to survive accumulation and detoxify metals in the shoots by compartmentation of metals in the vacuole, by complexation of metals by organic ligands such as organic acids, amino acids, and metal-binding peptides (Baker, 1981; Clemens, 2001; Hall, 2002). Metal exclusion is by far the most common strategy in metal tolerant species. De Vos et al. (1991) compared copper uptake in roots of two copper-tolerant Silene vulgaris populations from copper mines and a copper sensitive population from a nonmetalliferous site. The net copper uptake at the same external copper concentration was found to be inversely related to the copper tolerance level. This suggests that a reduced uptake and/or an increased efflux of copper across the root plasma membrane might contribute to the higher tolerance of the mine plants. However, if compared at their own no-effect concentration (NEC), 50%-effect concentration (EC50) or 100%-effect concentration (EC100) values, this relationship is reversed meaning that the maximum tolerable Cu uptake rates are manifold higher in hypertolerant S. vulgaris than in sensitive one (Schat and Kalff, 1992). Therefore, adaptative Cu hypertolerance must rely on an increased capacity to sequester the metals inside the plant (Verkleij, 2008). On the other hand, metal accumulation is a rare phenomenon on terrestrial higher plants. To date, about 450 plants have been identified as metal hyperaccumulators, representing less than 0.2% of all angiosperms.

25.3.1  Mechanisms of Metal Tolerance in Plants 25.3.1.1  Cellular Antioxidant Defense against Oxidative Stress Induced by Metals The induction of the activity of particular groups of enzymes as well as reducing metabolites is considered to play an important role in the cellular defense strategy against oxidative stress.

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Hydrophilic antioxidants such as reduced glutathione (GSH) and ascorbate (AsA) participate in the defense in the aqueous phase, while the lipophilic tocopherols and carotenoids fulfill essential antioxidant action in membranes. These antioxidants can be regenerated through the glutathioneascorbate cycle which utilizes NADPH as reducing agent (Sgherri and Navari-Izzo, 1995). In addition, lipoic acid (LA), due to its solubility in both water and lipid phases, connects the activity of antioxidants in the cell membrane (tocopherols) with antioxidants in the cytoplasm (AsA and GSH), strengthening the antioxidant network (Navari-Izzo et al., 2002; Sgherri et al., 2002) (Figure 25.1). LA is unique among antioxidant molecules in that it retains protective functions in both its reduced (DHLA) and oxidized forms (LA) although DHLA is the more effective in performing antioxidant functions (Navari-Izzo et al., 2002). DHLA acts directly by scavenging O2•−, hydroperoxyl, and hydroxyl radicals and can also donate an electron to oxidised glutathione (GSSG) and dehydroascorbate (DHA).

Membrane lipids

PUFA ROS

Scavenging

PUFA· (A) PUFA

Tocopherol

Interplay

GSSG

Interplay

DHLA

GSH

Tocopheroxyl radical

LA

Membrane lipids

PUFA ROS

Scavenging

PUFA·

Tocopherol

PUFA

Tocopheroxyl radical

Interplay

Disproportionation

MDA

MDA

AsA

DHA Interplay

LA

(B)

DHLA

Membrane lipids

PUFA ROS PUFA·

Tocopherol

MDA APX MDHAR

PUFA

Tocopheroxyl radical

AsA

MDA DHA DHAR

DHLA (C)

LA

GSH GSSG

GR

NADP+ NADPH

FIGURE 25.1  Diagrams showing the scavenging of lipid peroxyl radicals formed in membrane, the regeneration of tocopheroxyl radical by glutathione (A) or ascorbate (B) and the interplay of lipoic acid in the regeneration of ascorbate and glutathione (C). APX, ascorbate peroxidase; AsA, reduced ascorbate; DHAR, dehydroascorbate reductase; DHA, dehydroascorbate; DHLA, dehydrolipoic acid; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; LA, lipoic acid; MDA, monodehydroascorbate; MDHRA, monodehydroascorbate reductase; PUFA, polyunsaturated fatty acids; PUFA., lipid peroxyl radical.

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SOD, ( EC 1.15.1.1), the first enzyme in the detoxification process, converts O2•− to H2O2. Catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.7) and a variety of general peroxidases catalyze the breakdown of H2O2 (Navari-Izzo et al., 1997; Navari-Izzo and Rascio, 1999). To regenerate oxidized ascorbate, plant cells use monodehydroascorbate reductase (MDHA, EC 1.6.5.4), dehydroascorbatereductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2). Glutathione peroxidases (GPs, EC 1.11.1.9) and glutathione transferases (GSTs, EC 2.5.1.18), also contribute to the redox status maintenance of the cells. Under normal conditions the balance between the generation of ROS and the mechanisms that protect cells from the action of ROS is tightly controlled, but in presence of metal excess an increase in the level of ROS and a decrease in the detoxification mechanisms can lead to degradation of biomembranes (Quartacci et al., 2000, 2001; Berglund et al., 2002; Calucci et al., 2003). In the presence of excess metals, cells may attempt to prevent the formation of ROS by increasing the production of metal-binding compounds and/or by scavenging them via antioxidative defense systems. In hyperaccumulator plants evidences for a role of antioxidative defenses are scanty and do not provide a basis sufficient for defining direct relationships (Cuypers et al., 1999; Schickler and Caspi, 1999). In recent years however, in metal-accumulator species, the activities of SOD, CAT, and POD were shown to be enhanced upon Cd exposure (Mobin and Khan, 2007; Sun et al., 2007a,b) and Wang and collaborators (2008) found that compared to tobacco plants, metal accumulator plants are equipped with superior antioxidative defense to adapt to the oxidative stress induced by Cd toxicity. Overexpression of enzymes involved in antioxidative defenses seems a useful approach to obtain metal tolerance. The transformed accumulator Indian mustard (Brassica juncea) overexpressing glutathione synthetase increased the biosynthesis of glutathione enhancing Cd tolerance and accumulation more than the nontransformed plants (Zhu et al., 1999), while GR overexpression in the plastids led to increased Cd tolerance at chloroplast level but decreased GSH accumulation in the shoots (Pilon-Smith et al., 2000). In Arabidopsis the overexpression of glutathione transferase and peroxidase resulted in enhanced Al tolerance (Ezaki et al., 2000), and increased transcript levels of APX, CAT, and GP in Hordeum vulgare subjected to Cd stress have been reported (Finkemeier et al., 2003; Metwally et al., 2003; Sharma et al., 2004). There are conflicting reports about nonhyperaccumulator plants; these reports depend on the plant species and tissue analyzed, growth conditions, the metal used for the treatment and its concentrations as well as the organelle taken into consideration (Table 25.1). Therefore, the observations reported throughout the text may depend on the experimental design and are likely to be element- and species-dependent. Subsymptomatic concentrations of Cu or Cd increased CAT activity in a sensitive cv of wheat, while a more tolerant cv responded with the induction of POD (Sgherri et al., 2001; Milone et al., 2003). Increase in CAT activity was also observed in pea plants grown for 18 and 33 days under toxic concentrations of Mn or Zn (Del Rio et al., 1985). In P. vulgaris, Cd induced POD but decreased CAT activities (Shaw, 1995). CAT activity decreased also in Bacopa monnieri where the activities of SOD and POD were instead enhanced by Cd (Singh et al., 2006). In pea the Mn-SOD and CAT of peroxisomes were found to be higher in Cu-tolerant than in Cu-sensitive plants, suggesting a protective role of these enzymes in Cu-induced oxidative stress in pea leaf peroxisomes (Palma et al., 1987). On the contrary, in pea plants both SOD and CAT activities as well as accumulation of transcripts were decreased by Cd, while POD did not change (Romero-Puertas et al., 2007). However, in Solanum nigrum the activities of the three enzymes were enhanced by Cd exposure (Mobin and Khan, 2007; Sun et al., 2007) suggesting species-specific differences as response to Cd toxicity. In both thylakoids and PSII particles of wheat treated with 50 μM Cu, a higher production of superoxide was monitored together with an induction in thylakoid-bound CuZn-SOD and APX. The activity of the stromal enzymes SOD, APX, and MDHA was also increased, thus suggesting that Cu might have induced a general response in the biosynthesis of both thylakoids and stromal •− enzymes by directly influencing their gene expression (Navari-Izzo et al., 1998). In this way, O2 and H2O2 could be efficiently removed, and possible formation of •OH, highly toxic for biological membranes, avoided. In Helianthus annuus Fe, Cu or Cd decreased the activity of CAT, APX, GR,

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TABLE 25.1 Trend of Activities of Antioxidative Enzymes in Different Plant Species Subjected to Toxic Metals Metal As

Cu

Cd

Species

Enzyme Activitya

Holcus lanatus (As-tolerant) Holcus lanatus (As-sensitive) Hydrilla verticillata Nephrolepsis exaltata Pteris eusiformis Pteris vittata Brassica juncea Helianthus annuus Triticum durum (drought-sensitive) Triticum durum (drought-tolerant) Triticum durum

SOD (+) SOD (−) APX (+), CAT (+), GPX (+), GR (+), SOD (+) APX (=), CAT (+), GPX (+), GR (+), SOD (+) APX (+), CAT (+), GPX (+), GR (+), SOD (+) APX (+), CAT (+), GPX (=), GR (=), SOD (+) APX (+), CAT (−), GPX (+), SOD (+) APX (−), CAT (−), DHA (−), GR (−), SOD (+) APX (−), CAT (+), GPX (=), SOD (−)

Hartley-Whitaker (2001) Hartley-Whitaker (2001) Srivastava et al. (2007) Srivastava et al. (2005) Srivastava et al. (2005) Srivastava et al. (2005) Wang et al. (2004) Gallego et al. (1996) Sgherri et al. (2001)

APX (+), CAT (−), GPX (+), POD (+), SOD (=) Stromal-APX (+), -MDHA (+), -SOD (+), Thylakoid-APX (+), -Cu-ZnSOD (+) APX (+), GR (−), SOD (+) CAT (−), POD (+), SOD (+) CAT (+), SOD (+) CAT (+), POD (+), SOD (+) APX (−), CAT (−), DHA (−), GR (−), POD (+), SOD (−) CAT (−), POD (+), SOD (+) APX (+), CAT (−), GPX (+), GR (+), POD (+) APX (+), CAT (−), GPX (+) CAT (−), POD (=), SOD (−)

Sgherri et al. (2001)

Alyssum argenteum Bacopa mounieri Brassica juncea Brassica juncea Helianthus annuus Nicotiana tabacum Phaseolus vulgaris Phaseolus vulgaris Pisum sativum

Fe Hg

Mn Ni Zn

Solanum nigrum Thlaspi caerulescens Triticum durum (drought-sensitive) Triticum durum (drought-tolerant) Helianthus annuus Atriplex codonocarpa Lycopersicon esculentum Phaseolus aureus Pisum sativum Alyssum argenteum Alyssum maritimum Phaseolus vulgaris Pisum sativum

References

Navari-Izzo et al. (1998) Schickler and Caspi (1999) Singh et al. (2006) Wang et al. (2008) Mobin and Khan (2007) Gallego et al. (1996)

CAT (+), POD (+), SOD (+) CAT (+), SOD (+) APX (−), CAT (+), GPX (=), SOD (−)

Wang et al. (2008) Chaoui et al. (1997) Shaw (1995) Romero-Puertas et al. (2007) Sun et al. (2007) Wang et al. (2008) Milone et al. (2003)

APX (+), CAT (+), GPX (+), SOD (=)

Milone et al. (2003)

APX (−), CAT (−), DHA (−), SOD (−) APX (+), GR (−), SOD (+) CAT (+), SOD (+), POD (=) CAT (+), POD (+) CAT (+) APX (+), GR (+), SOD (−) APX (+), GR (+), SOD (+) APX (+), CAT (−), GPX (+), GR (+), POD (+) CAT (+)

Gallego et al. (1996) Lomonte et al. (2010) Cho and Park (2000) Shaw (1995) Del Rio et al. (1985) Schickler and Caspi (1999) Schickler and Caspi (1999) Chaoui et al. (1997) Del Rio et al. (1985)

Note: APX, ascorbate peroxidase; CAT, catalase; DHA, dehydroascorbate reductase; GPX, guaiacol peroxidase; GR, glutathione reductase; MDHA, monodehydroascorbate reductase; POD peroxidase; SOD, superoxide dismutase. a In comparison to control: +, increase; −, decrease; =, no change.

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and DHAR, while enhanced LOX activity, thus provoking lipid peroxidation. In the same plants, Fe and Cd ions caused a decrease in SOD activity, while Cu raised SOD levels (Gallego et al., 1996). Upon Cd exposure, lipid peroxidation was raised in pea plants (Lozano-Rodriguez et al., 1997), in Phaseolus aureus (Shaw, 1995) and in P. vulgaris (Chaoui et al., 1997), whereas in Daucus carota no lipid peroxidation was observed (Sanità di Toppi and Gabbrielli, 1999). The enzymes of the ascorbate-glutathione pathway take part in the defense against oxidative stress as they increased after application to bean seedlings of Cu and Zn, metals with different chemical behavior, indicating that both induced oxidative stress (Cuypers et al., 1999). However, striking differences in the relative induction time of these enzymes suggest that the chemical properties of the metals play a pivotal role in the induction of oxidative stress as well as in the induction of defense mechanisms. Some studies have been conducted to analyze induction of the antioxidant system under As stress (Hartley-Whitaker et al., 2001a; Srivastana et al., 2005). In Pteris vittata exposed to As, higher activities of antioxidant enzymes and lower peroxidation corresponded to a higher As hyperaccumulation and lack of toxicity symptoms in comparison to other ferns such as P. ensiformis and Neprholepis exaltata (Srivastava et al., 2005). Only a few studies compared the responses of antioxidants to As+3 and As+5 (Mylona et al., 1998; Srivastana et al., 2007). It appears that upon exposure of the plants to As+5 SOD, APX, and GR activities were stimulated to higher levels compared to As+3, thus maintaining the function of the ascorbate-glutathione cycle. The increase in the activities of antioxidant enzymes may be due to the induced transcription of their genes mediated by ROS. Varying responses are also likely related to concentration of –sulfhydryl (SH) groups present in the plants or induced by metal treatment. Thiols have strong antioxidative properties and it is important to know to what extent GSH is involved in metal tolerance. GSH not only directly reacts with free radicals and protects the thiol status of protein via thiol-disulfide exchange, but is also involved as substrate for GP, which reduces H2O2 and organic peroxides, thus protecting cell proteins and cell membranes against oxidation (Navari-Izzo and Izzo, 1994). Biosynthesis of GSH was enhanced in the presence of Cd and other heavy metals, but a low GSH concentration was instead found in the plants (Ruegsegger and Brunold, 1994; Xiang and Oliver, 1998). These results are consistent with the idea that GSH, besides being itself an antioxidant and a metal chelator, is also a precursor for the synthesis of metal-binding phytochelatins. In addition, the oxidation of GSH in response to oxidative damage is also important for protection of plasma membrane from lipid peroxidation. The hypothesis that in Nicotiana rustica constitutive GSH might chelate Cd ions present in the cytosol under low-level exposure (5 μM) is intriguing (Vögeli-Lange and Wagner, 1990), but the drop in GSH observed in the first hours of exposure to Cd could be due to its utilization against oxidative stress caused by the metal. May be that higher levels of exposure require additional response mechanisms, like synthesis of PCs. A complex defense system, comprising of antioxidant enzymes and PCs, occurred in the roots of Lupinus luteus exposed to Ca, Cu, and Pb (Gwozdz et al., 1997). In Raphanus sativus subjected to increased concentration of Cu or Cd, total glutathione (GSH + GSSG) did not increase, whereas oxidized glutathione (GSSG) rose with the increase in metal concentration (Cosi, 2001; Sgherri et al., 2003), indicating an enhancement of oxidative processes and an involvement of GSH in counteracting oxidative stress. The lack of increase in GSH could be due to the utilization of the newly synthesized GSH, besides as an antioxidant, in PCs synthesis as already outlined (Cobbett, 2000). Heavy metals activate the PC synthase activity thus inducing the PCs biosynthesis, resulting in a depletion of GSH level (Zenk, 1996). In fact, in the experiment outlined before, PCs amounts increased in Cd- or in Cu-grown plants. In the same plants total ascorbate (AsA + DHA) and reduced ascorbate (AsA) contents, as well as phenolic acid amounts, increased with the rise in metal concentration (Cosi, 2001). Phenolic acids are simple polyphenols. Polyphenolic structure has the ability to inhibit free radical formation and the propagation of free radical reactions through the chelation of metal ions, especially Cu and Fe, thus preventing Fenton reactions. The phenolics may also act as antioxidants by acting as hydrogen donor, leading to the formation of phenoxyl radicals. In the presence of metals, however, it is not clear to what relative

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Cytosol NADP+

NADPH GR

PCS

GSH DHA

NADPH

NADP+

MDA MDAR AsA

GSSG

DHAR

AsA

PhO• + H2O

AsA

POX

MDA DHA

PhOH + H2O2

H2O APX H 2 O2

FIGURE 25.2  Proposed interrelation between peroxidases/phenols/AsA and the NADPH/GSH/AsA cycle. APX, ascorbate peroxidase; AsA, reduced ascorbate; DHA, dehydroascorbate; DHAR, dehydroascorbatereductase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; MDA, monodehydroascorbate; MDAR, monodehydroascorbate reductase; PCs phytochelatins; PhO •, phenoxyl radicals; PhOH, phenolic compounds; POX, peroxidases. (Redrawn from Sgherri, C. et al., Physiol. Plant., 118, 21, 2003.)

extent radical scavenging or metal chelation might contribute to their antioxidative properties. Their effect could be likely dictated by structural features and their propensity to interact and penetrate lipid bilayers (Brown et al., 1998). A peroxidase/phenol/ascorbate system can represent an efficient detoxification mechanism of hydrogen peroxide in the vacuole (Takahama and Oniki, 1997). A cycle, that can occur in both the apoplast and in the vacuole, and where H2O2 is scavenged by phenolics through a peroxidase, has been hypothesized (Sgherri et al., 2003). Phenolics are oxidized to phenoxyl radicals which can be reduced by AsA (Figure 25.2). DHA produced from AsA oxidation in the vacuole should be transported back into the cytosol to be regenerated to AsA. In R. sativus grown in excess of Cu or Cd, the amount of ASC produced was sufficient to replenish its amount that was reduced to form phenoxyl radicals to explain the increase in phenolic compounds (Cosi, 2001; Sgherri et al., 2003). An accumulation of phenolics, due to the induction of shikimate dehydrogenase (EC 1.1.1.25) and peroxidase (EC 1.11.1.7), was also observed in the hypocotyl of pepper grown in excess copper (Diaz et al., 2001). An interrelation between the peroxidase/phenol/ ascorbate system of the vacuole and the NADPH/glutathione/ascorbate system of the cytoplasm can strengthen the removal of H2O2. This removal and the synthesis of PCs from GSH could not be ruled out and might increase the tolerance of plants to heavy metals. 25.3.1.2  Metal Compartmentation Once inside the root cells, excess metals are translocated into a place where they can do the least damage to cellular metabolism. At the cellular level this involves storage in the vacuole or in the cell walls and at tissue level accumulation in the epidermal cells and in the trichomes. Trichomes apparently play a major role in storage and detoxification of metals. In B. juncea, Cd accumulation was found to be more than 40-fold higher in trichomes than in the total leaf (Salt et al., 1995a) and in tobacco a preferred storage of excess Cd in trichomes has also been observed (Choi et al., 2001). Trichomes accumulate other toxic metals, including Mn (Blamey et al., 1986) and Pb (Martell, 1974). Expression of a gene encoding a type 2 metallothionein (a metal binding protein) was localized in trichomes of bean plants (Foley and Singh, 1994). The distribution pattern of metals varies in the different leaf cell types and with plant species and metal. In roots and leaves of bush bean Cd ions seem to be mostly bound to pectic sites and histidyl groups of the cell wall (Leita et al., 1996), while in tomato suspension cultures and in root cell walls of Silene cucubalus a negligible amount of Cd was found (Inhoue et al., 1991) and no differences in cell wall binding were observed between

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normal and Cd-tolerant plants (Verkleij et al., 1990). Most of Cd is also sequestered by cell wall anionic groups in roots of rice (Rascio et al., 2008) and in leaves of the aquatic macrophyte Elodea canadensis (Dalla Vecchia et al., 2005). Higher concentrations of Cu bound to the cell wall and uronic acid in the roots of Sorghum sudanense L. were speculated to be the main reason to restrain Cu translocation from roots to shoots (Wei et al., 2008). The vacuole of root cells has been identified as the major site of sequestration for Zn, Cd, and Ni, and malate and citrate were found as relevant ligands within the vacuole (Krämer et al., 2000; Ma et al., 2005), although it is not clear how these ligands are sequestered (Haydon and Cobbett, 2007). Leaf compartmental analysis of buckwheat revealed that 80% of the Al—as Al-oxalate complex— was stored in the vacuole (Shen et al., 2002). A direct evidence of Zn vacuolar sequestration came from Zn uptake studies in Mg-ATP energized tonoplast vesicles isolated from Zn-hypertolerant and sensitive S. vulgaris (Verkleij et al., 1998). Zn-citrate was more effectively transported into the vacuole in the Zn tolerant ecotype although it is yet unknown which transporter is involved in the transport over the vacuolar membrane. Recently, Verkleij (2008) reported that the allocation patterns of Zn and Cd showed a preferential accumulation of these metals in the lower epidermis over storage in mesophyll cells, indicating that vacuolar compartmentation in leaves is less important. Furthermore, the allocation of metals in leaves could be explained by the effects of the metals on plant growth and leaf morphology and not as a specific result of tolerance mechanisms. Zn uptake in shoot vesicles of sensitive plants was higher than in root vesicles, suggesting that the enhanced tonoplast zinc transport, correlated with zinc tolerance in roots, is not very prominent in shoots (Chardonnens, 1999). From Schizosaccharomyces pombe cells it is known that PC–Cd complexes (LMW complexes) are transported in the vacuole by Hmt1, an half-size ABC-type transporter (Ortiz et al., 1995) and a “high molecular weight” (HMW) complex is formed. No protein responsible for this activity has so far been found in plants, but the fact that in mesophyll cells of tobacco most of Cd and PCs were found in the vacuole (Vögeli-Lange and Wagner, 1990) and isolated in oat tonoplast vesicles displaying ATP-dependent uptake of Cd–PC complexes (Salt and Rauser, 1995), suggests that a similar process might be operational also in plant cells. Some authors observed a disappearance of PC complexes after several days and in plant samples from metal-rich sites no PCs were detected (Leopold et al., 1999). The vacuolar complex may dissociate because of the acidic vacuolar pH, and Cd might be complexed by vacuolar organic acids (citrate, oxalate, malate) (Krotz et al., 1989) and, possibly, by amino acids. Another mechanism of vacuolar sequestration derives from studies on tonoplast-enriched vesicles of oat: a Cd2+/H+ antiport might be involved in the accumulation of free Cd2+ into the vacuole (Salt and Wagner, 1993). In addition, the transporter could be a CAX2 gene, identified as a low efficiency Ca2+/H+ exchanger (Hirschi et al., 2000). Furthermore, tobacco plants expressing CAX2 are able to accumulate more Ca2+, Mn2+, and Cd2+ and CAX2 was found to be the only vacuolar Mn2+-transporter. Also nicotinamine seems to be important for sequestration of Fe in vacuoles, and it was found by immunohistochemical detection that nicotianamine (NA) concentrations increased in pea and tomato vacuoles under excess of Fe supply (Pich et al., 2001).

25.4  Chelation of the Metals by Ligands 25.4.1  Organic Acids and Amino Acids Internal detoxification of accumulated metals by chelation of heavy metal by particular ligands containing S, N, and O, may be implicated in differential metal tolerance, metal transport through xylem, and vacuolar metal sequestration. Although chelating agents can increase the translocation of metals in the xylem, they are also able to keep free metal ions within certain limits, thus reducing their toxicity. The detoxification and homeostasis of metals such as Ni and Zn is potentially correlated to their hydroxy and/or amino acids binding capacity (for a review see Rauser, 1999; Clemens, 2001), although a clear correlation between amounts of those ligands and exposure to metals has not

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been presented yet. Due to their low association constant for complex formation and because high concentrations of these ligands are present in many plants independent from heavy metal tolerance, it is rather unlikely that these organic compounds are involved in tolerance mechanism. Thanks to the improved technologies, in vivo molecular biology studies allowed to identify the most important long-distance Cu transporters (Herbik et al., 1996; Pich and Scholz, 1996; Liao et al., 2000; Takahashi et al., 2003; Kim et al., 2005; Irtelli et al., 2009), which seems to be all amino acid compounds. The nonproteinaceous amino acid NA plays a key role in copper complexation in xylem sap. Much of the understanding of the physiological functions of NA comes from the NA-deficient tomato mutant chloronerva, which contained excess copper in roots but failed to transport normal amount of copper into mature leaves. Furthermore, xylem exudates from mutant plants showed unusually low levels of copper (Herbik et al., 1996; Pich and Scholz, 1996). These results were confirmed by Liao et al. (2000) who reported that NA is likely the most important Cu ligand in tomato and chicory xylem exudates. However, the xylem transport of Cu was efficient even in the absence of NA, provided that histidine (His) was present (Liao et al., 2000). Notably, as far as the stability constant is concerned, His (log Kst = 17.5) could compete with NA (log Kst = 18.6) as a ligand for Cu. Another important enzyme in the regulation of the level of NA is the nicotianamine aminotransferase (NAAT) that catalyses the amino group transfer of NA in the biosynthetic pathway of phytosiderophores. The gene that encodes NAAT from barley was introduced into the non-graminaceous plant, tobacco. Transgenic tobacco plants (naat tobacco) showed a lower concentration of Cu, Mn, Fe, and Zn in both young leaves and flowers than the wild type. This lower concentration, attributable to the depletion of endogenous NA, is a further confirmation of the role of NA in metals transport (Takahashi et al., 2003). Recently, NA and His/proline were found to be the most important Cu chelators in xylem sap of Brassica carinata under condition of Cu deficiency and excess, respectively (Irtelli et al., 2009). The increase in NA detected in conditions of copper starvation but not in copper excess seems indicate that NA is not involved in the response to excess of copper but participates in Cu transport to the shoots in conditions of deficiency. This could be explained when the biosynthetic pattern of NA is taken into consideration. NA is an intermediate in mugineic acids (MAs) biosynthesis formed via the NA synthase-catalyzed trimerization of S-adenosyl-l-methionine. MAs are naturally secreted from graminaceous plants in iron starvation conditions in order to solubilize Fe in the soil. Unlike MAs, NA is not secreted from the roots but it is not unreasonable to think that its increase is involved in internal copper transport when plants are in metal deficiency rather than in metal excess. NA may also be important for Ni tolerance. A number of recent studies shows that transgenic overexpression of nicotianamine synthase (NAS) in Arabidopsis or tobacco confers increased tolerance to Ni (Douchov et al., 2005; Kim et al., 2005; Pianelli et al., 2005).

25.4.2  Root Exudates The exudation of organic molecules from the roots seems to be linked to the exclusion mechanism, and the exudation of organic acids is considered one of the most important strategies by which plants can exclude metals such as Al, Cd, and Pb by chelating them in the rhizosphere or in the a­ poplastic space, thus preventing their entry into the symplast (Zheng et al., 1998; Matsumoto, 2000; Yang et al. 2000; Hill et al., 2002; Watanabe and Osaki, 2002). The efflux of organic substances is regulated by the electrochemical gradient existing across the plasma membrane (Ryan et al., 2001) due to the activity of ATP-driven proton pumps (H+-ATPases). Organic acids typically flow across the lipid bilayer at a slow rate in response to the electrochemical gradient. However, efflux may be greatly increased under stress conditions such as toxic metal excess due to the expression (coding) of anion channels embedded in the plasma membrane or to their upregulation (Rengel, 2002). In this way, some metal-tolerant species can restrict uptake and translocation of metals maintaining low metal level in the shoots over a wide range of external concentrations (Baker, 1981). The introduction of a Pseudomonas aeruginosa citrate synthase (CS)

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gene into tobacco and papaya, caused an increase in the CS activity in citrate exudation and in Al tolerance in transgenic plants (De la Fuente et al., 1997). High citrate, malate, and oxalate exudation from roots was found in Al-excluder plants such as Al-resistant cultivars of P. vulgaris (Miyasaka et al., 1991), Paraserianthes falcataria L. Neilson, Acacia mangium Wild. (Osawa et al., 1997), Fagopyrum esculentum (Ma et al., 1997, 2001b) and Brachiaria brizantha (Ishikawa et al., 2000), confirming the role of this organic acid in Al exclusion and tolerance. Current evidences suggest that efflux of malate affects the differential Al tolerance among genotypes of several crop species influencing their capacity of excluding Al from the apical tissues by chelating it in the apical rhizosphere or, more likely, in the apoplastic space. Several studies have shown that root tips of tolerant wheat genotypes exhibit an Al-stimulated efflux of malate that is minimal or absent in Al-sensitive lines (Delhaize et al., 1993; Basu et al., 1994a; Ryan et al., 1995a,b; Pellet et al., 1996). However, Parker and Pedler (1998) probing the “malate hypothesis” of differential aluminum tolerance in wheat by using other rhizotoxic ions as proxies for Al argued that malate efflux plays at most a minor role in the differential tolerance of wheat, and that a more integrative, multifaceted model of tolerance is needed. Qin et al. (2007) found that Al induced root exudation of oxalate and citrate, Cu induced exudation of oxalate, malate, and formate, and Zn induced exudation of formate from an aseptically grown poplar. However, in that study, no evidence of the organic acids involvement in heavy metals exclusion and tolerance was reported. Conversely, the release of oxalate was found to be responsible for a differential Pb tolerance among rice varieties (Yang et al., 2000). Molecules other than organic acids have been found in root exudates of plants exposed to Al. The high exudation rate of the phenolics catechol, catechin, quercetin, and curcumin, identified in three maize varieties, were found to be correlated with their different resistance to Al. However, in the same varieties the exudation of organic acids, besides being small, did not correlate with the resistance to Al shown by the three varieties (Kidd et al., 2001). Upon exposure to Ni, the nonhyperaccumulator Thlaspi arvense exuded histidine and citrate more than the hyperaccumulator Thlaspi caerulescens suggesting that the exudation of both compounds may be a strategy to exclude Ni but clearly it is not involved in Ni hyperaccumulation (Salt et al., 2000). On the contrary, in Norway spruce (Picea abies) (Heim et al., 2000) and in four tropical woody species (Nguyen et al., 2003) exposed to Al toxicity, the phenolic and amino acid exudates did not account for interspecific differences in Al tolerance. Cultivars of Triticum aestivum differing in resistance to Al were grown in the presence or absence of Al. Differences in exudation of total polypeptides, enhanced accumulation of specific polypeptides, and the greater association of Al to high molecular mass fraction from Al-resistant cultivars in response to Al stress, suggested that root exuded polypeptides may play a role in plant response to Al toxicity (Basu et al., 1994b, 1997). In conclusion, unequivocal evidence for a function of these compounds in plant metal tolerance has been difficult to obtain.

25.4.3  Phytochelatins Phytochelatins (PCs) are a family of small nonprotein metal-binding polypeptides, broadly classified as Class III metallothioneins (MTs), first sequenced by Grill et al. (1985) in Rausolvia serpentina. These compounds are synthesized, in the cytoplasm, from reduced glutathione (GSH) by a γ-glutamyl-cysteine dipeptidyl transpeptidase, called phytochelatin synthase (PCS), which is activated and post-translationally regulated by metal ions (Oven et al., 2002) and have a general structure (γ-Glu-Cyst)n-Gly with n = 2−11, although PC2 and PC3 are the most common (Cobett, 2000; Schmoger et al., 2000). The amino acid glycine (Gly) may be substituted by β-alanine, serine or glutamine, suggesting the existence of corresponding PC homologs, homophytochelatins (h-PCs), in different plant species. Structure and synthesis of PCs have been extensively reviewed and will not be discussed here (Steffens, 1990; Rauser, 1999; Cobbet, 2000; Sanità Di Toppi et al., 2002). There has been considerable debate concerning the functions of PCs. A wide range of metals, among which the most effective seem to be Ag, As+3, Cd, Cu, Hg, and Pb, has been seen to induce

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the formation of PCs, but there is only limited evidence supporting their potential role in heavy metal tolerance (Ernst et al., 1992; Schat and Kalff, 1992; Meharg, 1994; Zenk, 1996; Cobbet, 2000; Goldsbrough, 2000). As yet, the only unambiguous established function of PCs seems to be Cd, Hg, and As detoxification. The evidence of this came from the characterization of the PC-synthase-deficient Arabidopsis cad1 mutants, hypersensitive to Cd and Hg but no or hardly sensitive to any essential heavy metal micronutrient (Howden et al., 1995; Ha et al., 1999; Hall, 2002). Inhibition of PC synthesis by buthionine sulfoximine (BSO), a γ-glutamyl cysteine synthase (γ-ECS) inhibitor, enhanced Hg sensitivity in Vallisneria spiralis and Hydrilla verticillata (Gupta et al., 1998) and BSO-treated cell cultures of tobacco and Rauvolfia serpentina showed hypersensitivity to As (Nakazawa et al., 2000; Schmoger et al., 2000). Disruption of the PCS gene in S. pombe resulted in hypersensitivity to Cd (Clemens et al., 1999; Ha et al., 1999). In Saccharomyces cerevisiae the expression of PCS cDNA from wheat, Arabidopsis, and S. pombe increased Cd tolerance and Cd-hypersensitive Arabidopsis mutants were impaired in PC synthesis (Howden et al., 1995; Cobbett et al., 1998). In addition, tomato cell lines selected for hypertolerance to Cd exhibited enhanced PC synthesis under Cd exposure (Chen and Goldsbrough, 1994). However, Schat and collaborators (2002) found that in nonmetallicolous and metallicolous population, the ability to induce PC accumulation decreased in the order As/Cd/Cu > Zn > Ni/Co, and was consistently higher in nonmetallicolous plants than in hypertolerant ones, except for the case of As. The sensitivities to Cu, Zn, Ni, and Co were unaffected by BSO treatments suggesting that PC-based sequestration is not essential for constitutive tolerance to these metals. Cd sensitivity was considerably increased by BSO only in nonadapted plants, whereas BSO increased As sensitivity both in nonadapted and As-hyper-tolerant plants. In conclusion, PC-based sequestration seemed to be essential only for constitutive tolerance to Cd, while it is essential for both normal constitutive tolerance and adaptative hyper-tolerance to As. Cd-treatments are also known to stimulate sulfur assimilation required for sulfide formation in plants (Nussbaum et al., 1998; Robinson, 1989). CdCl2 (10 μM) in the nutrient solution induced a 100% increase in sulfate uptake by maize roots (Nocito et al., 2002). Further evidence against direct roles of PCs for Cu or Zn tolerance has been presented. In S. vulgaris in vivo inhibition of PC synthesis by BSO decreased Cd tolerance but did not affect Zn and Cu tolerance (De Knecht et al., 1992; Schat and Vó’ijs, 1997). Although Cu in vitro activates PC synthase and PCs can chelate Cu in vitro, PCs do not seem involved in Cu homeostasis in vivo. Higher level of PCs in tolerant plants than in sensitive ones has been reported (Steffens, 1990; Rauser, 1999), and at equal external Cu concentrations PC production was significantly higher in the metalliferous clones than in nonmetalliferous clones of H. lanatus, S. vulgaris, and S. cucubalus (Schat and Kalff, 1992; De Vos et al., 1992; Hartley-Whitaker et al., 2001b). However, the comparison of PC production at Cu NEC or EC50 revealed that PCs were equal at the same level of stress. Therefore, it has been concluded that PCs are not decisively involved in differential copper tolerance in S. vulgaris, S. cucubalus, and H. lanatus (Schat and Kalff, 1992; De Vos et al., 1992; Hartley-Whitaker, 2001a). In addition, Cu hypertolerance is associated with strongly decreased PC synthesis under Cu exposure (De Vos et al., 1992). More recently it has been shown that in Escherichia coli or S. cerevisiae Cd tolerance of cells increases with higher levels of expression of PCS gene (AtPCS1) (Matsumoto et al., 2004); however, the over expression of this gene does not lead to increased Cd tolerance and accumulation in Arabidopsis thaliana (Lee et al., 2003). Although suppression of the high-affinity phosphate uptake system is doubtlessly the major genetic determinant of As (V) hypertolerance (Meharg and MacNair, 1992; Meharg, 1994; Wang et al., 2002; Bleeker et al., 2006), it cannot be excluded that the precise level of tolerance is influenced by other additional determinants besides the rate of As(V) uptake. Schmöger et al. (2000) have clearly demonstrated the formation of PC-As complexes in vitro and in vivo, but indications for a potential role of PCs in As hypertolerance came from studies which demonstrated that PCS-deficient A. thaliana showed hypersensitivity to As(V) (Ha et al., 1999), and that in hypertolerant H. lanatus, which exhibited elevated PC-thiol: As ratios, BSO abolished As tolerance (Hartley-Whitaker et al., 2001b), indicating that PCs are essential for As(V). The first step in As(V) detoxification could be

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the reduction to As(III) catalyzed by the plant arsenate-reductase (Bleeker et al., 2006). Several studies have suggested the reduction of arsenate to arsenite by endogenous arsenate reductase in plant cells (Pickering et al., 2000; Dhankher et al., 2002). Bleeker et al. (2006) demonstrated that an enhanced arsenate reductase activity improves arsenate internal detoxification, promoting the formation of As(III)-GS3 or As(III)-PC complexes (Raab et al., 2004, 2005), which are then transported in the vacuole, likely by an ABC-type transporter. Recently, the presence of an arsenate reductase activity from a root extract of P. vittata that reduces arsenate to arsenite in in vitro assays has been reported (Duan et al., 2005) and a gene encoding a putative endogenous arsenate reductase from A. thaliana that reduces As(V) to As(III) has been identified in plants (Dhanker et al., 2006). However, the As hyperaccumulator fern, P. vittata, which accumulates As as As(III) produced only very low amounts of PCs upon exposure to As(V) (Zhao et al., 2003). However, little is known about As tolerance and the detoxification mechanism in this hyperaccumulator. More recently it was shown that arsenate tolerance in Silene paradoxa, from a mine site enriched in As and in other heavy metals, did not rely on arsenate reduction and subsequent PC-based sequestration (Arnetoli et al., 2008). Schulz et al. (2008) instead found that the toxicity of As(V) in six plant species with different As sensitivity under sufficient phosphate nutrition is dependent on PCs production but arsenic tolerance is not associated with an extended biosynthesis of long-chain PCs. Generally, in As-tolerant plant species, PCs with a shorter chain length dominate, PC2 being the dominant phytochelatin. With increased As sensitivity, the production of PC3 and PC4 are increased. The previous authors suggest that the PC2/PC3/PC4 ratio in roots of those plants could be useful for early recognition of arsenic exposure and as a tool to assess the degree of As sensitivity. Further studies are, therefore, required to unequivocally understand the mechanism involved in As(III) and As(V) tolerance. Recently, it has been reported that soybean plants showed a notably high contribution of homoglutathione (hGSH)—a compound which in some legumes replaces GSH synthesizing homo-­phytochelatins (h-PCs) instead of PCs—to the pool of thiols in shoots under both Cd and arsenate exposure. However, a higher level of hPCs in Cd-treated soybeans compared to PCs in lupins did not prevent growth inhibition. In contrast, the highest thiol concentrations in soybean exposed to arsenate were associated with reduced growth inhibition of roots; therefore, the role of hPCs in the arsenate detoxification mechanisms of this species seems to be clearer, showing higher thiol concentrations and lower growth reduction than those present in lupin plants (Vα´ zquez et al., 2009). In conclusion, an overproduction of PCs in plants exposed to heavy metals does not seem a general likely mechanism for metal tolerance, owing to the energy required for sulfate reduction to support PC synthesis (Steffen, 1990). Aside from detoxification, PCs are considered to have an important role in cellular essential metal-ion homeostasis (Steffen, 1990). Some metal ions, such as Zn2+ and Cu2+ are part of catalytic proteins or structural elements to allow for metabolism in plants. While there is, as yet, no direct evidence that PCs play a role in essential metal homeostasis, in vitro experiments have shown that PC–Cu and PC–Zn complexes could reactivate the apo forms of the Cu-dependent enzyme diamino oxidase and the Zn-dependent enzyme carbonic anhydrase, respectively (Thumann et al., 1991). Thus, the production of PCs and of metal-PC complexes may be seen as a first and transient response of the cells since their production generally decreases or even disappears with duration of time of heavy metals exposure (Leopold et al., 1999; Wó’jicik and Tukiendorf, 2003). PC complexes have been also identified as a possible transport form of Cu from roots to leaves in Cu-exposed Larrea tridentata (Polette et al., 2000). Therefore, according to other recent studies further roles have been proposed for PCs. It is conceivable that PCs may also be involved in the long-distance transport of Cd, and possibly other heavy metals. In transgenic A. thaliana and in grafted Arabidopsis plants it has been shown that PCs undergo long-distance transport in the root-to-shoot and shoot-to-root directions (Gong et al., 2003; Chen et al., 2006), and that in addition to the directional xylem transport, the phloem results a major vascular system for long-distance source to sink transport of Cd as

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PC-Cd and glutathione-Cd complexes (Mendoza-Cózatl et al., 2008). However, future researches are needed to understand the mechanism by which thiol, Cd and/or thiol-Cd complexes are loaded and unloaded from the phloem.

25.4.4  Metallothioneins Metallothioneins (MTs) are small protein (MW 6–8 kDa) cysteine (Cys) rich proteins that can bind metals via the thiol groups of their Cys residues. The first demonstration that plants could produce, in addition to PCs, these metal-binding proteins comes from the purification of the wheat embryo Ec protein, the first MT purified from a plant tissue, and characterized as a Zn-binding protein involved in the regulation of Zn homeostasis during early seed germination (Lane et al., 1987). Since this discovery, Mt genes have been found throughout the plant kingdom (Cobbett and Goldsbrough, 2002). MTs have been divided into two classes based on amino acidic sequence. Class I includes mammalian MTs, Class II includes Mts from plants and fungi as well as invertebrate animals. Plant MTs are further classified into four types according to the arrangement of Cys residues (Cobbett and Goldsbrough, 2002). Based on the analysis of MT RNA expression in a number of plant species, type 1Mt genes are, in general, expressed more in roots than in shoots, whereas type 2Mt genes are expressed primarily in leaves (Hsieh et al., 1995, 1996). Type 3 genes are expressed in leaves or in ripening fruits (Ledger and Gardner, 1994), while expression of type 4 MTs appears to be restricted to developing seeds (White and Rivin, 1995). For the genome of A. thaliana seven putative MT genes have been annotated (plus one pseudogene), including the previously characterized genes AtMT1, AtMt2a/2b, and AtMT3 (Guo et al., 2003). The diverse patterns of expression of different MT genes suggest that plant MT isoforms may differ not only in their amino acidic sequence but also in the function they perform in specific tissues. However, expression studies have revealed that different MT isoforms exhibit overlapping expression patterns, pointing to partial function redundancy (Guo et al., 2003). In the aim of enhancing metal tolerance, sequestration, or accumulation of plants, the high metalbinding capacity of MTs has been widely exploited. Plant MT1 and MT genes have been shown to complement MT-deficient yeast (cup 1 D), demonstrating their function in copper detoxification (Zhou and Goldsbrough, 1994). Expression of plant MT1 and MT2 in the yeast mutant cup 1 D also increased cadmium tolerance. Likewise, expression of plant MTs in E. coli led to increased tolerance toward copper and cadmium (Evans et al., 1992; Zhigang et al., 2006). Based on these findings, plant MTs are thought to play a major role in cellular copper and cadmium homeostasis. However, when MT2 gene from A. thaliana was expressed in a Synechoccus mutant deficient in its Zn-metallothionein gene smtA, a partial complementation was achieved, pointing to a possible role for MT2 in plant zinc homeostasis (Robinson et al., 1996). Only few studies have exploited the in vivo functions of MTs in plants. Progresses were hampered by the difficulties in detecting these proteins because of their instability and tendency to oxidize under normal protein isolation conditions (Murphy et al., 1997). In E. coli and A. thaliana, over expressing a MT-like gene from pea, PsMTA, an up to eight-fold accumulation of copper was reported (Evans et al., 1992). MT2a and MT3 of A. thaliana were transiently expressed in Vicia faba guard cells (Lee et al., 2004), which showed an enhanced resistance to cadmium exposure. A MT2 protein from B. juncea, BjMT2, was over expressed in A. thaliana under the control of the 35S promoter. The transformed seedlings exhibited an increased tolerance to copper and cadmium on the basis of shoot growth and chlorophyll content. Analysis of transiently transformed cells of A. thaliana leaves revealed exclusive cytosolic localization of BjMT2:EGFP (Enhanced Green Fluorescent Protein) fusion protein in control and heavy metal-exposed plant cells. Remarkable, ectopic expression of BjMT2 reduced root growth in the absence of heavy metal exposure, whereas in the presence of copper root growth in control and transgenic lines was the same indicating that in A. thaliana, root and shoot development are differentially affected by ectopic expression of BjMT2 (Zhigang et al., 2006). Although MT2 mRNAs increase in response to a great variety of stresses, MT2 gene expression seems to be required for tolerance to heavy metals, especially copper (Rauser, 1999). MT2 expression was

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the primary determinant of ectopic differences in the copper tolerance of Arabidopsis seedlings (Murphy and Taiz, 1995) and MT expression was induced by Cu and, though less effectively, by Cd and Zn (Zhou and Goldsbrough, 1995). However, in some plant species no induction of MT2 transcript levels was found after copper exposure (Schafer et al., 1997; Thomas et al., 2003). A study on S. vulgaris showed that in copper-tolerant ecotypes, a MT gene was much more expressed (SvMT2b) than in the sensitive ecotypes, independent of Cu exposure (Van Hoof et al., 2001). This constitutive MT expression contrasts with an inducible MT2b expression in A. thaliana and points out that Cu tolerance mechanism is fundamentally different from Cu detoxification (Verkleji, 2008). A similar mechanism was observed in S. paradoxa. The levels of copper tolerance and constitutive MT2b expression were increased in cupricolous populations as compared to the serpentine and nonmetallicolous populations (Mengoni et al., 2003). The results of the latter author, together with those of Van Hoof et al. (2001) allow to argue that the MT2b locus represents a major target for natural selection imposed by soil copper toxicity. However, the over-expression of SvMT2b did not produce Cu tolerance per se but merely enhanced the degree of hypertolerance in already tolerant plants. Therefore, MT2b seems to act as a hypostatic enhancer, rather than as a primary tolerance gene (Van Hoof et al., 2001). Although to date, the relationship of plant MTs in metal detoxification has been widely studied only for copper and cadmium, there is a growing body of evidence that these cys-rich proteins might be involved in the tolerance of other metals. Arsenic is able to induce MT synthesis in mice and humans (Liu et al., 2000; Garret et al., 2001) and the As binding to mammalian MTs has been demonstrated as well (Toyama et al., 2002; Jiang et al., 2003). However, the role of plant MTs in As binding and detoxification is still unexplored. Trivalent As bound to MTs from Fucus vesciculosus has been reported and five arsenic-MT complexes with increasing As to protein ratio have been characterized (Merrifield et al., 2004). In addition, it has been demonstrated that A. thaliana MT1 proteins were stabilized by metals among which As (Zimeri et al., 2005). Zhang et al. (2005), studying the heavy metal tolerance mechanism of Allium sativum L., firstly demonstrated that the transcript level of AsMT2a in roots was significantly increased by Na3As O4. Recently, preliminary details of three Prosopis juliflora Mt genes have been provided (Usha et al., 2009). All three PjMTs demonstrate the ability to bind Cd, Zn, and Cu with PjMT1 showing maximum binding to the three heavy metals. PjMT1 and PjMT2 seems to be involved in copper and zinc homeostasis, respectively, while PjMT3 might be involved in heavy metal detoxification. In conclusion, many decades of intensive reports have addressed MT structure, functions, and gene expression, but despite the increasing experimental data, several topics remain to be clarified, and the true function of this elusive protein (Palmiter, 1998) has yet to be disclosed.

25.5  Mycorrhizas and Heavy Metal Tolerance Mycorrhizal fungi are a major component of the rhizosphere, where they establish mutual association with the roots of most plant species. Numerous studies provided evidence that these fungal symbionts can be effective in the heavy metal tolerance by helping the host plant to cope with the metal toxicity, therefore, root mycorrhization can be seen as one of the means that allow metallophytes to thrive on metal polluted soils (Hildebrandt et al., 2007; Rodriguez and Redman, 2008). The highlighting of the ameliorating effects of root-fungus associations on plant heavy metal tolerance strengthened the interest in mycorrhizal fungi and in understanding the mechanisms whereby they can alleviate the heavy metal stress in the host plant; this in view of the potential use of plant mycorrhization in ecological reclamation of contaminated soils and in revegetation of mine tailing that usually can hardly support any plant growth (Göhre and Paszkowski, 2006). Large diversities in metal response and metal specificity have been found among different mycorrhizal fungi; therefore, the ameliorating effects strongly depend on the fungal species or genotype. The most efficient in plant protection turn out to be the fungal isolates from methallicolous soils, which are more adapted to the heavy metals (Adriaensen et al., 2003).

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As regards ectomychorrizas, common among forest trees, it has been shown, for instance, that Zn or Cu tolerant isolates of Suillus bovinus provide excellent insurance against these heavy metals in Scot pine (Pinus sylvestris) seedlings (Van Tichelen et al., 2001; Adriaensen et al., 2003) and that specific genotypes of Suillus luteus are very efficient in protecting pines from Cu (Adriaensen et al., 2005) or Cd (Krznaric et al., 2009). Most mechanisms proposed for ectomycorrhizas to explain their ameliorating effects involve exclusion processes that restrict metal movement to the host roots. The tolerant fungi retain the heavy metals by means of strategies similar to those employed by higher plants, namely through extracellular sequestration (binding them to slimes, cell walls or extruded ligands) and intracellular detoxification (storing them in the vacuolar compartment or complexing to chelators such as GSH and metallothioneins) (Bellion et al., 2006). Indeed, the Cu and Zn tolerance of ectomycorrhizal fungus Pisolithus tinctorius relies on the heavy metal absorption by extrahyphal slime (Tam, 1995) whereas in Paxillus involutus Cd is both bound to cell walls and stored in vacuoles (Blauzer et al., 2000), and Cd and Cu are also sequestered in the cytosol as metallothionein complexes (Bellion et al., 2007). Interestingly, Cd-tolerant Suillus-Pinus and Paxillus-Pinus mycorrhizal associations show an enhanced symbiosis defense system with high GSH concentrations (Schützenbdübel and Polle, 2002). Similarly, the Cd exposure strengthens the antioxidative system of Paxillus evolutus with a rise of SOD- and GSH-related enzyme activities (Ott et al., 2002). Thus, it seems that ectomycorrhizal fungi can also provide the host plant with protection by “arming” the roots with physiological defenses against the heavy metal oxidative stress (Bellion et al., 2006). The endomycorrhizal arbuscular fungi (AMFs), which are the most widespread among the mycorrhizal fungi, have been commonly reported in metal contaminated soils (Göhre and Paszkowski, 2006). Recent studies give strong evidence that AMFs can filter out toxic heavy metals by keeping them away from the roots (Hildebrandt et al., 2007) and that they can help the host plant to overcome the difficulties in acquisition of P and other mineral nutrients (Dong et al., 2008). Lin et al. (2007) reported that colonization by the AMF Glomus mosseae positively affected growth and Cd/Zn tolerance of three leguminous plants (Sesbania rostrata, Sesbania cannabina, Medicago sativa) through mycorrhizal immobilization of heavy metals and decreased translocation to the shoot. Moreover, the mycorrhization stimulated the root nodule formation, thus leading to increased N2 fixation. Glomus mosseae was found to reinforce Cu, Cd, and As tolerance in other plants (Trifolium repens, Lolium perenne, Coreopsis drummondii, and P. vittata) as well as to improve the P nutrition and to curtail the shoot amount of toxic elements (Chen et al., 2007). Dong et al. (2008) showed that Trifolium repens and Lolium perenne heavily depended on mycorrhizas for surviving the As toxicity in an arsenic contaminated soil. In Medicago truncatula (Xu et al., 2008) and in barley (Christophersen et al., 2009) AMFs enhanced the As tolerance by restricting the root As uptake through the suppression of high affinity arsenate/phosphate transporters and the upregulation of the phosphate uptake system responsible for transfer of Pi from the symbiotic interface to cortical cells. Recently, genes responsive to heavy metals (Cu, Cd or Zn) exposure were studied in the AMF Glomus intraradices mycelium and in Medicago truncatula mycorrhizal roots. The finding that genes encoding proteins (GSH S-transferase and HSP90) which potentially counteract the ROS damage are upregulated by heavy metals in mycorrhizal roots seems to indicate that a primary concern of the fungal partner in the symbiosis is the heavy metal oxidative stress (Hildebrandt et al., 2007).

25.6  Heavy Metal Hyperaccumulation 25.6.1  Hyperaccumulator Plants High tolerance to a range of heavy metals has evolved in many species exposed to elevate metal concentrations in native soils. Although the majority of these heavy metal-tolerant plants behaves as “excluders,” a series of tolerant plants exists, which deal with heavy metals in just an opposite way. This kind of plants,

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defined “hyperaccumulators” (Brooks et al., 1977), actively take up the potentially toxic metals, which are then translocated from root to shoot and accumulated in the aboveground tissues where they reach exceptional concentrations of several percent of dry weight (usually 100–1000-fold higher than those found in normal plant species), without giving rise to phytotoxic effects (Rascio, 1977; Reeves, 2006). Although a number of hypotheses have been proposed (Boyd and Martens, 1992), the evolutionary reason for metal hyperaccumulation still remains unclear. One of the most attractive proposals is that a huge concentration of toxic elements can provide the plant with an “elemental defense” against pathogens and herbivores (Boyd, 2007). Tolerance and hyperaccumulation are two distinct features; however, the hyperaccumulators also have the characteristic of tolerance (or hypertolerance) which, according to Chaney et al. (1997), is the key property that makes the hyperaccumulation possible. The first discovery of a plant with such an extreme ability to accumulate heavy metals goes back to 1948, when Minguzzi and Vergnano found 10,000 μg Ni g−1 of dry weight in shoots of Alissum bertolonii from serpentine soils of Italy. Besides the ecological and physiological interest, hyperaccumulator plants have attracted considerable attention for practical applications, because of their potential utilization in phytoremediation of metal contaminated soils (Pilon-Smits, 2005) as well as in phytomining (Li et al., 2003). Recently, the possibility of exploiting the accumulation traits of these plants for strategies of food crop biofortification has also sparked great interest (Palmgren et al., 2008). The better understanding of hyperaccumulation mechanisms, in fact, would assist the development of genetically engineered plants with improved nutritional value for the world’s populations suffering from mineral element deficiency (especially Zn) in their vegetarian diet (Maret and Sandstead, 2006). Over 450 species of plants, belonging to a wide range of unrelated families, have been identified as hyperaccumulators of heavy metals (Ni, Zn, Cd, Cu, Co, Mn), and also of metalloids (As, Se). Most of them are endemic to metalliferous soils, but some species, classified as facultative metallophytes, although preferring metal-rich soils, can also grow on nonmetalliferous ones (Assunção et al., 2003b). Hyperaccumulators are generally minor vegetation components in most European and North American habitats, but they are relatively abundant in some locations from New Caledonia, Cuba, and South Africa (Boyd, 2004). The majority of taxa (nearly 400) hyperaccumulate Ni, whereas there are fewer hyperaccumulators of other metals, such as Zn, Pb, Cu, and Co, and only four species hyperaccumulate Cd (Reeves, 2006; Verbruggen et al., 2009). There are also rather few plants that hyperaccumulate As (Wang et al., 2007) and Se (Terry et al., 2000). Sebertia acuminata (Sapotaceae), a rare rainforest tree, endemic to the New Caledonia ultramafic serpentine soils, is the most extreme example of a hyperaccumulator. Its latex concentrates the highest Ni quantities found in an organism (up to 26% of dry mass) and a single tree may contain 37 kg of the metal (Jaffré et al., 1976; Sagner et al., 1998). The common definition of hyperaccumulator plants meets the requirement that the metal concentration in the shoot must be higher (as % of dry weight) than a threshold, which is different for each metal, depending on its phytotoxicity. According to such a criterion, hyperaccumulators are plants that accumulate >10,000 μg g−1 (1%) Zn or Mn, >1,000 μg g−1 (0.1%) Ni, Pb, Cu, Cr, Co, and also As or Se, and >100 μg g−1 (0.01%) Cd, when grown on native soils (Baker and Brooks, 1989; Terry et al., 2000; Wang et al., 2006). Other requirements proposed for classifying a hyperaccumulator are that the metal concentration in plant grown on heavy metal-rich soils must be 10–500 times higher than in the same plant species from non-polluted environments (Yanqun et al., 2005) and that the ratios of both shoot to root and shoot to soil metal concentration must be >1 (McGrath and Zhao, 2003; Yanqun et al., 2005). However, these last criteria have recently been debated, since they may exclude rare and strictly metallophyte plants which cannot be compared with plants from unpolluted environments as well as species able to accumulate very high quantities of earth abundant metals such as Al or Fe in aboveground tissues (Branquinho et al., 2007).

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The Brassicaceae family, and particularly the Thlaspi and Alyssum genera, are rich in hyperaccumulator species (approximatively 25% of the documented taxa). The greatest number of Ni hyperaccumulators (about 50 taxa) belongs to the genus Alyssum (Brooks, 1998). The genus Thlaspi also includes a high number of Ni hyperaccumulators and species have also been found in genera of different other families (Baker and Brooks, 1989). The list of Zn hyperaccumulators is shorter (10,000 μg g−1 (dry weight) without suffering phytotoxicity (Lombi et al., 2000). In 2001, the fern P. vittata was identified as the first plant able to hyperaccumulate arsenic in its fronds (up to 2.3% of dry weight) (Ma et al., 2001a) and then it was demonstrated that the As tolerance of P. vittata results from both constitutive and adaptative traits and that this species is also able to constitutively accumulate Zn and Pb (Zhao et al., 2002a; Wu et al., 2009). After P. vittata other Pteris taxa were found to hyperaccumulate over 1,000 μg As g−1 (dry weight) in fronds under field conditions (Wang et al., 2006, 2007). Selenium hyperaccumulation has been observed in plants of different families, among which species belonging to the genus Astragalus, such as Astragalus bisulcatus (Fabaceae), and in Stanleria pinnata (Brassicaceae), which can accumulate Se in a range of 2,000–16,000 μg g−1 of shoot dry weight (Terry et al., 2000; Galeas et al., 2007). In hyperaccumulating plants the concentration of exceptionally high quantities of heavy metals (or metalloids) in their aboveground tissues resisting their toxic effects depends on three basic hallmarks that distinguish these plants from the related nonhyperaccumulating nontolerant species: • A much greater ability to take up metals from the soil • A much more rapid and efficient root to shoot translocation of the absorbed metals • A much greater capability to detoxify and to store high metal amounts in the leaf cells Physiological as well as genomic and transcriptomic studies carried out by comparing hyperaccumulating with related nonhyperaccumulating plants have paved the way for the understanding of metal hyperaccumulation mechanisms at functional and molecular levels. Most studies focused on Zn, Ni, and Cd hyperaccumulation having T. caerulescens and A. halleri as model plant systems. However, analyses performed on other heavy metal or metalloid hyperaccumulators also provided useful information on the hyperaccumulation strategies. Comparative transcriptomic studies revealed that most genes thought to be involved in hyperaccumulation and

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hypertolerance key steps are not novel but rather common to hyperaccumulating and nonhyperaccumulating plants, being only differently expressed and regulated in the two kinds of plants. In particular, it was found that a large gene array shows a constitutive overexpression in hyperaccumulators (Verbruggen et al., 2009).

25.6.2  Enhanced Heavy Metal Uptake In roots of T. caerulescens and A. halleri a key role in enhanced Zn uptake seems to be played by the overexpression of genes belonging to the ZIP (Zinc-regulated transporter Iron-regulated transporter Proteins) family, which encodes putative plasma membrane-located cation transporters common to plants and animals (Grotz et al., 1998). In roots of T. arvense, like in the other nonhyperaccumulating plants, these genes are all expressed at a detectable level only under zinc deficiency, being strongly downregulated at normal Zn supply. By contrast, in T. caerulescens roots the expression of two ZIP genes (ZTN1 and ZTN2) is barely Zn-responsive and still persists at sufficient Zn nutrition, suggesting that this high expression, irrespective of Zn availability, is the major reason for the enhanced Zn uptake in this hyperaccumulator plant (Pence et al., 2000; Assunção et al., 2001). Similarly, the lack in A. halleri roots of a Zn-dependent transcriptional regulation of ZIP6 and a barely Zn-regulated expression of ZIP9, which are both strongly Zn-responsive in nonhyperaccumulator A. thaliana, lead to regard these genes as candidates for a role in Zn hyperaccumulation (Weber et al., 2004; Filatov et al., 2006). Other overexpressed but Zn-regulated ZIP genes have been identified in roots and shoots of T. caerulescens and A. halleri. Nevertheless, their involvement in Zn hyperaccumulation is not clearly established (Becher et al., 2004; Weber et al., 2004; Talke et al., 2006). Despite the chemical similarity between Zn and Cd, physiological evidence exists that the mechanisms of Cd uptake may be different from those of Zn uptake in roots of the Zn/Cd hyperaccumulator T. caerulescens and that multiple absorption systems operate in a specific way in ecotypes with different Cd hyperaccumulation capacity (Lombi et al., 2001; Zhao et al., 2002b). In the lower Cd-accumulating Prayon ecotype (from Belgium) the Zn and Cd influxes depend on a system with a strong preference for Zn over Cd. The Cd uptake significantly decreases with the increase of Zn concentration, suggesting that the Cd absorption is largely mediated by Zn transporters (Zhao et al., 2002b). Plausibly this Cd uptake involves ZTN1 which has been shown to mediate a high affinity Zn and low affinity Cd transport (Pence et al., 2000). Moreover, the inhibition of Cd absorption by increasing Ca concentrations suggests that in this T. caerulescens accession the Cd uptake might also occur via Ca channels (Zhao et al., 2002b). Differently, in the higher Cd hyperaccumulator Ganges ecotype (from southern France) surviving concentrations of more than 3,000 μg Cd g−1 (dry weight) in the aboveground tissues (Lombi et al., 2000), the Cd uptake occurs with the same Km but a Vmax fivefold higher than the Prayon one. Furthermore, it is not inhibited by Zn or other metals nor by increasing Ca concentrations. This strongly suggests the existence of a highly selective Cd transport system in root cell membranes of this high Cd accumulating accession of T. caerulescens (Lombi et al., 2001; Zhao et al., 2002b; Assunção et al., 2008). Interestingly, the existence of a specific and highly efficient Cd transport system, coupled with the Cd-increased plant growth (Roosens et al., 2003), raised the question as to whether Cd might play any physiological role in the high accumulating Ganges ecotype, as it occurs in the marine diatom Thalassiosira weissglogii where a Cd-requiring carbonic anhydrase exists (Lane et al., 2005). In a recent study, Liu et al. (2008) provide evidence of a positive correlation between carbonic anhydrase activity and Cd concentration in the shoots of Ganges plants, suggesting that Cd might actually play a physiological role in this high accumulating ecotype by enhancing the activities of some enzymes. In the other Zn/Cd hyperaccumulator, A. halleri, the Cd amounts in roots and shoots dramatically decrease with the supply of high Zn concentrations, thus implying that Cd and Zn uptake is mediated by the same transport system with a higher Zn preference (Küpper et al., 2000). Besides the ability to hyperaccumulate Cd in the aboveground tissues, A. halleri also shows a Cd hypertolerance

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due, at least in part, to a lower short-term Cd uptake rate and a very efficient sequestration of Cd ions in root cells (Bert et al., 2003; Weber et al., 2006). Finally, in S. alfredii, a high Zn supply enhances the Cd concentration in roots and aerial organs, suggesting the existence, in this species, of synergic Cd and Zn interactions for both absorption and transport (Yang et al., 2004). Hitherto candidate transporters mediating the Ni uptake in the hyperaccumulating plants have not been identified (Verbruggen et al., 2009). Since several serpentine populations of Zn/Ni hyperaccumulators prefer Zn over Ni under equimolecular supply of the two metals (Assunção et al., 2001, 2008) the involvement of a Zn transport system in Ni uptake has been supposed. However, the finding that at low Ni concentrations the rates of Ni uptake and root-to-shoot translocation are similar in both the Ni-hyperaccumulator Thlaspi goesingense and the non-Ni-hyperaccumulator T. arvense, led Krämer et al. (1997) to suggest that the Ni hyperaccumulator phenotype relies primarily on a remarkable Ni tolerance achieved through efficient leaf systems of metal detoxification rather than on the enhanced heavy metal uptake/translocation. As regards the arsenic hyperaccumulation, ample evidence exists that plants take it up as arsenate via the transport system of the chemically analog phosphate (Meharg and Hartley-Whitaker, 2002) and that the hyperaccumulator fern P. vittata is much more efficient in arsenate uptake than other nonhyperaccumulating species (Caille et al., 2005; Lou et al., 2009). This enhanced As absorption seems to result from a greater density of phosphate/arsenate transporters due to the gene overexpression in the root cells (Caille et al., 2005). Furthermore, the lower Km of P. vittata compared with the nonhyperaccumulating fern Nephrolepis exaltata, shows a higher arsenate affinity of the hyperaccumulator transport proteins (Poynton et al., 2004). In Se hyperaccumulating plants, such as A. bisulcatus, and Stanleya pinnata, the selenium uptake, mainly as selenate, occurs through sulfate transporters, due to the chemical similarity between selenium and sulfur (Terry et al., 2000). More precisely, the selenate absorption involves specifically high-affinity sulfate transporters localized exclusively in the root cells (Shibagaki et al., 2002; Sors et al., 2005b). In nonhyperaccumulating species transporters of this kind are inducible and regulated by the S status of the plant (Hirai et al., 2003). The finding in hyperaccumulator plants of Se/S ratios 100-fold higher than in related nonhyperaccumulating species strongly suggests that one or more sulfate transporters may have evolved into Se-specific transporters unresponsive to the plant S status (Sors et al., 2005b; Galeas et al., 2007).

25.6.3  Improved Root to Shoot Heavy Metal Translocation A common trait of heavy metal (or metalloid) hyperaccumulation, after the uptake from the soil into the root, is the fast and efficient translocation from root to shoot of the absorbed heavy metals. This is a key step requiring a symplastic radial flow followed by an active loading into the xylem vessels. The enhanced translocation to the shoot entails a limited sequestration into and a fast release from the root cell vacuoles to make higher metal amounts readily available for the xylem loading. This could indicate differences in root tonoplast characteristics accounting for a smaller influx and a larger efflux of metals (Lasat et al., 2000). It has been found, indeed, that 2.7-fold less Zn is stored in root vacuoles of the hyperaccumulator S. alfredii compared with the nonhyperaccumulating ecotype (Yang et al., 2006b). A much lower Zn amount (2.5-fold) is also sequestered in root vacuoles of T. caerulescens when compared to T. arvense and, furthermore, the rate of Zn efflux out of the vacuoles is about twofold faster in the hyperaccumulator plant (Lasat et al., 2000). Recently, Xing et al. (2008) reported an inverse relation between Cd storage in root vacuoles and translocation efficiency in T. caerulescens accessions differently able to accumulate Cd. In enhanced xylem loading of hyperaccumulators, the key role is played by some types of transporters among which P1B-type ATPases, also known as heavy metal transporting ATPases (HMAs), a class of proteins mediating the heavy metal ion transport in plants, with roles in homeostasis and metal tolerance (Axelsen and Palmgren, 1998). The expression of the HMA3 and HMA4 genes, encoding two HMAs of the bivalent cation transporter group, is significantly higher in both T. caerulescens

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and A halleri than in A thaliana, with transcript levels 2–3-fold higher in roots than in shoots of the two hyperaccumulators (Papoyan and Kochian, 2004; Talke et al., 2006; Courbot et al., 2007). Furthermore, the HMA4, which is localized at the plasma membrane of xylem parenchyma cells, where it mediates the active heavy metals efflux, is upregulated in T. caerulescens (Papoyan and Kochian, 2004) and, conversely, downregulated in A. thaliana (Mils et al., 2003) upon plant exposure to high Zn and Cd concentrations. Interestingly, in T. caerulescens, HMA4 is also induced by Zn deficiency, supporting the idea of a role in both heavy metal hyperaccumulation and Zn nutrition (Papoyan and Kochian, 2004). HMA4 is constitutively overexpressed also in A. halleri, where it possibly mediates both Zn and Cd transport from roots to shoots (Talke et al., 2006). Another gene with a constitutive higher expression in roots of T. caerulescens and A. halleri compared to A. thaliana is FRD3 encoding a member of the MATE (Multidrug And Toxin Efflux) family of small organic molecule transporters (Verbruggen et al., 2009). In A. thaliana FRD3, localized at the plasma membrane of root pericycle cells, mediates the efflux into the vascular tissue of citrate, a low molecular weight chelator necessary for root-to-shoot Fe translocation (Durrett et al., 2007). The overexpression of FRD3 in T. caerulescens and A. halleri is supposed to contribute to Fe homeostasis and mobility in presence of higher xylem Zn concentrations requiring an improved metal chelation capacity. However, it cannot be ruled out that FRD3 might be involved in homeostasis and translocation of other metals, including Zn (Krämer et al., 2007). The hyperaccumulator plants exhibit constitutively elevated concentrations of organic acids, mainly citrate and malate (Montargès-Pellitier et al., 2008), which are assumed to play a role as heavy metal chelators in hyperaccumulation strategies. However, the necessity of organic acids as heavy metal ligands in roots and during the long-distance xylem transport is rather controversial. This is because of the low association constants of organic acids with metals that seem to limit their role to the metal storage in cell vacuoles, where the metal-organic acid complexation is favored by the acidic environment (Haydon and Cobbett, 2007). Sarret et al. (2002) found Zn-malate and Zn-citrate in A. halleri roots, whereas Zn-organic acid complexes were not detected in roots of T. caerulescens (Salt et al., 1999). Only 20% of the xylem sap Zn in T. caerulescens is complexed with citrate, the remaining 80% being present as free ion (Salt et al., 1999); in the xylem sap of A. halleri about half of the Zn is associated with citrate while most Cd is transported in the free ionic form (Ueno et al., 2008). Moreover, only one-third of Ni is complexed with citrate in the xylem of the Ni-hyperaccumulator Stackhousia tryoni (Bidwell, 2001). By contrast, Schaumlöffel et al. (2003) showed that most Ni was bound to citrate in the latex of the Ni-hyperaccumulator S. acuminata and Callahan et al. (2008) identified a number of other organic acids as Ni ligands in the latex of this plant. In heavy metal hyperaccumulation and transport, a role as metal chelators is played by other small organic compounds, as free amino acids, mainly histidine (His), and NA, which are known to form stable complexes with several bivalent cations (Callahan et al., 2006). NA is present at higher concentrations in roots of both T. caerulescens and A. halleri than in those of the nonhyperaccumulators T. arvense and A. thaliana (Weber et al., 2004; Mari et al., 2006). This for the constitutive overexpression in hyperaccumulating plants of the NAS2 gene encoding one isoform of NA synthase (Weber et al., 2004; van de Mortel et al., 2006). In A. halleri, the enhanced NA synthesis is essentially related to Zn hyperaccumulation (Weber et al., 2004), while it seems to be involved in Ni hyperaccumulation in T. caerulescens where a strong correlation occurs between NA and Ni but not between NA and Zn (Vacchina et al., 2003; Callahan et al., 2007). The transport of NA-metal complexes, required for loading into and unloading from vascular tissues, is mediated by the YSL (Yellow Strip 1-Like) family of proteins (Colangelo and Guerinot, 2006). Transcriptomic studies led three YSL transporter genes to be identified, namely TcYSL3, TcYSL5, and TcYSL7, constitutively overexpressed in roots and shoots of T. caerulescens, with the expression of TcYSL3 and TcYSL7 localized around the root vascular tissues. Evidence supports a role of these YSLs in metal hyperaccumulation by participating in vascular transport of NA-metal (particularly NA-Ni) complexes (Gendre et al., 2006). In roots of the Ni hyperaccumulator Alyssum lesbiacum a His-coupled

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Ni xylem loading occurs and the Ni-His complexation is favored by a high endogenous His pool (Krämer et al., 1996; Kerkeb and Krämer, 2003) due to the constitutively overexpression of ATPphosphoribosyltransferase, the first enzyme of the His biosynthetic pathway (Ingle et al., 2005). Elevated free His concentrations have also been reported in roots of T. geosingense (Persans et al., 1999) and a Ni induced His accumulation has been observed in T. caerulescens, suggesting that a similar mechanism might operate in all these Ni-hyperaccumulating species (Assunção et al., 2003b). However, the transport system involved in Ni-His xylem loading has not been identified yet. Recent evidence suggests that the Ni-His complexation, besides sustaining the heavy metal release into the xylem, may also prevent the trapping of Ni in the root cell vacuoles maintaining the ions in a detoxified form available for translocation to the shoots (Verbruggen et al., 2009). In arsenic hyperaccumulators, such as P. vittata and Pteris cretica, the higher rate of uptake into the roots and the lower sequestration within the cell vacuoles contribute to the greater As translocation to the shoot, when compared with non accumulating ferns (Poynton et al., 2004). A percentage of As taken up as arsenate (AsV) is rapidly reduced to arsenite in the root cells and translocated to the shoot as AsIII (Poynton et al., 2004; Vetterlein et al., 2009). In the xylem sap of P. vittata AsIII accounts for 93%–98% of the total As (Su et al., 2008), in agreement with the finding by Duan et al. (2005) of arsenate reductase activity only in the roots of the hyperaccumulating fern. The reduction of AsV to AsIII renders necessary transport systems other than that for phosphate to translocate arsenite to the shoot, and the most likely candidates seem to be aquaporins that are known to mediate transmembrane AsIII transport in mammals (Liu et al., 2002) and yeasts (Wysocki et al., 2001). Indeed, plasma membrane-localized aquaglyceroporins of NIP (Nodulin 26-like Intrinsic Protein) subfamily, permeable to arsenite but not to arsenate, have recently been show to operate in the AsIII transport into the xylem in plants (Ma et al., 2008; Kamiya et al., 2009). It is conceivable that a high expression of this kind of transporters might account for the arsenite efflux from root cortical cells toward xylem in As hyperaccumulators (Zhao et al., 2009). Differently, in Se hyperaccumulator plants such as A. bisulcatus and Stanleya pinnata most of Se uptaken into the roots is translocated to the shoots as selenate through the sulfate transport systems (Sors et al., 2005a), the young leaves being the major site for SeO 4 2− reduction and metabolism (Freeman et al., 2006).

25.6.4  Heavy Metal Detoxification in Leaves The concentration of huge quantities of heavy metals (or metalloids) in the aboveground parts (especially leaves) in hyperaccumulating plants without suffering from phytotoxicity requires the efficient activity of detoxification mechanisms in the cells mainly based on metal complexation with ligands and/or sequestration in extracytosolic sites, such as vacuoles or cell walls (Küpper et al., 2001; Cosio et al., 2005). The ability of hyperaccumulators to remove high heavy metal amounts from active cellular sites by storing them in inactive compartments is a key mechanism of tolerance which relies, at least in part, on the constitutive overexpression of transporters localized at the cell tonoplast and/or plasma membrane. A role in heavy metal hyperaccumulation has been recognized for CDFs (Cation Diffusion Facilitators), also called MTPs (Metal Transporter Proteins), a class of proteins which mediate bivalent cation efflux from the cytosol (Mäser et al., 2001). It has been found that the transcript levels of the MTP gene ZTP1 are higher in leaves of T. caerulescens compared with T. arvense (Assunção et al., 2001; Hammond et al., 2006), and that the tonoplast MTP1 protein, with a plausible role in vacuolar Zn sequestration, is more expressed in shoots of A. halleri than in the Zn-sensitive Arabidopsis lyrata (Dräger et al., 2004). Furthermore, in the Zn/Ni hyperaccumulator T. goesingense the localization of the constitutively overexpressed TgMTP1 at the tonoplast and plasma membrane is consistent with its role in both Zn and Ni import into the shoot vacuoles (Persans et al., 2001) and export from the cytoplasm to the cell wall (Kim et al., 2004). Comparative transcriptome analyses between shoots of A. halleri and A. thaliana (Becher et al., 2004) and T. caerulescens and T. arvense (Hammond et al., 2006) suggest the involvement in Zn hyperaccumulation of HMA3,

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a vacuolar P1B-type-ATPase overexpressed in the hyperaccumulating species. In leaves of A. halleri Elbaz et al. (2006) found a constitutively high expression of MHX, a vacuolar metal/proton exchanger belonging to the superfamily of Ca2+/cation (CaCA) exporters. Moreover, constitutively overexpressed genes coding for some cation exchangers (CAXs ) of another CaCA subfamily have been identified in A. halleri and T. caerulescens and are supposed to mediate the Cd compartmentation (Cracium et al., 2006; van de Mortel, 2006, 2008). More recently the activity of a vacuolar Ni/H+ antiport has also been evidenced by Ingle et al. (2008) in leaves of the Ni hyperaccumulator Alyssum lesbiacum. All these classes of transporters are important for heavy metal sequestration and detoxification, thereby generating a metal sink in the shoot that could be one of the driving forces in metal hyperaccumulation (Becher et al., 2004). A further strategy to detoxify heavy metals in leaves of hyperaccumulating plants is by complexing them in the cytoplasm with low molecular weight chelators, principally organic acids, to avoid binding to physiologically active proteins and to facilitate the subsequent transport and storage within vacuoles. Cd is mainly sequestered as malate complexes in leaves of T. caerulescens (Ueno et al., 2008) while the majority of Zn is bound to citrate in this plant (Salt et al., 1999) and to malate in A. halleri (Sarret et al., 2002). Citrate is the main Ni ligand in a range of Alyssum hyperaccumulating species (Lee et al., 1978) and in T. goesingense (Krämer et al., 2000). Hitherto the identity of transporters mediating the vacuolar storage of the chelated heavy metals remains unknown. Differently from nonhyperaccumulating plants the detoxification strategies in hyperaccumulators do not rely on heavy metal binding with high molecular mass chelators, such as phytochelatins (Ebbs et al., 2002; Schat et al., 2002). As regards metallothioneins (MT), an overexpression of some members of the MT gene family has been noticed in high Cd hyperaccumulating populations of T. caerulescens (Roosens et al., 2005; Hassinen et al., 2007). However, no clues exists currently about a role in heavy metal hypertolerance of these MTs, which appear to be more involved in Cu homeostasis in the context of exceptional Cd and Zn concentrations in the hyperaccumulator tissues (Roosens et al., 2005). In various Thlaspi species, the ability to hyperaccumulate Ni is related to high glutathione levels in shoot tissues, driven by constitutive activation of enzymes of sulfur assimilation pathway and GSH metabolism (Freeman et al., 2004). This enhanced GSH synthesis, also induced in shoots of T. caerulescens (van de Mortel et al., 2008) and S. alfredii (Sun et al., 2007) by Cd exposure, suggests an involvement of GSH antioxidant activity in hyperaccumulation. Higher antioxidationrelated gene expressions (Chiang et al., 2006) and antioxidative enzyme activities (Table 25.1) (Schickler and Caspi, 1999; Wang et al., 2008) occur in other Ni and Cd hyperaccumulators compared with relative nonhyperaccumulating species. The essential role played in hyperaccumulation strategies by the heavy metal exclusion from metabolically active sites of leaves accounts for their preferential sequestration in leaf epidermis where they do least damage to photosynthesis. In leaves of T. caerulescens, the highest Zn and Cd concentrations are present in the vacuolar sap of the epidermal cells (Küpper et al., 1999; Ma et al., 2005) but, interestingly, Zn appears to be largely absent from within the subsidiary and guard cells of the stomatal complexes, plausibly to protect the function of stomata against metal toxicity (Frey et al., 2000; Cosio et al., 2005). A similar absence of Ni in the guard cells has been reported in the Ni hyperaccumulator Thlaspi montanus by Healt et al. (1997) who suggest that the heavy metal exclusion might be due to unique ion-transport properties of the guard cell plasma membrane. A lower Ni concentration in the stomatal complex than in the other epidermal cells also occurs in the hyperaccumulators T. goesingense (Küpper et al., 2001) and Alyssum murale (Broadhurst et al., 2004). Different ecotypes of T. caerulescens accumulate Cd both inside the vacuoles and in the cell walls of the epidermis (Cosio et al., 2005) while in A. halleri the highest concentrations of Cd and Zn are found in the leaf trichomes (Küpper et al., 2000). Ni was distributed predominantly in vacuoles of the leaf epidermal cells also in several hyperaccumulator species of Alyssum (Küpper et al., 2001; Broadhurst et al. 2004; Asemaneh et al., 2006) and in Hybanthus floribundus (Bidwell et al., 2004). The Ni hyperaccumulator Berkheya coddii behaves in a peculiar manner in that it accumulates Ni

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at significantly higher concentrations in the cuticle of the upper epidermis than in the rest of the leaf thus protecting the mesophyll cytoplasms from the heavy metal toxic effects (Robinson et al., 2003). In fronds of the arsenic hyperaccumulator fern P. vittata up to 90% of the total As is present as inorganic arsenite stored in vacuoles (Pickering et al., 2006; Su et al., 2008). The vacuolar sequestration results as the key mechanism of As detoxification in the hypertolerant ferns, but how it occurs and what the As transporters are at the tonoplast deserve further investigation (Zhao et al., 2009). Also in As hyperaccumulators, differently from nonhyperaccumulators, phytochelatins do not seem to have a significant role in As detoxification (Zhang et al., 2004; Vetterlein et al., 2009). Raab et al. (2004) showed that less than 1% of the As is complexed with phytochelatins in fronds of P. cretica. According to Zhao et al. (2002a), this might be explained with the prohibitive cost of a PC-dependent As hyperaccumulation, which would require a S amount exceeding that normally accumulated inside the plants. In the As tolerance of hyperaccumulating ferns, instead, an ­inherently high antioxidant potential plays a role against ROS produced by arsenate to arsenite reduction. This is based on an ascorbate-GSH pool (Sing et al., 2006) and an antioxidative enzyme activity (Table 25.1) much greater than in nonhyperaccumulating species (Srivastava et al., 2005; Kertulis-Tartar et al., 2009). As regards the selenate, it is known that its assimilation, which occurs in plastids through the sulfur assimilation pathway, leads to formation of selenoamino acids, such as selenocysteine (Se-Cys), whose misincorporation into proteins accounts for the Se toxicity (Terry et al., 2000). Thus, in hyperaccumulator plants the ability to tolerate very high Se concentrations in leaves relies on the activity of the enzyme selenocysteine methyltransferase (SMT) that specifically methylates SeCys to produce the harmless nonprotein amino acid methylselenocysteine (MeSeCys). The SMT is ­constitutively expressed in roots and leaves of Se hyperaccumulators (Pickering et al., 2003) and the SMT activity is closely correlated with the Se hyperaccumulation in species with ­varying capacity to accumulate Se (Sors et al., 2005a). In the young leaves of the hyperaccumulator A. bisulcatus containing the greatest amount of Se, the MeSeCys accounts for 99% of the accumulated Se (Pickering et al., 2003). Conversely, in the non-Se-hyperaccumulators, such as Astragalus drummondii, the synthesis of MeSeCys cannot occur due to the inability of a SMT-like enzyme to catalyze the SeCys methylation (Sors et al., 2009). Interestingly, also Se, like the heavy metals, is concentrated predominantly in the leaf epidermis of hyperaccumulators, while in nonhy­peraccumulators it is distributed throughout the mesophyll cells (Freeman et al., 2006). This finding strongly suggests that actively directed transport mechanisms for hyperaccumulated elements may be a unifying theme in hyperaccumulating species (Freeman et al., 2006).

25.7  Conclusions Chemical, technological, and agricultural activities pour into the environment ever greater quantities of heavy metals thus polluting wild and cultivated lands and exposing plants growing on poisoned soils to the risk of phytotoxic damage. Moreover, the possible entrance of these heavy metals into the food chain via plants represents a serious threat to animals and human health. The serious problem of heavy metal pollution led, in the last two decades, to a real explosion of research dealing with the harmful effects of heavy metals in plants and with the defense strategies that plants accomplish to tolerate this kind of dangerous chemicals. Great interest was also taken in highlighting the mechanisms allowing metallicolous species to hypertolerate and hyperaccumulate heavy metals. This because of the potential application of the acquired knowledge in strategies useful for removing unwanted heavy metals from the biosphere as well as for obtaining biofortified food crops. The information gained led to a substantial clarification of physiological, biochemical, molecular, and genetic mechanisms of heavy metal tolerance in both excluder and hyperaccumulator plants, but, despite the recent progress achieved by the application of new technological methods, a number of “tesserae” are still waiting to be unraveled in order to complete the whole puzzle of metal tolerance. However, the great and increasing scientific and applied interest will surely allow most black boxes of this fascinating research field to be unlocked in the next few years.

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Sgherri, C., Milone, M.A., Clijsters, H., Navari-Izzo, F. 2001. Antioxidative enzymes in two wheat cultivars, differently sensitive to drought and subjected to subsymptomatic copper doses. J. Plant Physiol. 158: 1439–1447. Sgherri, C., Quartacci, M.F., Izzo, R., Navari-Izzo, F. 2002. Relation between lipoic acid and cell redox status in wheat grown in excess copper. Plant Physiol. Biochem. 40: 591–597. Sgherri, C., Cosi, E., Navari-Izzo, F. 2003. Phenols and antioxidative status of Raphanus sativus grown in copper excess. Physiol. Plant. 118: 21–28. Sgherri, C., Quartacci, M.F., Navari-Izzo, F. 2007. Early production of activated oxygen species in root apoplast of wheat following copper excess. J. Plant Physiol. 164: 1152–1160. Sharma, S.S., Kaul, S., Metwally, A., Goyal, C., Finkemeier, I., Dietz, K.J. 2004. Cadmium toxicity to barley (Hordeum vulgare) as affected by varying Fe nutritional status. Plant Sci. 166: 1287–1295. Shaw, B.P. 1995. Effects of mercury and cadmium on the activities of antioxidative enzymes in the seedlings of Phaseolus aureus. Biol. Plant. 37: 587–596. Shen, R., Ma, J., Kyo, M., Iwashita, T. 2002. Compartmentation of aluminium in leaves of an Al-accumulator, Fagopirum esculentum Moench. Planta 215: 394–398. Shibagaki, N., Rose, A., McDermott, J.P. et al. 2002. Selenate-resistant mutants of Arabidopsis thaliana identity Sultr1;2, a sulfate transporter required for efficient transport of sulfate into roots. Plant J. 29: 475–486. Sing, N., Ma, L.Q., Srivastava, M., Rathinasabapathi, B. 2006. Metabolic adaptations to arsenic-induced oxidative stress in Pteris vittata L. and Pteris ensiformis L. Plant Sci. 170: 274–282. Singh, S., Eapen, S., D’Souza, S.F. 2006. Cadmium accumulation and its influence on lipid peroxidation and antioxidative system in aquatic plant, Bacopa monnieri L. Chemosphere 62: 233–246. Somashekaraiah, B.V., Padmaja, K., Prasad, A.R.K. 1992. Phytotoxicity of cadmium ions on germinating seedlings of mung bean (Phaseolus vulgaris): Involvement of lipid peroxidation in chlorophyll degradation. Physiol. Plant. 85: 85–89. Sors, T.G., Ellis, D.R., Na, G.N. et al. 2005a. Analysis of sulfur and selenium assimilation in Astragalus plants with varying capacities to accumulate selenium. Plant J. 42: 785–797. Sors, T.G., Ellis, D.R., Salt, D.E. 2005b. Selenium uptake, translocation, assimilation and metabolic fate in plants. Photosynth. Res. 86: 373–389. Sors, T.G., Martin, C.P., Salt, D.E. 2009. Characterization of selenocysteine methyltransferases from Astragalus species with contrasting selenium accumulation capacity. Plant J. 59: 110–122. Srivastava, M., Ma, L.Q., Singh, N., Singh, S. 2005. Antioxidant response in hyperaccumulator and sensitive fern species to arsenic. J. Exp. Bot. 56: 1335–1342. Srivastava, S., Mishra, R.D., Tripathi, S., Dwivedi, P.K., Trivedi, P.K., Tandon, P.K. 2007. Phytochelatins and antioxidant system respond differentially during arsenite and arsenate stress in Hydrilla verticillata (L.f.) Royle. Environ. Sci. Technol. 41: 2930–2936. Steffens, J.C. 1990. The heavy metal-binding peptides of plants. Ann. Rev. Plant Physiol. Plant Mol. Biol. 41: 553–557. Stohs, S.J., Bagchi, D. 1995. Oxidative mechanisms in the toxicity of metal ions. Free Radic. Res. 18:321–336. Su, Y.H., McGrath, S.P., Zhu, Y.G., Zhao, F.J. 2008. Highly efficient xylem transport of arsenite in the arsenic hyperaccumulator Pteris vittata. New Phytol. 180: 434–441. Sun, Q., Ye, Z.H., Wang, X.R., Wong, M.H. 2007a. Cadmium hyperaccumulation leads to an increase of glutathione rather than phytochelatins in the cadmium hyperaccumulator Sedum alfredii. J. Plant Physiol. 164: 1489–1498. Sun, R.L., Zhou, Q.X., Sun, F.H., Jin, C.X. 2007b. Antioxidative defense and proline/phytochelatin accumulation in a newly discovered Cd-hyperaccumulator, Solanum nigrum L. Environ. Exp. Bot. 60: 468–476. Takahama, U., Oniki, T.A. 1997. A peroxidise/phenolic/ascorbate system can scavenge hydrogen peroxide in plant cells. Physiol. Plant. 101: 845–852. Takahashi, M., Terada, Y., Nakai, I. et al. 2003. Role of nicotianamine in the intracellular delivery of metals and plants reproductive development. Plant Cell 15: 1263–1280. Talke, I.N., Hanikenne, M., Krämer, U. 2006. Zinc-dependent global transcriptional control, transcriptional deregulation, and higher gene copy number for genes in metal homeostasis of the hyperaccumulator Arabidopsis halleri. Plant Physiol. 142: 148–167. Tam, P.C.F. 1995. Heavy metal tolerance by ectomycorrhizal fungi and metal amelioration by Pisolithus- tinctorius. Mycorrhiza 5: 181–187. Terry, N., Zayed, A.M., Souza, M.P., Tarum, A.S. 2000. Selenium in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51: 401–432. Thomas, J.C., Davies, E.C., Malick, F.K. et al. 2003. Yeast metallothionein in transgenic tobacco promotes copper uptake from contaminated soils. Biotechnol. Prog. 19: 273–280.

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Metals and 26 Heavy Plastid Metabolism Katalin Solymosi and Martine Bertrand Contents 26.1 Introduction........................................................................................................................... 675 26.2 Metals Inside Plants............................................................................................................... 676 26.2.1 Metal Transporters..................................................................................................... 676 26.2.2 Interactions between Different Metals...................................................................... 680 26.3 Metals and Plastid Metabolism............................................................................................. 681 26.3.1 Impact of Unbalanced Metals on Nongreen Plastids................................................ 682 26.3.2 Impact of Unbalanced Metals on Photosynthesis...................................................... 685 26.3.2.1 Ultrastructural Alterations in Chloroplasts................................................ 688 26.3.2.2 Molecular and Metabolic Alterations in Chloroplasts under Heavy-Metal Deficiency................................................................... 691 26.3.2.3 Molecular and Metabolic Alterations in Chloroplasts under Heavy-Metal Excess......................................................................... 693 26.3.3 Some Unusual Phenomena Associated with Heavy-Metal Stress............................. 698 26.3.3.1 When the Excess of a Metal Alleviates the Stress Caused by Another Metal........................................................................................ 698 26.3.3.2 When Nonessential Metals Added at Low Concentrations Have a Stimulating Effect........................................................................... 699 26.4 Conclusion.............................................................................................................................700 Abbreviations.................................................................................................................................. 701 References....................................................................................................................................... 701

26.1  Introduction Heavy metals (HMs) are in general defined as metals with a specific gravity greater than 5.0 or with high atomic mass. Often, toxicity is associated with their definition; however, several HMs are necessary for the proper functioning and metabolism of living organisms. Such elements essential for plant development are Cu, Co, Fe, Mn, Mo, Ni, and Zn. One type of plant stresses is nutrient deficiency, with which most plants can barely cope; therefore their growth and crop productivity are impaired when the phytoavailability of any of these metals is low. Unfortunately, in several agricultural fields, essential-metal deficiency is a serious concern, especially in the case of Fe and Zn (Guerinot 2000). Another plant stress is represented by the excess of essential HMs and by the presence of nonessential metals. These nonessential HMs (e.g., Cd, Cr, Hg, Pb) are in general toxic or neutral for plant metabolism. The increased presence of HMs in air, soil, and water is also a global problem that represents a growing threat to the environment and to humankind and requires immediate attention. There are hundreds of natural and anthropologic sources of HMs, including industry, atmospheric deposition, use of agrochemicals, and waste disposal. The pollutants can enter plants via various mechanisms and in this way can easily reach intracellular compartments of plants, such as plastids. In this chapter, the stress caused by essential-HM deficiency and by excess 675

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of HMs on plastid metabolism is discussed. In addition, the interesting phenomena of HM stress alleviation by the excess of another HM and the stimulatory effect of low concentration stressors on photosynthesis is briefly reviewed.

26.2  Metals Inside Plants For healthy plant growth and development, a range of HMs must be acquired from the soil, transported along the plant, distributed, and compartmentalized in different tissues and cells. Clearly, membrane transport systems are likely to play a key role in these events. The genes encoding these plant nutrient transporters appear to be transcriptionally regulated by a feedback mechanism that reduces their expression when the plant reaches an optimal level of nutrition (reviewed by Amtmann and Blatt 2009, Burkhead et al. 2009, Kawachi et al. 2009). In most terrestrial plants, metals are absorbed by the roots and transported via the xylem into the aerial parts. Essential HMs are in general highly mobile and therefore approximately one third of them is translocated to the aboveground organs, while two-thirds are retained in the roots (e.g., Kawachi et al. 2009). However, when present or added in excess, most of the surplus essential metal(s) also accumulates in the roots, similar to most nonessential or toxic metals (e.g., Brunner et al. 2008, Zehra et al. 2009), but rarely the contaminating metal principally accumulates in the leaves (e.g., Vázquez et al. 1990). This way, in most cases, the root plastids are the first targets of HM toxicity or deficiency. Therefore, root plastids are in general less prone to metal deficiency, but more often affected by high levels of metals. Aquatic plants and some epiphytes absorb the essential nutrients via their whole surface from the water or the air, respectively. In these cases, HMs may reach the plastids directly. Similarly, atmospheric deposition of pollutants contributes to toxic metal levels in aerial plant parts (leaves, fruits, flower parts, and stems) of terrestrial plants, and in this case, chloroplasts or other types of plastids (i.e., chromoplasts, amyloplasts) can be directly affected by relatively high concentrations of HMs, although not much is known about the uptake mechanisms and transportation of airborne metal pollutants within plant tissues. Nonessential metals (Cd, Ag, and Pb), generally intrude into plant cells or into organelles at the expense of essential inorganic ions on account of similar properties, such as ionic radii (PerfusBarbeoch et al. 2002), for example, Cd uses Ca ion channels to enter plant cells.

26.2.1  Metal Transporters After the first phase of extracellular metal adsorption—rapid and nonspecific binding of the cations to the negatively charged cell wall components and mobilization of the soil-bound HMs by secretion of organic acids by the roots—metals have to enter the symplasm by metal transporters or ion channels (Table 26.1). Most of the metals enter cells as cationic elements (e.g., Zn2+), whereas others cross the plasma membrane as anionic groups (e.g., AsO 43−) or included in small organic compounds (e.g., methyl-mercury, CH3Hg+) (reviewed by Kucera et al. 2008). The HMs are taken up as hydrated ions or in metal–chelate complexes through channel proteins and/or carrier proteins. These include the (1) heavy-metal (P1b-type or CPx-type) ATPases (HMA) that can pump a variety of essential and nonessential HMs across the plasma membrane; (2) the natural resistance associated macrophage proteins (NRAMP), which are H+-coupled transporters implicated in the transport of divalent ions (e.g., Cu2+, Fe2+, Mn2+, and Cd2+); (3) the cation diffusion facilitators (CDFs) involved in Zn2+, Co2+, Mn2+, and Cd2+ transport (Williams et al. 2000); (4) the ZRT, IRT-like protein (ZIP) family transporting mostly Fe2+, Mn2+, Cd2+, and Zn2+ (Cohen et al. 1998, Guerinot 2000, reviewed by Grotz and Guerinot 2006); (5) the cation/H+ antiporters exchanging Na+ or H+ to Ca2+ or Cd2+; and (6) ATP-binding cassette (ABC) transporters that have a role in intracellular transport of Cd, Fe, or Mn (Table 26.1, reviewed by Clemens 2001, Hall and Williams 2003, Kucera et al. 2008, Poirier et al. 2008). Although these transporters are rather specific for a single element, their specificity is not absolute, and other metals can also be transported (Table 26.1). However, knowledge about

Proposed Metal Specificity (++ Indicates the Primarily Transported Metal) Carrier Type ABC-type transporter Cation diffusion facilitator (CDF)

Cation/H+ antiporters Conserved copper transporter (COPT)

Carrier

Cd

Co

STA1 ZAT1/MTP1 and MTP3

ShMTP1 CAX2 (Ca) CAX4 (Ca) COPT1, COPT2

CNGC1 NtCBP4 HMA1 HMA2 HMA3 HMA4 HMA5

Fe

Mn

Ni

Pb

Zn

+ +

+ + +

+

+

+ ++ ? ++ +

+

++

+ + +

+ +

+

+

+

Localization M T

T T T PM

+

COPT? COPT? Cyclic nucleotide gated channels (shaker type) Heavy-metal ATPases (HMA)

Cu

+

T PE

++

PM PM PE

+ +

+ +

++ ++ ++

PM T PM PM, G

References Briat et al. (2007) Van der Zaal et al. (1999), Broadley et al. (2007), Puig and Penarrubia (2009), and Kawachi et al. (2009) Delhaize et al. (2003) Hirschi et al. (2000) Cheng et al. (2002) Sancenon et al. (2003), Puig et al. (2007), and Burkhead et al. (2009) Yruela (2009) Sancenon et al. (2003), Burkhead et al. (2009), and Yruela (2009) Sunkar et al. (2000) Arazi et al. (1999) and Talke et al. (2003) Seigneurin-Berny et al. (2006), Yruela (2009), and Kim et al. (2009) Eren and Argüello (2004) Pilon et al. (2009) and Morel et al. (2009) Eren and Argüello (2004) Andres-Colas et al. (2006) and Yruela (2009)

Heavy Metals and Plastid Metabolism

TABLE 26.1 Heavy-Metal Transporters and Ion Channels Characterized In Vivo or In Vitro and Localized Experimentally or Only Computer Predicted to Different Plant Membranes

(continued)

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678

TABLE 26.1 (continued) Heavy-Metal Transporters and Ion Channels Characterized In Vivo or In Vitro and Localized Experimentally or Only Computer Predicted to Different Plant Membranes Proposed Metal Specificity (++ Indicates the Primarily Transported Metal) Carrier Type

Magnesium-selective ion channel Mg2+ (Zn2+)/H+ antiporter NRAMP

Cd

Co

Cu

HMA6/PAA1

++

HMA8/PAA2

+

OsHMA9 RAN1/ HMA7 TaLCT1 (Ca)

+ +

Fe

Mn

Ni

Pb

Zn +?

+

+

PE PI

+

+

+

MGT1 (Mg)

Localization

+

+

+

MHX (Mg) NRAMP1

+ +

+ ++

NRAMP3 TcNRAMP3

+

++

+

PM ER, PG PM

PM +

T PE

+

T

References Shikanai et al. (2003), Abdel-Ghany et al. (2005), Puig et al. (2007), and Yruela (2009) Shikanai et al. (2003), Puig et al. (2007), and Burkhead et al. (2009) Lee et al. (2007) Yruela (2009) and Kim et al. (2009) Schachtman et al. (1997), Antosiewicz and Hennig (2004), Wojas et al. (2007), and Szczerba et al. (2009) Li et al. (2001) Shaul et al. (1999) and Berezin et al. (2008) Curie et al. (2000), Thomine et al. (2000), Grotz and Guerinot (2006), and Puig et al. (2007) Grotz and Guerinot (2006) and Oomen et al. (2009)

Handbook of Plant and Crop Stress

Low-affinity cation transporter

Carrier

Oligopeptide transporter (OPT) Permease

YSL1, YSL3 ZmYS1 PIC1

Vacuolar iron transporter ZRT, IRT-like proteins (ZIP)

VIT1 IRT1

IRT3 ZIP2 and ZIP4 ZIP4

+

+ Cu-NA

+

Zn-NA

+

+ + +

+

Fe-NA Fe-PS + + ++

+

+

+

T PM PM PE

+

T PM

+ ± +

PM PM? PE

Hall and Williams (2003), Grotz and Guerinot, (2006), and Oomen et al. (2009) Waters and Grusak (2008) Curie et al. (2001) Duy et al. (2007) and Puig and Penarrubia (2009) Kim et al. (2006) and Briat et al. (2007) Cohen et al. (1998), Connolly et al. (2002), Fodor (2006), Puig et al. (2007), and Mills et al. (2008) Broadley et al. (2007) Burkhead et al. (2009) Grotz et al. (1998), Hall and Williams (2003), and Grotz and Guerinot (2008)

Heavy Metals and Plastid Metabolism

NRAMP4 TcNRAMP4

Notes: Carriers are listed in alphabetic order. For more details see the cited references. ++ indicates the main transported HM, and when the transporter is primarily responsible for the transport of another (non-heavy) metal, the symbol of the metal is indicated in parenthesis after the name of the carrier. Abbreviations: ABC, ATP-binding cassette; ER, endoplasmic reticulum; G, Golgi; M, mitochondrial membrane; NA, nicotianamine; PE, plastid envelope; PG, post-Golgi membranes; PI, plastid inner membranes; PM, plasma membrane; PS, phytosiderophore; T, tonoplast. ? Question marks indicate uncestainties.

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these transporters is still rather scarce and many of them are only characterized in vitro. Some of them are expressed in an organ-specific manner, and their intracellular localization also varies, i.e., some are located in the plasma membrane, others are involved in other endomembranes such as chloroplast or mitochondrial envelopes, Golgi, and tonoplast membranes (Table 26.1, reviewed by Hall and Williams 2003). As a consequence, some of them have a role in metal uptake, extracellular detoxification, or long-distance transportation of HMs within the plants (from those located in the plasma membrane); others have roles in the sequestration of excess HMs in the vacuole or in their remobilization from this organelle (these are found in the tonoplast) or in metal delivery to other intracellular compartments. While transporters require active transport through symporters and antiporters, and can transport ions against an electrochemical potential gradient, passive ion transport occurs through channels, which are membrane proteins with ion-selective pores that allow ion movement down an electrochemical gradient. Among cationic channels, some are not highly selective and can therefore participate in the transport of different toxic cations (reviewed by Demidchik et al. 2002, Table 26.1).

26.2.2  Interactions between Different Metals The different HMs can interact in the soil. Positive and negative synergisms, competition, protection, and sequential additivity are observed among the interactions. The nature of interactions varies considerably with concentration levels, soil pH, soil texture, level of soluble Ca in soil, presence of salinity, differential distribution in soil of the metals present in high quantities, presence or absence of chelating agents, soil organic matter levels, and other factors (reviewed by Wallace et al. 1992). HMs bind organic ligands with different stability constants or may form precipitates with inorganic anions in the soil solution of the rhizosphere. This way, they might mutually influence each other’s solubility or compete for different binding ligands, including those secreted out by the plant to improve the solubility of essential metals (e.g., phytosiderophores for Fe; Delhaize and Ryan 1995, Hinsinger et al. 2003). As illustrated in Table 26.1, some HMs use the same ion channels, metal transporters or chelators, and therefore they have an impact on each other’s uptake and intracellular concentration. Therefore, toxic HMs often cause reduced productivity and biomass in crop plants indirectly, by inducing essential-metal deficiency in the plants. It is generally assumed that for nonessential elements such as Cd, there are no specific uptake mechanisms. Cd ions compete with nutrients such as K, Ca, Mg, Fe, Mn, Cu, Zn, and Ni (Table 26.1, reviewed by Pál et al. 2006, Clemens et al. 2009). Some data suggest that Cd can be taken up via the phytosiderophore pathway as well (reviewed by Reichman and Parker 2005, Fodor 2006). This outlines that, besides alleviating nutrient deficiency of Fe, Zn or Ni, phytosiderophores also increase the bioavailability of toxic metals and thus increase the potential for food-chain transfer hazards for them (e.g., for Cd), and they also increase the competition between Fe and other metals, leading then to physiological Fe-deficiency. In Cd-treated leaves, Cd can enter guard cells via Ca channels (Perfus-Barbeoch et al. 2002). It was also observed that Pb can enter the cells via Ca and Ni transport systems (Table 26.1, Arazi et al. 1999, Sunkar et al. 2000, Wojas et al. 2007). However, data are relatively scarce in the literature about the uptake of nonessential HMs. Besides the competition of metals in the soil and during metal uptake in the roots, HMs also compete in their translocation from the roots to the shoots. Since very little metal in plants is assumed to exist as free ions, several small organic molecules have to be implicated in metal ion homeostasis as metal ion ligands or chelators, in order to improve acquisition and transport of metal ions with low solubility and to enhance immobilization for metal tolerance and storage. Citrate, mugineic acid, avenic acid, deoxymugineic acid (Suzuki et al. 2008) and nicotianamine have been shown to participate in the intra- and intercellular transport of essential metals such as Cu, Fe, Mn, Ni, or Zn (reviewed by Fodor 2006, Puig et al. 2007, Chen et al. 2009, Yruela 2009). In vitro, nicotianamine is able to form stable complexes with Mn, Fe, Co, Zn, Ni, and Cu, in increasing order of affinity (Curie et al. 2009). The pH stability of these complexes suggests their occurrence in symplasm or

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apoplasm, indicating that nicotianamine should complex Cu, Fe, and Zn in the phloem, and Cu and Zn in the xylem for their translocation from roots to shoots (reviewed by Yruela 2009). The Zn/Cd-transporting ATPases, HMA2 and HMA4, essential for root-to-shoot Zn translocation, facilitate the transport of Cd (Table 26.1, Wong and Cobbett 2009). Citrate was found to be the principal compound chelating metals in the xylem sap (Cd: reviewed by Fodor 2006, Hasan et al. 2009; Pb: reviewed by Fodor 2002, 2006). Finally, after their xylem unloading, essential HMs must enter the symplasm of the cells in the aerial parts of the plant by different membrane transport systems (Table 26.1) and have to reach the intracellular compartment where they will be used. The mechanisms of intracellular HM trafficking is still not very well understood. In the next part of the chapter, we review the effect of unbalanced metal concentrations on the structure and function of different plastids.

26.3  Metals and Plastid Metabolism The plastid is a unique, semiautonomic organelle characteristic of photosynthetic eukaryotic cells and evolved from the endosymbiosis of free-living cyanobacteria with an ancient eukaryotic cell (reviewed by Solymosi and Schoefs 2008). All plants contain plastids. These organelles are widespread within the plants, because with a few exceptions, all cells possess plastids in one form or another. Despite their diversity, plastids have several common features. Their boundary to the cytoplasm is a double membrane called the plastid envelope, which encircles the protein-rich stroma. The outer membrane is permeable to molecules up to a molecular mass of ca. 6 kDa due to the presence of porins, while the inner membrane is highly selective and contains different membrane transporters (e.g., Table 26.1, reviewed by Weber et al. 2005, Johnson et al. 2006, Aronsson and Jarvis 2008). Besides the more or less developed inner membrane system, the plastids often contain spherical bodies that contain lipids, carotenoids, plastoquinone, and proteins and others that are called plastoglobuli that contain lipids, carotenoids, plastoquinone, and proteins (Austin et al. 2006). In addition, the plastids contain nucleoids (DNA-containing structures), procaryotic ribosomes and, as semiautonomic organelles, they can synthesize at least part of their proteins. Different inclusions are also frequently seen in the stroma of the plastids. One example for them is the Fe-containing phytoferritin or simply ferritin (reviewed by Briat et al. 1999). Depending on their physiological function, chemical composition and internal structure, the plastids are divided into different groups (reviewed by Solymosi and Schoefs 2008). Chloroplasts are present in many different types of cells and organs (i.e., in ripening or mature fruits, in green colored parts of flowers like the calyx and the gynoecium, in green stems and leaves). Their presence is essential because they provide energy and oxygen to the biosphere via photosynthesis. Their inner membrane system is laterally segregated into two major functional domains, the appressed (stacked) granal membranes and the interconnected, non-appressed stromal membranes (reviewed by Solymosi and Schoefs 2008). Besides chloroplasts, several nongreen plastid types exist and are developmentally interrelated. Proplastids are characteristic in meristematic cells (e.g., in the root and shoot apical meristems), and in dedifferentiated and/or reproductive cells (reviewed by Solymosi and Schoefs 2008). Proplastids are small and have only a poorly developed inner membrane structure. They can differentiate into any other plastid type. Under natural light conditions, and in photosynthesizing organs, they differentiate into chloroplasts. However, in angiosperm plants, in the absence of light, normal chloroplast development is impaired, and proplastids differentiate to so-called etioplasts (e.g., Solymosi et al. 2004, 2006a, 2007, reviewed by Solymosi and Schoefs 2008). The formation of etioplasts is a natural phenomenon observed in different crops (e.g., cabbage heads—Solymosi et al. 2004) and in seeds germinating in the soil in agricultural systems (e.g., sunflower: Solymosi et al. 2007; bean: Schoefs and Franck 2008, reviewed by Solymosi and Schoefs 2008). These plastids contain special inner membrane system consisting of lamellar prothylakoids and a paracrystalline membrane network called prolamellar body.

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During senescence of the tissues, and also before defoliation of the leaves, the conversion of chloroplasts to the so-called gerontoplasts (or senescing chloroplasts) can be observed (reviewed by Thomas 1997). During this transformation, chlorophyll (Chl) is degraded, the inner membrane system of the chloroplasts is disorganized and large, often electron-transparent plastoglobuli appear (e.g., Solymosi et al. 2004). The leucoplasts are colorless plastids with poorly developed inner membranes; they are specialized in storage of either starch (amyloplasts) or lipids (elaioplasts) or proteins (proteinoplasts) and function therefore as storage organelles. These plastids are heterotrophic and convert photosynthates derived from source tissues into storage compounds. Amyloplasts are characteristic of the parenchymatic tissues of storage organs (tubers, rhizomes, roots, fruits, seeds) but can also be found in root cap cells, where they are associated with geotropism (reviewed Solymosi and Schoefs 2008). The chromoplasts accumulate carotenoids and are responsible for the bright yellow, orange, and red colors of petals, fruits like tomatoes, pepper, roseberry and for that of some roots, i.e., carrot (reviewed by Solymosi and Schoefs 2008). Thus, in addition to photosynthesis, plastids harbor many more vital biosynthetic functions, such as nitrogen and sulfur assimilation or the biosynthesis of fatty acids, (aromatic) amino acids, lipids, pigments (Chls and carotenoids), purines, pyrimidines and secondary metabolites including terpenoids, and other important compounds used in pharmaceutical or perfume industry. In consequence, these functions require an active solute exchange across the outer and inner envelope membranes surrounding the chloroplast stroma (reviewed by Weber et al. 2005). Metal transport proteins in both membrane systems thus provide a bottleneck to the control of metal homeostasis in the chloroplast as well as in the plant cell (Table 26.1). The transition metals Fe, Cu, and Mn play a vital role in photosynthetic electron transport in chloroplasts and in stroma-located reactions of CO2 fixation (Table 26.2). Plastid localized Fe and Cu/Zn superoxide dismutases scavenge reactive oxygen species (ROS). In addition, Zn is known to function as a cofactor (in RNA polymerase, Zn finger domains) in plastid transcription (Table 26.2), while among others, Fe is required for heme and for Fe-S clusters (Cornah et al. 2002) and for enzymes of Chl biosynthesis (Myśliwa-Kurdziel and Strzałka 2002, Duy et al. 2007). Several other enzymes functioning in other plastid types also require HMs (Table 26.2). Among all plastid types, the effect of metal stress and also the functioning of metal uptake machineries are best characterized in chloroplasts, but there are also a few data indicating changes induced by excess metals in other plastid types (for details see below). Both essential-metal deficiency and excess influence plastid metabolism. Similarly, various concentrations of nonessential metals also affect plastid structure and function. In this chapter, these processes are briefly summarized. Since the Chl concentration may fundamentally influence the functioning of the photosynthetic apparatus and thus affect the whole plant metabolism, it is a really important factor in assessing the impact of metal stress in chloroplasts and on plant productivity. One of the most usual symptoms of metal deficiency or metal excess is chlorosis, i.e., the decrease of the Chl content. This further outlines, that plastids—and especially chloroplasts—are central targets in metal stress.

26.3.1  Impact of Unbalanced Metals on Nongreen Plastids As discussed earlier, except in the case of airborne HMs in terrestrial or epiphytic plants, or water polluting elements for aquatic plants, HMs generally first affect the cells of roots or other underground organs (such as potato tubers, onion bulbs) and may interact with their plastids. Unfortunately, there are almost no data about the effect of HM deficiency or excess on nongreen plastids. Although excess HMs and essential-metal deficiency also interfere with the metabolism of nongreen plastids, much less data are available on structural and metabolic alterations of these plastids (Table 26.3) than chloroplasts. For instance, besides chloroplasts, heme biosynthesis mostly occurs in root plastids and etioplasts, and is affected by Fe-deficiency (Cornah et  al. 2002). Nongreen plastids play important roles in several important plant metabolic processes including amino acid

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TABLE 26.2 Nonexhaustive List of Key Molecules Requiring Essential Metals in the Chloroplast Metals Cu

Fe

Mg Mn

Mo

Zn

Proteins Cu/Zn-superoxide dismutase Cytochrome oxidase Plastocyanin Polyphenol oxidase Ascorbate peroxidase Cytochrome b6-f Cytochrome c6 Ferredoxin Ferredoxin–thioredoxin reductase Ferritin Ferrochelatase Fe-SOD Glutamine-2-oxo-glutarate amido transferase NADPH-plastoquinone oxidoreductase Nitrite reductase Pheophorbide a oxygenase Sirohydrochlorin ferrochelatase Sulfite reductase Tic55 Chlorophylls Glutathione synthetase Isocitrate dehydrogenase Malic enzyme Mn-SOD Phenylalanin ammonia lyase Water oxydase Aldehyde oxidase Sulfite oxidase Xanthine dehydrogenase Carbonic anhydrase Cu/Zn-SOD Enzymes involved in RNA editing Metalloendopeptidase Stromal processing peptidase Zn finger Zn-metalloprotease FtsH Zn-protease degrading RUBISCO

References Grace (1990) Hänsch and Mendel (2009) Abdel-Ghany (2009) Kieselbach et al. (1998) Raven et al. (1999) Raven et al. (1999) Raven et al. (1999) Tognetti et al. (2007) Duy et al. (2007) Briat et al. (1999) Cornah et al. (2002) Allen (1995) Duy et al. (2007) Raven et al. (1999) Briat and Vert (2004) Duy et al. (2007) Duy et al. (2007) Duy et al. (2007) Duy et al. (2007) Shaul (2002) Shaul (2002) Elias and Givan (1977) Takeuchi et al. (2000) Grace (1990) Nishizawa et al. (1979) Grace (1990) Weigel et al. (1986) Eilers et al. (2001) Borner et al. (1986) Randall and Bouma (1973) Grace (1990) Hänsch and Mendel (2009) Moberg et al. (2003) Hänsch and Mendel (2009) Sasaki et al. (1989) Bailey et al. (2001) Bushnell et al. (1993)

biosynthesis, hormone synthesis, sugar homeostasis, storage of different metabolites, carotenoid synthesis, and secretion. The secondary metabolite production of nongreen plastids is important in medicinal plants and crops also for human health and nutrition. Often, edible parts of crops contain nongreen plastids (e.g., carrot roots contain chromoplasts; celery, potato tubers, onion bulbs, garlic, and radish contain amyloplasts and/or proplastids; the inner tissues of cabbage heads, avocado and different cucumber fruits contain etioplasts or etio-chloroplasts) (reviewed by Solymosi and Schoefs 2008). However, molecular interactions of these plastids and the effect of metals on plastid metabolism in these organs remain poorly understood. Root plastids seem to be a primary target

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TABLE 26.3 Examples of Alterations in the Ultrastructure of Nongreen Plastids Caused by Excess of Heavy Metals Metal Cd

Species

Plastid Alterations

Bean (Phaseolus vulgaris)

Decreased starch content and reduced internal membrane system in root plastids Ferritin-like deposits in root plastids No ultrastructural effect on etioplasts and on etioplast–chloroplast conversion Disruption of the prolamellar body structure in etioplasts, retarded etioplast–chloroplast conversion (delayed grana formation, swollen intrathylakoidal spaces in greening plastids) Amoeboid plastids in root tip cells, appearance of amyloplasts in upper parts of the roots Increased starch content in etio-chloroplasts of stems Amyloplast-leucoplast transition is induced in roots, decrease in starch content, dark globular inclusions and many small droplets of an electron translucent substance Changes in the unit cell size of prolamellar bodies (PLBs) of etioplasts, formation of vesicles on the PLB surface Amoeboid plastids, different forms of plastid inclusions (“engulfment” of cytoplasm or a mitochondrion)

Maize (Zea mays) Soybean (Glycine max)

Cr (VI)

Bean (Phaseolus vulgaris)

Cu

Oregano (Origanum vulgare)

Hg

Wheat (Triticum aestivum)

99

Tc

Common bean (Phaseolus vulgaris)

References Barceló et al. (1988) Vázquez et al. (1992) Ghoshroy and Nadakavukaren (1990) Ghoshroy and Nadakavukaren (1990)

Vázquez et al. (1987)

Panou-Filotheou and Bosabalidis (2004)

Solymosi et al. (2006b)

Vázquez et al. (1990)

of metal excess. The detailed review of Barceló and Poschenrieder (2006) came to the conclusion that except for Cd, no visible ultrastructural damage is observed in the organelles of the roots, but the metals rather disturb the polar zonation of the organelles within the cells. Changes in the amyloplasts and their arrangement in root columella cells (Table 26.3) may directly influence root gravitropism and growth direction and seem to be associated with, for example, Al stress–induced root growth defects. Sometimes, alterations in plastid shape were also reported, for example, in Cr6+ and 99Tc-treated roots, where amoeboid plastids occurred in the roots (Table 26.3, Vázquez et al. 1987, 1990). Similarly, metal stress altered sugar metabolism and often, changes in the starch contents of plastids have been reported (Table 26.3). The Cd-induced ferritin accumulation in bean root plastids may be related to disturbed Fe homeostasis of these plants (Vázquez et al. 1992). Cu excess–induced the formation of dark globular inclusions of unknown nature in plastids (PanouFilotheou and Bosabalidis 2004). Another important, but poorly studied field is the effect of metals on plastid differentiation, on the biosynthesis of the photosynthetic apparatus and on chloroplast biogenesis. These studies are even more important, as seedlings germinating in polluted soil have to cope with metal stress at this level, and seedlings that fail to develop functionally active chloroplasts do not survive. Chloroplast differentiation may proceed directly from proplastids, or in case of agricultural systems, seedlings germinating from seeds buried in the soil often develop etioplasts before reaching the soil surface (reviewed by Solymosi and Schoefs 2008). Therefore, studies related to the impact of excess metals on proplastids, etioplasts or young chloroplasts differentiating from these two plastid types are crucial to understand the molecular interactions of metals with these organelles and also to enhance seedling survival in polluted areas.

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Several metals (Cd, Na, K and Hg) influence the etioplast to chloroplast transformation (Table 26.3). These elements induce slight structural alterations in etioplast structure, which are in some respect similar to those observed in chloroplasts (see later, i.e., swelling of intrathylakoidal space, formation of vesicles, regularly spotted bodies indicating possible osmotic stress). Chl biosynthesis and etioplast–chloroplast transition (the reorganization of the inner membrane system and the development of the thylakoids and the photosynthetic apparatus) is impaired under different metal stresses under in vitro conditions with short-term, high-concentration treatments (excess Cd: Ghoshroy and Nadakavukaren 1990; excess Na and K: Abdelkader et al. 2007; excess Hg: Solymosi et al. 2006b). This might indirectly indicate that these processes may be also partially inhibited in the light.

26.3.2  Impact of Unbalanced Metals on Photosynthesis Chloroplasts are key organelles for plant development, growth and biomass production because of their ability to produce sugars via photosynthesis. On the other hand, photosynthesis is a process that requires several metalloproteins containing different HMs (Table 26.2). The different essential metals present in plastids, besides being structural constituents of various molecules, are also important in modulating reactions or cellular processes, or in maintaining the ion homeostasis of the organelles or the cells. For instance, plastids seem to have a role in Ca (Seigneurin-Berny 2000), Cu (Abdel-Ghany 2009), and Fe (Izaguirre-Mayoral and Sinclair 2005, 2009) storage, Fe being often stored in the metabolically inactive form of phytoferritin, which prevents photooxidation reactions caused by “free” metal ions (Briat et al. 1999, Arosio and Levi 2002, Ravet et al. 2009). As discussed earlier, plants possess different uptake systems to transport essential metals into the plastids (Table 26.1). Part of these transporters can also deliver nonessential elements. However, the knowledge about plastid metal transporters in respect to metal stress is relatively scarce and it is difficult to determine the exact concentration of metals inside chloroplasts. Most of such studies have been done on algae, in which the accumulation of different metals can be easily observed in thylakoid membranes (reviewed by Barceló and Poschenrieder 2006), but they are not relevant for terrestrial plants. Only rough estimations exist, that assume that 1% of total Cd content of plants is probably transported to plastids (reviewed by Siedlecka and Krupa 1999). Some data about metal concentrations in chloroplasts are summarized in Table 26.4. Unfortunately, even if available, often other literature data about plastid metal contents are not comparable, because they are expressed in different and non-interconvertible units or on different basis (e.g., Cd: Ramos et al. 2002; Cu: Baszyński et al. 1978; Zn, Mg, Cu: Kim et al. 2009) or were determined only in thylakoids (e.g., Mn: Lidon and Teixeira 2000a, Lidon et al. 2004) or were measured after different durations of metal treatments (e.g., Ramos et al. 2002).

TABLE 26.4 Intraplastidial Concentration of Some Essential Metals Metal Ca Cu Fe K Mg Mn Zn

Concentration within the Plastid

References

Total: 4–23 mM, free: few μM 60 μM 0.13 mM 150 mM 5 mM 33 μM 0.13 mM

Johnson et al. (2006) Joyard and Douce (1976) Joyard and Douce (1976) Neuhaus and Wagner (2000) Neuhaus and Wagner (2000) Joyard and Douce (1976) Joyard and Douce (1976)

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At the same time, the mode of HM pollution is also important in determining the interactions of the metals with plastids. In the leaves of lettuce seedlings treated with Cd solutions for 16 days through their roots, the lowest intracellular Cd concentration (6–16 μg g−1 FW, 12%–14% of total Cd content of leaves) was found in the chloroplasts (Ramos et al. 2002). Interestingly, the Cd content of the chloroplasts was 10–22 μg g−1 FW (8%–9% of total leaf Cd content) when lettuce leaves were incubated for 24 h in Cd-containing solutions, a model experiment mimicking airborne metal pollution (Ramos et al. 2002). In both cases, most Cd was accumulated in the apoplasm. These studies indicate that, in case of airborne metals, atmospheric metal deposition may have a stronger influence on plastid metabolism than soil pollution (Ramos et al. 2002). Developing methods of electron microscopy combined with analytical techniques such as energy dispersive x-ray microanalysis (EDXA), laser microprobe mass analysis (LAMMA), electron energy loss spectroscopy (EELS), Synchrotron x-ray fluorescence (SXRF) microbeam analyses, secondary ion mass spectrometry or cytochemical methods are powerful tools to characterize primary mechanisms of metal toxicity and tolerance on the cellular and molecular level (reviewed by Barceló and Poschenrieder 2006). Similarly, atomic force microscopy, laser scanning optical microscopy using confocal microscopy or multiphoton excitation provide important information about metal stress on the cellular and tissue level. New methods are also under development (e.g., laser-induced breakdown spectroscopy and laser-ablation inductively coupled plasma mass spectrometry—Kaiser et al. 2009). Unfortunately, data about microlocalization of metals in plastids with these imaging methods are relatively scarce, probably because of the low concentrations accumulating in these organelles being under the actual detection limit of these methods. However, direct interaction of the metals with chloroplast membranes and metabolism cannot be excluded even in cases when the amounts of metals were below the detection limit in the plastids. Therefore, we overview some data that reported direct connections between the absence or the presence of metals in plastids, and observed metabolic alterations in vivo. Chl biosynthesis requires several metals; therefore, decreased Chl content is a general symptom of essential-HM deficiency (Cu: Burkhead et  al. 2009, Hänsch and Mendel 2009, Yruela 2009; Fe: Puig et al. 2007; Mn: Simpson and Robinson 1984, González and Lynch 1999, Yu et al. 1999, Henriques 2003, 2004; Zn: Singh et al. 2005). There are almost no data about metal concentrations in plastids under HM deficiency. Different estimations indicate that about 80% of foliar Fe is present in the plastids (Terry and Low 1982, Thoiron et al. 1997), and in Arabidopsis green tissues, 40% of Fe is found in the thylakoids (reviewed by Briat et al. 2007), so these organelles can be expected to be one of the first targets of Fe-deficiency. Chlorotic leaves of Fe-deficient plants have yellowish color because of decreased Chl content due to impaired Chl biosynthesis and the symptoms appear first in the interveinal areas (Thoiron et al. 1997, Misra et al. 2006, Mahmoudi et al. 2007, Timperio et al. 2007). Some authors have postulated that Fe-deficiency induced chlorosis is related to decreased Fe and Chl concentrations in thylakoid lamellae, and this way to retarded or disturbed thylakoid formation rather than to inhibited Chl biosynthesis per se (Terry and Low 1982). Moreover Fe-deficiency would impact photosystem I (PSI) functioning because of the reduced number of Fe-S clusters (Doan et al. 2003, Duy et al. 2007). The effect of excess HMs is often also reflected by reduced plant growth and chlorosis. In these cases, often the observed phenotypic alterations could be directly linked with the presence of the excess metals in the chloroplasts. For instance, the toxic effects of Ni on chloroplast structure of cabbage plants (Molas 1997, 2002) are in good agreement with the observed Ni accumulation inside the organelles. The histochemical techniques of Ni localization show that in cabbage plants the important sites for Ni accumulation in the leaf are mesophyll cells located on leaf edges, near the vascular bundles and intervascular bundles affected with chlorosis (Molas 2002). The chloroplast, the cell walls and the nucleus are the most important Ni-accumulation sites at the cellular level. In rice plants treated with increasing concentrations of Mn, the accumulation of this metal was found in the leaves and thylakoid membranes, which indicates that this element is highly mobile, enters the chloroplasts and accumulates up to a certain level in these organelles (Lidon et al. 2004). Similarly, it has been demonstrated that large part of Mn quantity entering the cytoplasm moves and

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binds on the outer side of thylakoid membranes of chloroplasts (González and Lynch 1999, Lidon and Teixeira 2000b, Lidon et al. 2004) affecting their structure and photosynthesis. Moreover leaf chlorosis induced by excess Mn was positively correlated to Mn content of chloroplasts (González and Lynch 1999). Therefore, in this case, the observed ultrastructural symptoms might be at least partially linked to direct interactions of Mn with thylakoid membranes. Chloroplasts are one of the main sites of Cu accumulation (Maksymiec 1997). Data indicate that under Cu treatment, this ion can enter the cytoplasm and the plastids also by Ca channels especially in younger plants and at the initial stages of the stress (Maksymiec and Baszyński 1999). Cu can bind directly to the thylakoids where it may induce oxidative stress (Figure 26.1, reviewed by Phytochelatins n Gly

(m)

GSSG proline

PCS ?

GR

Metals

Cd, Co, Cu

NADP+

GSH

n (GSH)

NADPH Cu Cd

(a)

RUBISCO

H2O2

O2

Reduced redox metals

Metals (e.g., Cu, Zn) H+

(b) 2O 2

Oxidized redox metals

O2–

s-SOD

(f )

Lipoxygenase Cu P U F A

(c) R

Thylakoid membrane

R C H O O O + O H C R'

Metals

OH– OH Proline

(g)

Stroma

As, Cd, Cr Cu, Hg, Ni, Zn

H2O2

(h) Fd

Calvin cycle (l) enzymes

(i)

H2O2

A

AP

m-SOD

Cd

MDHA H2O

?

Epoxidase

Acc PAA2/ HMA8

(d)

Lumen

Cu

e– PS

(e)

(f )

Cu Cd

(j) Xantophyll cycle

O2–

RC Cd, Cu, Hg Ni, Pb, Zn

PC

De-epoxidase

(k) Hg, Cd

FIGURE 26.1  Oxidative stress and defense reactions occurring in the chloroplast, when excess of metal is present. (a) Depletion of sulfhydryl groups by metals on reduced glutathione (GSH) regenerated by glutathione reductase (GR) from its oxidized form (GSSG); (b) Activation of lipoxygenase by metals; (c) Peroxidation of polyunsaturated fatty acids (PUFA) to saturated fatty acids (RCOOH + R′COOH) in membranous phospholipids; (d) Cu transporter PAA2/HMA8; (e) Electron transfer from the reaction center (RC) to the acceptor (Acc) of PSI or PSII; (f) univalent oxygen reduction by PSII; (g) Electron transfer through ferredoxin (Fd) and NADP+ (specific to PSI); (h) Fenton and Haber–Weiss reactions: one-electron oxidoreductions performed by redox metals leading to hydroxyl radicals (OH•); (i) Spontaneous and/or superoxide dismutase (SOD) catalyzed disproportionation of superoxides (O2−). Superoxide radicals generated by PSI and PSII are dismutated by the membrane-bound m-SOD, and the formed hydrogen peroxide (H2O2) is reduced to water by the thylakoid-bound ascorbate peroxidase (AP). Monodehydroascorbate (MDHA) is reduced back to ascorbate (A) by the photoreduced ferredoxin (Fd). O2− ions escaped from the thylakoid system are scavenged by a stromal s-SOD; ( j) Xanthophyll cycle associated to the enzymes epoxidase and de-epoxidase, preventing superoxide formation at high light intensity; (k) Inactivation of plastocyanin (PC) by nonessential metals after its release from the membrane or the release of its Cu; (l) Inactivation of Calvin cycle enzymes; (m) Chelation of metals by phytochelatins either imported from the cytoplasm or synthesized through the activity of a putative phytochelatine synthetase (PCS). → activation by metals; ┤ inhibition by metals.

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Maksymiec 1997). Under excess Cu, the Cu content of leaf chloroplasts showed more than tenfold increase on the protein or Chl content basis (Baszyński et al. 1988), which indicates that this element might directly interact with chloroplast components or might induce oxidative stress directly. In case of poorly mobile, nonessential metals such as Cr, present in the soil, the ultrastructural changes induced in the plastids and chlorosis are probably due to indirect effects of the metal only, because they appear without substantial increase in the metal concentration in the leaves (e.g., Moustakas et al. 1997, reviewed by Barceló and Poschenrieder 2006). As Table 26.2 lets it assume, each essential HM has specific functions and is needed in appropriate amounts within chloroplasts. Thanks to efficient ion homeostasis, plant development and functioning may be optimal also under moderate HM stress, but large metal unbalance is a disaster for plastids both on the structural and metabolic levels. 26.3.2.1  Ultrastructural Alterations in Chloroplasts As some HMs are crucial for the functioning of several proteins and structural components of chloroplasts (Table 26.2), it is not surprising that too low concentrations of HMs may cause chlorosis, functional alterations of photosynthesis and other ultrastructure. Before discussing the effects of toxic concentrations of metals on chloroplast structure, we briefly review the symptoms of essential-metal deficiency. Ultrastructural alterations are easy to be observed, but are more complicated to be interpreted on the metabolic level. Since unbalanced metal concentrations can be the result of several phenomena (including competition of essential and nonessential metals for transport systems in plants and this way to indirect effects), and to interaction of different metals with each other on several levels, including metabolic levels, the most reliable data about the direct ultrastructural effects of essential-metal deficiency are those that were obtained in nutrient transporter mutants impaired in chloroplast metal uptake or homeostasis (Henriques et al. 2002, Song et al. 2004, Duy et al. 2007). The disturbances in thylakoid biosynthesis can be in this case directly related to nutrient deficiency, outlining the importance of essential metals in plastid differentiation and in the maintenance of the active photosynthetic apparatus. Some recently characterized knockout mutants impaired in Fe and Zn uptake (irt1) or in their transport into the plastids (pic1) show several ultrastructural alterations at the chloroplast level. Irt1 mutants have reduced thylakoid system and stacking (maximum 2–3 thylakoids per grana), smaller starch grains and increased number of plastoglobuli (Henriques et al. 2002). Pic1 knockouts have unchanged Fe level, but changed ion homeostasis, Cu content, and their internal membrane system is significantly reduced (Duy et al. 2007). No grana, just simple and parallely arranged thylakoids or even no thylakoid characterize the plastids. Vesicle formation and increased ferritin content are also characteristic in these mutants (Duy et al. 2007). There are several other data in the literature that describe the ultrastructural alterations of chloroplasts of plants grown under essential-HM deficient conditions (Figure 26.2, Table 26.5).

(a)

1

2

3

(b)

4

5

(c)

6

7

8

FIGURE 26.2  Scheme summarizing the most important ultrastructural alterations of chloroplasts (b) induced by essential metal deficiency (a) or by excess of essential or nonessential metals (c). Note the swelling of the organelle and the alterations of the different structural elements of chloroplasts (1: plastoglobuli, 2: swollen intrathylakoidal space, 3: peripheral vesicles, 4: starch grains, 5: grana, 6: regularly spotted bodies, 7: dark deposits on the thylakoid surface 8: ferritin clusters).

HMs Cd

Cr Cu Cu

C Number

Mn

Disturbed C Shape

DE

TD

ST

Size of PG

+

+

+

+

↑

+ +

+ +

↑

↑

+

+

↑

↓

+

↓

↓

+ ↓ ↑

Fe Fe Mn

C Size

↓

+

Zn

↑

S

V

F

SB

DD

↑

+

+ + +

+ +

References

↑

+

Baszyński et al. (1980), Barceló et al. (1988), Ouzounidou et al. (1997), Baryla et al. (2001), McCarthy et al. (2001), Sandalio et al. (2001), Carrier et al. (2003), and Djebali et al. (2005) Vázquez et al. (1987) Baszyński et al. (1978) and Henriques (1989)

↑ ↓

↓

+

+

↑

↓ ↑

+

+

+

↑

↓

+

+

+

↑

↑

↑

+ +

+

↑ ↑

↑

↑ ↑

Baszyński et al. (1988), Eleftheriou and Karataglis (1989), Maksymiec et al. (1995), Ciscato et al. (1997), Quartacci et al. (2000), Panou-Filotheou et al. (2001), and Bernal et al. (2006) Platt-Aloia et al. (1983), Ji et al. (1984), Thoiron et al. (1997), Briat et al. (1999), and Henriques (2003) Izaguirre-Mayoral and Sinclair (2005) Mercer et al. (1962), Possingham et al. (1964), Weiland et al. (1975), Henriques (2003, 2004), Izaguirre-Mayoral and Sinclair (2005), and Papadakis et al. (2007) Lidon and Teixeira (2000a), McQuattie and Schier (2000), and Papadakis et al. (2007) Molas (1997, 2002)

+ ↓

Ni Pb Tc Zn

+

Number of PG

↓ +

+

+

+

↑ ↑

↑

↓

+

+

+

Heavy Metals and Plastid Metabolism

TABLE 26.5 Chloroplast Alterations of Crop Plants Caused by Essential Metal Deficiency (on White Background) or by Excess of Heavy Metals (HMs) (on Gray Background)

Woźny et al. (1995), Weryszko-Chmielewska and Chwil (2005), Vázquez et al. (1990) Thomson and Weier (1962), Henriques (2001), and Chen et al. (2008) Doncheva et al. (2001)

Notes: Plus signs indicate that the alteration was described by authors, upward (↑) and downward (↓) arrows indicate increase and decrease, respectively. Abbreviations: C, chloroplast; DD, dark deposits at thylakoids; DE, disruption of the envelope; F, ferritin; PG, plastoglobuli; S, starch content; SB, regularly spotted body; ST, swollen intrathylakoidal space; TD, thylakoid disorders; V, unusual peripheral vesicles.

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Chloroplasts are often swollen in such plants (Zn deficiency: Chen et al. 2008), or sometimes have smaller size than in plants grown under optimal metal concentrations (Fe deficiency: Platt-Aloia et al. 1983; Mn deficiency: Papadakis et al. 2007). Rarely, the disruption of the plastid envelope has been observed in such plants (Zn-deficient rice: Chen et al. 2008). Abnormal chloroplasts with almost no grana and disrupted or disorganized internal membranes (Cu deficiency: Thoiron et al. 1997, Briat et al. 1999, Henriques 2003; Mn deficiency: Possingham et al. 1964, Weiland et al. 1975, Izaguirre-Mayoral and Sinclair 2005; Zn deficiency: Thomson and Weier 1962, Henriques 2001, Chen et al. 2008), the swelling (dilatation) of the intrathylakoidal space (Cu deficiency: Baszyński et al. 1988, Henriques 1989; Fe deficiency: Thoiron et al. 1997; Mn deficiency: Weiland et al. 1975, Izaguirre-Mayoral and Sinclair 2005, Papadakis et al. 2007; Zn deficiency: Chen et al. 2008), the appearance of clusters of unusual, peripheral vesicles (Fe deficiency: Platt-Aloia et al. 1983, Briat et al. 1999; Mn deficiency: Mercer et al. 1962, Possingham et al. 1964), increased number (and size) of plastoglobuli (Cu deficiency: Henriques 1989; Fe deficiency: Ji et al. 1984, Henriques 2003; Mn deficiency: Weiland et al. 1975, Izaguirre-Mayoral and Sinclair 2005; Zn deficiency: Chen et al. 2008) are usual symptoms of metal deficiency. The starch contents of plastids also vary with different essential-HM availability; however, these changes are not consistent, i.e., Fe deficiency causes decreased starch content in apple chloroplasts (Ji et al. 1984), while starch content increases in chloroplasts of Fe-deficient pecan (Henriques 2003). Decreased starch size and content is observed in plastids of Mn-deficient lemon (Papadakis et al. 2007) and soybean (Weiland et al. 1975, IzaguirreMayoral and Sinclair 2005). Several data are available about the ultrastructural alterations caused by the excess of various HMs added in various treatments, under various experimental conditions and in various plant species. However, interestingly the symptoms are quite similar (Table 26.5). Most studies have been conducted under laboratory conditions (e.g., hydroponic cultures, etc.), but sometimes the plastid ultrastructure of plants grown in naturally contaminated soils has been also described (wheat plants grown in Cu-contaminated soils: Eleftheriou and Karataglis 1989). These plants contain a reduced number and a reduced size of chloroplasts, less developed plastid inner membrane system; they also have impaired grana formation, decreased starch content and decreased number of plastoglobuli (Eleftheriou and Karataglis 1989). In some cases, different steps of plastid ultrastructural alterations were distinguished as a function of increasing concentrations and/or duration of metal exposure (e.g., Na: Rahman et al. 2000; Zn: Doncheva et al. 2001). In this latter case, the first symptoms of Zn stress observed at lowest HM concentrations is the disintegration of stromal thylakoids, and the reduction of grana number, swollen thylakoids (i.e., the swelling of the intrathylakoidal space), decreased starch content, increase in size and number of plastoglobuli appear subsequently with increasing Zn concentrations (Doncheva et al. 2001). In the leaves of plants grown in experimental conditions with excess of HMs, the number of chloroplasts may decrease (Table 26.5), which is probably due to metal interference with chloroplast replication (reviewed by Kucera et  al. 2008). Besides this phenomenon, damage to chloroplasts is the most frequently observed ultrastructural effect of toxic metals in leaves. The inhibition of normal plastid development in HM-treated plants may be indicated by the appearance of amoeboid plastids observed occasionally (Vázquez et al. 1990) and is further supported by the fact that, in general, chloroplasts of young leaves are more affected by metal stress than old leaves (e.g., Maksymiec et al. 1995, Skórzyńska-Polit and Baszyński 1997, Maksymiec and Baszyński 1999). However, contradictory data have been also published (Barceló et al. 1988), and these observations might not be generalized, they also depend on the developmental stage at which metal pollution occurs. The most common ultrastructural symptoms are the swelling of the organelle, distortion of thylakoids leading to the loss of the parallel arrangement of the thylakoid membranes, reduction or increase of the thylakoid surface area, and swelling of the intrathylakoidal space (Table 26.5, reviewed by Barceló and Poschenrieder 2006). There are contradictory data about the changes in the starch content upon HM excess, which may increase or decrease in the plastids (Table 26.5). Often, the

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senescence of the chloroplasts, i.e., the chloroplast-gerontoplast conversion, is induced by the excess of metals (Table 26.5), which is not only indicated by the degeneration of grana and disorders in the thylakoid system but also by the increase in the number and size of plastoglobuli. These symptoms may be a result of metal-induced alteration of the hormone balance (e.g., Cd: Vassilev et al. 2004, Rodríguez-Serrano et al. 2009, reviewed by Maksymiec 1997, for more details about all metals, see Fodor 2002, Maksymiec 2007) leading to enhanced senescence. Most studies have dealt with plastid ultrastructure in leaves, however, some data indicate that chloroplasts in the green stems are also similarly damaged by excess of metals (Barceló et al. 1988). The careful comparison of the ultrastructural effects induced by essential-HM deficiency and excess of HMs (Figure 26.2, Table 26.5) indicates that besides several similar structural alterations, there are specific differences. Regularly spotted bodies, appearance of dark electron dense deposits at grana surfaces and ferritin were only related to the presence of excess HMs and might therefore be specific for this type of stress, while unusual peripheral vesicles appear under metal-deficient conditions (Figure 26.2, Table 26.5). Disruption of the plastid envelope was more often observed in plastids of plants treated with excess HMs. These results further outline the complicated interrelations and the difficulties in interpreting the effects of HMs at the molecular and cellular level. The observed effects have been mostly interpreted as the excess or deficiency of HMs on membranes or osmotic disturbances. However, it is unclear to what extent these ultrastructural effects are due to direct toxicity of the metal ions in the chloroplast, to metal-induced membrane disturbances, to metal-induced enhancement of ROS, to osmotic problems, or to consequences of metal-induced deficiency of essential nutrients. In the following sections we briefly review the possible reasons of the observed ultrastructural alterations. 26.3.2.2  M  olecular and Metabolic Alterations in Chloroplasts under Heavy-Metal Deficiency Essential HMs are needed for normal plastid ultrastructure, homeostasis, and functioning (i.e., they represent functional components of the thylakoids and the stroma, take part in plastid protein synthesis, DNA replication, … etc., Table 26.1), therefore, it is not surprising that their structure is strongly influenced in plants grown under nutrient-deficient conditions (Figure 26.2, Table 26.5). Nutrient-deficiency symptoms are usually expressed as reduced growth, biomass, and physiological functions. Chl biosynthesis requires several metals (Table 26.2); therefore decreased Chl content is a general symptom of essential-HM deficiency (Cu: Burkhead et al. 2009, Hänsch and Mendel 2009, Yruela 2009; Fe: Puig et al. 2007; Mn: Simpson and Robinson 1984, González and Lynch 1999, Yu et al. 1999, Henriques 2003, 2004; Zn: Singh et al. 2005). Not only because of the decreased Chl content, but due to direct interactions with photosynthetic reactions, the rate of CO2 fixation and biomass also decrease (e.g., Zn deficiency: Srivastava et al. 1997; Cu-deficiency: Droppa et al. 1984, Maksymiec 1997). Oxidative stress is also one of the most important components of mineral-nutrient-deficiency stresses (e.g., Zn, Mn, Fe, Cu, B, Mg, and K) (Yu et  al. 1999). This is not surprising as some micronutrients, such as Zn, Fe, and Cu are important components of ROS scavenging systems of plastids (Cu/Zn and Fe superoxide dismutases, Table 26.2), therefore, plants deficient in these metals may exhibit symptoms of oxidative stress. Below, we briefly overview some metabolic processes impacted by the deficiency of the most important essential HMs. Fe-deficiency affects not only Chl biosynthesis (Duy et al. 2007, reviewed by Myśliwa-Kurdziel and Strzałka 2002), but the functioning of the photosynthetic electron chain, the Calvin cycle (Siedlecka and Krupa 1996, reviewed by Myśliwa-Kurdziel and Strzałka 2002), plastid protein import (Duy et al. 2007), enzymes of chloroplast-localized nitrogen fixation machinery (Briat and Vert 2004), sulfur assimilation, siroheme biosynthesis, amino acid metabolism (Duy et al. 2007), and Fe-involving antioxidative response (Allen 1995) (Table 26.2). The different molecular alterations of the photosynthetic apparatus induced by Fe-deficiency (Sárvári 2005, Briat et al. 2007) and the adaptation mechanisms of photosynthesis under Fe starvation (Sharma 2007) are summarized

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elsewhere. Some recently characterized mutants impaired in Fe uptake (irt1) or in its transport into the plastids (pic1) have similar ultrastructural damage as reported in Fe-deficient plants (Figure 26.2) (Henriques et al. 2002, Duy et al. 2007). The disturbances in thylakoid biosynthesis and plastid differentiation can be in this case directly related to nutrient deficiency, outlining the importance of essential metals in plastid differentiation and in the maintenance of the active photosynthetic apparatus. Zn deficiency also leads to chlorosis and decreased photosynthetic activity in leaves (Cakmak 2000, Henriques 2001, Wang and Jin 2005, Chen et al. 2008). Leaves become then light sensitive. The leaves of Zn-deficient sugar beet plants have disorganized chloroplasts with senescence process leading to cell death and, ultimately, to necrotic blade lesions, thus reducing the photosyntheticallyactive leaf area, which explains the lower CO2 fixing capacity and decreased biomass of Zn-deficient plants (Henriques 2001). The observed symptoms are probably associated with oxidative stress (for a review see Cakmak 2000) and decreased antioxidant enzyme levels (e.g., Cu/Zn-superoxide dismutase = Cu/Zn-SOD) in Zn-deficient leaves (Wang and Jin 2005, Chen et al. 2008, reviewed by Cakmak 2000, Rengel 2006). In Zn-deficient chickpea plants, disturbances in stomatal conductance and water status were also observed (Khan et al. 2004). The reduced growth and productivity of Zn-deficient plants are often associated with decreased levels of indole-3-acetic acid (reviewed by Cakmak 2000). Zn deficiency also alters membrane lipid composition and fatty acid saturation and leads to lipid peroxidation. Zn deficiency induces a decrease in the activity of carbonic anhydrase, which catalyzes the reversible reaction of CO2 hydration and is therefore accompanied by reduced photosynthetic rates (reviewed by Rengel 2006). Cu is a crucial plastid component involved in photosynthetic electron transport (plastocyanin) but is also indispensable for photosystem II (PSII) and light-harvesting complex II (LHC II) and for the functioning of the antioxidant enzyme, Cu/Zn-SOD (reviewed by Maksymiec 1997). Fifty percent of total plastidic Cu is found in plastocyanin (reviewed by Hänsch and Mendel 2009). Therefore, it is not surprising that photosynthetic functions are highly impaired under Cu-deficiency. Decreased plastocyanin, Chl, and carotenoid contents observed in Cu-deficient leaves can be responsible for the lower rates of photosynthesis (Baszyński et  al. 1978, Henriques 1989, Shikanai et  al. 2003, reviewed by Burkhead et al. 2009). PSI activity and cyclic photophosphorylation (Baszyński et al. 1978) as well as PSII activity are also targets of Cu-deficiency (reviewed by Myśliwa-Kurdziel and Strzałka 2002). Cu deficiency is often accompanied with oxidative stress probably due to decreased Cu/Zn-SOD levels (reviewed by Rengel 2006). The lipid composition of Cu-deficient chloroplasts is altered (reviewed by Barón et al. 1995). The galactolipid content decreases and the fatty acid unsaturation levels are also altered in the different lipid fractions (they increase in galactolipids). A secondary effect of Cu deficiency can be insufficient water transport caused by a decrease in cell wall formation and lignification in several tissues, including xylem tissue (for a review, see Burkhead et al. 2009). Mn-deficiency symptoms include reduced growth, pale young leaves that subsequently develop interveinal chlorosis, and ultimately small necrotic spots (Yu et  al. 1999, Henriques 2003, Henriques 2004). Mn deficiency also induces oxidative stress that could be partially prevented in transgenic tobacco plants overexpressing Mn-SOD in chloroplasts (Yu et al. 1999). Mn-deficient plants show the loss of most, but not all, functional PSII reaction centers in grana, with no alteration in light-harvesting complex of PSI, which is linked to the disruption of the oxygen-evolving complex (Simpson and Robinson 1984). However, upon Mn resupply, the leaves become able again to control Mn levels after 2 days. Mn deficiency depresses leaf photosynthetic capacity primarily by reducing the number of PSII units in spinach leaves (e.g., Simpson and Robinson 1984). Recent investigations in pecan leaves have shown that the reduced number of PSII units per leaf area unit is achieved by decreasing the number of chloroplasts, but not the number of PSII per individual chloroplast, and that the remaining PSII possess photochemical abilities similar to those of control leaves (Henriques 2003, 2004), their Mn content being similar to that of chloroplasts isolated from control plants (Henriques 2004).

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26.3.2.3  Molecular and Metabolic Alterations in Chloroplasts under Heavy-Metal Excess Several visible symptoms have been observed in plants grown in the presence of excess essential or nonessential HMs. Leaf expansion is inhibited, leaf tissues can become deformed and chlorosis often occurs (e.g., Cd: Djebali et al. 2005, Ebbs and Uchil 2008, Ben Ghnaya et al. 2009; Cr: Vázquez et al. 1987; Cu excess: Baszyński et al. 1988, Ciscato et al. 1997, Panou-Filotheou et al. 2001; Mn excess: González and Lynch 1999, reviewed by Barceló and Poschenrieder 2006; Pb: Woźny et al. 1995; Zn excess: Doncheva et al. 2001, Ebbs and Uchil 2008, Wang et al. 2009). The inhibitory effect of excess metals on Chl biosynthesis (Myśliwa-Kurdziel and Strzałka 2002) and on photosynthesis (Fodor 2002, Myśliwa-Kurdziel et  al. 2004, Sárvári 2005, Briat et  al. 2007) is reviewed elsewhere in more detail. In this section, we briefly summarize the different direct and indirect effects of metals on plastid metabolism with emphasis on metal-specific effects and observations. Clearly, the thylakoid membranes and plastids are much influenced by metal excess (Figure 26.2, Table 26.5); however, it is not clear if they are really the primary targets of metal stress—and therefore, their alteration is responsible for the observed decrease in photosynthetic activity—or if they simply reflect the observed dramatic alterations of plastid metabolism. There are almost no data about the actual in organello concentration of excess metals, and therefore on the direct nature of their interactions. Furthermore several observed symptoms seem to be nonspecific. First, we briefly overview some examples for direct interactions of HMs with different plastid components (proteins, pigments, cofactors, and lipids), then we summarize data about metal-excess induced oxidative stress in plastids and the possible osmotic disturbances observed under excess metals. Finally, the essential metal (Fe, Cu, Zn, Mn)-deficiency induced by the excess of another HM is also discussed. 26.3.2.3.1  Molecules Impacted Directly by Metals Direct interactions of metals with chloroplast metabolism could be often only studied in vitro at relatively high concentrations that are unlikely to occur in the organelles under natural conditions. These effects are summarized below. 26.3.2.3.1.1   Pigments and Other Small Plastid Metabolites  Excess HMs may directly interact with pigments and may replace Mg in Chl in vivo or in vitro (Cd, Cu, Hg, Ni, Pb, Zn) (Figure 26.1) (Küpper et al. 1996, 1998, 2002, 2003). At high concentrations, different nonessential metals can also destabilize pigment precursors and may not substitute Mg, but induce its loss from the porphyrin ring (e.g., protochlorophyllide: Solymosi et al. 2004). There is much less data about the direct interaction of excess HMs with carotenoids. In general, carotenoid levels seem to be affected indirectly by HM stress as these molecules have an important role in scavenging ROS induced by HMs (see below). In diatoms, the xanthophyll cycle was shown to be altered by Cd (Bertrand et al. 2001). The observed decreased photosynthetic activity and disturbed plastid metabolism of HM-treated plants may be related to direct oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) by nonessential HMs (e.g., Cd: Figure 26.1, Böddi et al. 1995, reviewed by Pál et al. 2007; Hg: Lenti et al. 2002, Solymosi et al. 2004, Solymosi et al. 2006b), which might result in the inhibition of the activity of enzymes or metabolic processes that use NADPH as a hydrogen donor. Nonessential HMs have been also reported to cause high ATP content, and to change gene expression through DNA hypomethylation and DNA damage (reviewed by Poirier et al. 2008). This way, their carcinogenetic effect has also been reported (e.g., Monteiro et al. 2009). Hg-induced inhibition of photosynthesis occurs probably by inducing a severe loss of adenylate pool and decreasing thus the rate of cyclic and non-cyclic photophosphorylation. Hg also decreases PSII associated reactions, O2 evolution and CO2 fixation, probably due to the retardation of all ATP-dependent processes (reviewed by Romanowska 2002). 26.3.2.3.1.2   Lipids  The observed changes in plastid and chloroplast membrane structure (Figures 26.1 and 26.2, Tables 26.3 and 26.5) might be due, at least partially, to membrane lipid

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alterations in metal-exposed plants (reviewed by Devi and Prasad 2006). Nonessential HMs influence the lipid composition, the saturation, or even the chain length of the fatty acids of membrane lipids. Several metals (e.g., Cd: Skórzyńska-Polit et al. 1998, Jemal et al. 2000, Nouairi et al. 2006; Pb: Stefanov et al. 1995) but also excess micronutrients (e.g., Cu: Maksymiec et al. 1992, Quartacci et al. 2000; Mn: Lidon et al. 2004) decrease the monogalactosyl-diacylglycerol (MGDG) content of thylakoid membranes. This change is explained by increased galactolipase activity (Skórzyńska and Baszyński 1993, Stefanov et  al. 1995). Other lipid fractions have been also affected, but as MGDG and digalactosyl-diacylglycerol (DGDG) ratios influence membrane curvature, the changes in their ratios can explain the observed alterations in grana stacking and/or grana to stroma thylakoid ratios under metal stress (Table 26.5, Figure 26.2). Similarly, the swollen intrathylakoidal space has been related to decreasing MGDG levels caused by increased galactolipase activity (Skórzyńska et  al. 1991). MGDG is also required for proper functioning of photosynthesis (PSII complexes); therefore changes in this lipid fraction may have a detrimental effect on thylakoid functions and the total photosynthetic efficiency of plants. In addition, Ag, Cu, Pb, and Hg inhibit the plastidial phosphatidylcholine synthesis (Akermoun et al. 2002). The amount of highly unsaturated fatty acids (especially 18:2 and 18:3) has been shown to increase after Cd (maize: Pál et al. 2007), Cu (spinach: Maksymiec et al. 1994), and Pb (spinach: Stefanov et al. 1995) treatment. However, contradictory data indicating lower degree of fatty acid unsaturation have been also reported in other plants treated with Cd (pepper: Jemal et al. 2000; tomato: Djebali et al. 2005; Brassica napus: Nouairi et al. 2006, Ben Ammar et al. 2007). Increased fatty acid desaturation as well as decreased MGDG content can change membrane fluidity (Quartacci et al. 2000), which in turn leads to altered membrane physiological functions; it particularly influences the ionic permeability of the thylakoid membranes (for a review, Sandalio et al. 2001, see Devi and Prasad 2006). Different membrane permeabilities of the thylakoids might also explain the observed swelling of the intrathylakoidal space. Membrane injuries of metal-treated plants (e.g., disruption of the envelopes, Table 26.5) are often related to increased peroxidation of membrane lipids caused by highly toxic free radicals (ROS) (reviewed by Devi and Prasad 2006, Maksymiec 2007). As, Cr, Cd, Cu, Hg, Ni, and Zn have all been shown to induce lipid peroxidation (Figure 26.1c) (e.g., Cd: Djebali et al. 2005, SkórzyńskaPolit and Krupa 2006; Hg: Cho and Park 2000) as well as several of the Fe and Cu compounds that can catalyze the Haber–Weiss and Fenton reactions (Babu et al. 2001). Cd directly affects the lipid structure around LHCII, leading to lipid peroxidation and the release of several pigment–protein complexes, oxygen-evolving complex (OEC) and plastocyanin (Figure 26.1k), then blocking further electron transport processes (for reviews, see Siedlecka and Krupa 1999, Pál et al. 2006). All these observations indicate that potentially toxic metals enter the plastids and can influence directly the different plastid components and then plastid metabolism. 26.3.2.3.1.3   Proteins  One of the most often–reported direct toxic effects of metals on plastid proteins is attributed to the ability of some metals to bind to sulfhydryl-, histidyl-, and carboxylgroups of proteins or enzymes, inducing therefore conformational changes resulting often in protein inactivation or disturbed function (Cd, Pb, Hg: Vallee and Ulmer 1972, Lenti et al. 2002, Solymosi et al. 2004; Cu: Maksymiec 1997, Yruela 2009). Another possibility is that metals in excess may substitute essential metals in catalytic sites of enzymes (Figure 26.1). For instance, Hg can substitute Cu in plastocyanin (Radmer and Kok 1974), Co replaces Mg in the ribulose bisphosphate carboxylase oxygenase (RUBISCO), or Zn in transcription factors (reviewed by Poirier et al. 2008), and Cd replaces Ca in PSII reaction centre, causing the inhibition of PSII photoactivation (Faller et al. 2005, reviewied by Kucera et al. 2008). Cd can replace Zn and Ca in metalloenzymes (reviewed by Clemens et al. 2009). The substitution of Mn by Zn or Cd leads to the inactivation of OEC, and as a consequence, electron donation to PSII is inhibited (for reviews, see Bertrand and Poirier 2005, Pál et al. 2006, Kucera et al. 2008). Induced changes in the arrangement and structure of LHCII decrease the efficiency of excitation energy

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capture by PSII and reduce the rate of photosynthetic oxygen evolution. Photophosphorylation rates decrease due to PSII dysfunctioning, without evidence for a direct inhibition of the ATP-synthase by the metal (reviewed by Kucera et al. 2008). Hg ions can directly interact with some sites in the photosynthetic electron transport chain situated in D1 and D2 proteins, with the Mn cluster in the oxygen-evolving complex (reviewed by Romanowska 2002). Excess Cu inhibits several polypeptides of PSII and PSI (e.g., Lidon and Henriques 1993, Maksymiec et  al. 1994). A primary site of Cu inhibition was identified on the antenna Chl a molecules of PSII (Lidon et al. 1993). The enzymes RUBISCO, phosphoenolpyruvate carboxylase, alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and nitrate reductase have been described as sensitive to excess metal concentrations (Figure 26.2l) (reviewed by Siedlecka and Krupa 1999, Romanowska 2002, Myśliwa-Kurdziel et al. 2004, Pál et al. 2006, Kucera et al. 2008). One possible explanation of the metal-induced chlorosis is that excess metals directly interact with enzymes of pigment biosynthesis. Other possibilities involve enhanced pigment degradation (partially due to oxidative stress), direct interaction with pigment precursors or cofactors required for the process, or metal-induced Fe- or Mg-deficiency as these essential metals have key roles in the biosynthetic processes (reviewed by Myśliwa-Kurdziel and Strzałka 2002). Preferential loss of pigments (e.g., Chl b in case of Zn and Cd stress—Ebbs and Uchil 2008) and decreased levels of carotenoids (e.g., excess Cu: Baszyński et al. 1988) are often reported under metal stress and can lead to impaired photosynthetic activities. Cr6+, Fe3+, and Hg2+ have been shown to directly inhibit one of the key enzymes of Chl biosynthesis, NADPH: protochlorophyllide oxidoreductase, in vitro (Lenti et al. 2002, Solymosi et al. 2004, Myśliwa-Kurdziel and Strzałka 2005). Cd also inhibits Chl biosynthesis directly through ALA dehydratase and protochlorophyllide reductase (in vitro: Böddi et al. 1995, Myśliwa-Kurdziel and Strzałka 2005, for reviews, see Myśliwa-Kurdziel and Strzałka 2002, Poirier and Bertrand 2005). However, other scientists have found that neither the synthesis nor the photoreduction of protochlorophyllide is influenced by Cd treatment in greening barley leaves (Horváth et al. 1996). 26.3.2.3.2  Metal-Induced Oxidative Burst HM ions block the electron flow in PSII, leading to the formation of excited triplet Chl (3Chl*), which can react with an oxygen molecule with triplet electronic configuration and by this way induces the formation of singlet oxygen. Oxidative stress leads to an imbalance in the regeneration and removal of ROS, including singlet oxygen (1O2), superoxide radical (O2−.), hydrogen peroxide (H2O2), and the most damaging and reactive hydroxyl radical (OH.), which can lead to further lipid peroxidation and can damage membranes, proteins, and nucleic acids, leading to altered membrane fluidity, loss of enzyme function, and genomic changes, respectively (reviewed by Kucera et  al. 2008). Induction of free radicals and ROS by metals is very well documented and might be responsible for membrane injuries and some of the ultrastructural changes observed under metal stress (e.g., Babu et al. 2001, Zhang et al. 2005, Rodríguez-Serrano et al. 2009, reviewed by Sharma and Dietz 2008). However, some elements are considered to be redox-active metals (Cu, Fe) and can therefore directly elicit ROS generation (Gallego et al. 1996, Drazkiewicz et al. 2004), while others induce it only indirectly (Cd: Gallego et al. 1996, Pál et al. 2007; Cr and Cu: Yruela 2009; Hg: Cho and Park 2000; Mn: Lidon and Teixeira 2000a,b; Ni: Chen et al. 2009; Zn: Panda et al. 2003, Kawachi et al. 2009, reviewed by Sharma and Dietz 2008). The different antioxidant enzyme systems (such as catalase localized into peroxisomes and plastids, SOD in the cytosol, mitochondria and plastids, peroxidases in vacuoles, cell walls and cytosol, and the ascorbate–gluthation cycle in several plant cell compartments) as well as carotenoids (and particularly the xanthophyll cycle) may protect the plants under oxidative stress and can therefore be responsible for metal tolerance or can indicate metal stress (e.g., Drazkiewicz et al. 2004, reviewed by Kucera et al. 2008). Ascorbate takes part in growth processes, electron transport, photoprotection, regulation of photosynthesis, and preservation of the enzymatic activities that contain

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prosthetic transition metal ions. Similarly, carotenoids that can quench the oxidizing ROS and the triplet state of Chl are often affected by metal stress (reviewed by Kucera et al. 2008). The carotenoid content of metal-stressed leaves also changes, and particularly the xanthophyll cycle pigments that belong to a protection system for the photosynthetic apparatus from the nonphotochemical quenching of excited triplet Chl and from ROS (reviewed by Kucera et al. 2008). Therefore, high concentrations of antioxidative enzymes, together with enhanced pigment synthesis may allow a plant to completely overcome the harmful action of a toxic metal and to show normal healthy growth. However, the plant still accumulates fairly high amounts of the metal, a feature that can be useful for phytoremediation but dangerous in case of edible crops. 26.3.2.3.3  Metal-Induced Disturbances in Plastid Water and/or Ion Homeostasis It is well documented that several HMs influence gas exchange and transpiration of plants (by influencing root hair formation, membrane permeability, number and diameter of vascular bundles, stomatal conductance changes, and by inducing the closure of stomata) and therefore cause disturbances in respiration, in CO2 fixation, in the water and nutrient status of plants (e.g., Cd: Shi and Cai 2008, Nedjimi and Daoud 2009, Sayyad et al. 2009; Cr: Vázquez et al. 1987; Cu: Sayyad et al. 2009; Hg: Martínez-Ballesta et al. 2003; Mn: Lidon et al. 2004; Pb: Sayyad et al. 2009; Zn: Sayyad et al. 2009). The inhibitory effect of metals on the dark phase of photosynthesis is also a complex phenomenon. Increasing stomatal and mesophyll resistance leads to reduced CO2 uptake, because of a reduced number of stomata or stomatal closing (e.g., Moustakas et  al. 1996, Shi and Cai 2008), which might directly inhibit crop production. Cr6+ treatment in bean has shown that this metal delayed or inhibited the differentiation of stomata on leaves (Vázquez et al. 1987). Similarly, under excess Ni, fewer stomata developed in cabbage leaves and many stomata were defective (Molas 1997). Decreased respiration and transpiration definitely alter the water and nutrient status of the plants. These data taken together, indicate that it is not surprising that sometimes the symptoms of metal excess—even at the plastid level—resemble those observed under osmotic disturbances or changes in water relations. Some changes in the chloroplast shape and size (Figure 26.2, Table 26.5) or the disruption of the plastid envelope may indicate some osmotic disturbances of the membranes or changes in the envelope membrane permeability (Moustakas et al. 1997). In Cd- or Cu-stressed plants, chloroplasts often exhibited small, regularly spotted bodies, that were described as “pseudocrystalline bodies” (Cu: Ciscato et al. 1997) or “microtubule-like” structures (Cd: Ouzounidou et al. 1997) in wheat leaves. Similar structures have also been described in etioplasts of other species (barley: Wellburn et al. 1982, Wellburn 1984; wheat: Artus et al. 1990) and in greening plastids of Pb-treated barley leaves (Woźny et al. 1995) or salt-stressed etiolated wheat leaves (Abdelkader et al. 2007) or wheat leaves grown in unusual environments (Solymosi, unpublished results). These spotted bodies might resemble the so-called prothylakoid bodies of unstressed plants (Wellburn 1984) or the so-called stromacenters formed in leaves dehydrated by plasmolysis, wilting or grown in windy areas (Gunning 1965, Gunning et al. 1968, Gunning and Steer 1975) and showing therefore some symptoms of water and osmotic imbalance. It should be mentioned that direct osmotic effects are probable to occur in case of short-term treatments with very high, nonphysiological concentrations (0.1–1 M range). However, as most nonessential metal ions are toxic to plants from μM concentrations, the influence of such low concentrations on the osmotic potential of the plants is probably negligible, but other, indirect effects on the water status of the plants are probable to occur (reviewed by Poschenrieder and Barceló 2006). 26.3.2.3.4 Deficiency of Essential Heavy Metals Induced by the Excess of Another Heavy Metal It is well documented that indirect injury mechanisms caused by nonessential metals can be based on metal-induced deficiency of Fe, Mn, or other essential micro- or macronutrients. This process might be related to excess metal-induced changes in transpiration, root development, and as a

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consequence to decreased water and nutrient uptake of plants, or to substitution or replacement of essential metals in metalloproteins (physiological essential-metal deficiency) or to competitive interactions with nutrient uptake and transport components. According to the Irving–Williams series, the different metal ions can bind to organic ligands in a metal-binding site of a metalloprotein, metal chaperone or metal transporter with different affinities (Zn2+ < Cu+ > Cu2+ > Ni2+ > Co2+ > Fe2+ > Mn2+ > Mg2+ > Ca2+). At the same time, the binding affinity for a metal ion is also determined by other secondary factors such as the size of metal-bindingsite cavity in a protein, the geometry of ligand atoms, and other characteristics, but normally each metal ion can be replaced by other metal ions downstream in the Irving–Williams series (reviewed by Yruela 2009). The potential for Zn, and especially Cu, to displace other metals is relatively high. This replacement is not only true for metal-containing proteins, important for plant functioning, for metal-containing molecules such as Chls (e.g., see Table 26.2), but also for all metal transporters and ion channels within the cells. Similarly, toxic metals may also substitute these ions. This way, it is quite evident that excess metals may induce nutrient deficiency. However, these interactions depend also on the treatment (i.e., if roots or leaves are treated), the form of the metal added, the plant material, and the experimental conditions (concentration of ions, pH, presence of chelators); therefore the results in the literature are difficult to compare (reviewed by Fodor 2002). Different chelators can have a strong influence on uptake, transportation, and/or on apoplasmic or non-apoplasmic accumulation of different interacting metals in the nutrient solution (e.g., Fodor et al. 2005). One of the best known examples for nonessential metal-induced nutrient deficiency is the chlorosis caused by Cd, which has been shown to be related to Fe-deficiency, rather than to the direct inhibitory effect of Cd (e.g., Sárvári 2005, Fodor 2006, Pál et al. 2006). In plants grown on Fe-deficient nutrient solution, relatively more Cd was translocated into the shoot, and both PSs showed higher sensitivity to Cd (reviewed by Siedlecka and Krupa 1999). On the other hand, elevated Fe supply applied together or after Cd treatment could prevent most Cd effects (Siedlecka and Krupa 1996, Solti et al. 2008, reviewed by Siedlecka and Krupa 1999). Cd may also have indirect effects on PSI and influences the electron transport chain, ferredoxin-dependent NADP + photoreduction, and Chl biosynthesis, by causing Fe-deficiency (reviewed by Siedlecka and Krupa 1999, Pál et al. 2006). Cd also lowers the Chl a/b ratio (Sárvári et al. 1999) due to stronger reduction of PSI than LHCII (Szegi et al. 2007). Cd reduced the amount of Chl-containing complexes in the order of PSI > LHCII > PSII core (Sárvári et al. 1999) similar to that observed in Fe-deficient plants. The indirect nature of the Cd-induced inhibition of the light phase of photosynthesis and of the different Chl–protein complexes is confirmed by the fact that in Cd-treated plants exhibiting all symptoms of Cd toxicity, the photosynthetic activity could be restored at least partially with the addition of Fe (Solti et al. 2008). Chl fluorescence imaging has shown that the recovery of the photosynthetic activity started from the parts adjacent to the veins and gradually extended to the interveinal parts (Solti et al. 2008) indicating clearly that Cd interfered with Fe uptake and/or transportation in the plants. Therefore Fe-deficiency can be considered as a key factor in Cd-induced inhibition of photosynthesis. The molecular basis of Cd-induced Fe-deficiency in some plants is that phytosiderophores and other complexing agents excreted by plants can also chelate other metals including Cd and Pb (Strategy II plants; reviewed by Kochian 1995, Hill et al. 2002, Fodor 2006). In Strategy I plants (dicots and nongraminaceous monocots), metallic pollutants interfere with root ferric-chelate reduction (Cd, Pb, Cu, Ni, Mo, Zn) (reviewed by Fodor 2006). However, in some cases, there is no clear correlation between the Fe content of the leaves of metal-treated plants and the decrease in various physiological parameters, suggesting that the total Fe content in the tissues is not necessarily the same one as the active Fe pool (Sárvári et al. 1999, reviewed by Fodor 2002). Cd treatment also induced decreased levels of micronutrients (Cu, Zn, Fe, Mn, Mo, Ni) and macronutrients (Ca, K, Mg, N, P, S) in leaves and roots (reviewed by Fodor 2002, Pál et al. 2006, Chen et al. 2009, Hasan et al. 2009). This process might be related to the fact that Cd was shown to decrease water uptake and thereby the amount of all transported nutrients (cucumber roots: Varga et al. 1999). The Cd-induced Ca-deficiency in pea plants caused the downregulation of Cu/Zn-SOD, leading to the

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overproduction of the ROS hydrogen peroxide and superoxide, as monitored in vivo by confocal laser microscopy (Rodríguez-Serrano et al. 2009). The production of these ROS was mainly associated with vascular tissue, epidermis, and mesophyll cells, and the production of superoxide radicals could be prevented by exogenous Ca (Rodríguez-Serrano et al. 2009). The toxic effects of Cd were reduced in the presence of elevated amounts of Ca in runner bean plants (Skórzyńska-Polit et al. 1998) and in transgenic tobacco plants transformed with TaLCT1 (Antosiewicz and Hennig 2004). Addition of Ca to lettuce plants increased tolerance and accumulation of Cd, while La decreased Cd accumulation (reviewed by He et al. 2005). Interestingly, other authors found no effect of Cd on Ca accumulation in rice (Cui et al. 2008) or found positive effect on Ca accumulation in sugar beet (Larbi et al. 2002). Some experiments with lettuce have shown that increasing Cd concentrations increased the Mn uptake (Ramos et al. 2002), while in cucumber (Sárvári et al. 1999) and tomato (Baszyński et al. 1980) the opposite tendency was found. Addition of Mn to the nutrient solution of hydroponically grown Cd-treated tomatoes restored the photosynthetic pigment content and the photosynthetic activity of the seedlings and induced grana formation (Baszyński et al. 1980). Similarly to Cd, Pb was also shown to cause Fe-deficiency in plants (Wallace et al. 1992, Varga et al. 2002, Sinha et al. 2006) and chlorosis (Sinha et al. 2006, reviewed by Fodor 2002). Pb had an inhibitory effect on Fe-chelate reductase activity in sugar beet (Larbi et al. 2002). In this study, Pb had only a minor effect on other nutrient concentrations. Pb contamination leads to a drastic reduction of Ca accumulation in cucumber roots and a slight increment in the transport of the essential nutrients, especially Mn (Varga et al. 1999, Cseh et al. 2000). In different experiments, Pb decreased Ca, Mn, K, Zn (reviewed by Fodor 2002), and Ni (reviewed by Chen et al. 2009) contents of the plants. In cabbage, Pb decreased the Mn and Cu contents (Sinha et al. 2006). Cr3+ induces Fe-deficiency in cabbage (Pandey and Sharma 2003). Interestingly, in Cr6+-treated bean seedlings, higher Cr concentrations, decreased Chl content, and drastic changes in chloroplast structure were observed, while the plastid ultrastructure and Chl content of primary leaves that accumulated less Cr was almost not affected (Vázquez et al. 1987). Exposure of spruce plants to both organic and inorganic Hg resulted in a loss of K, Mg, and Mn and accumulation of Fe, indicating that essentialnutrient deficiency may also contribute to the toxic physiological effects observed under excess Hg (for a review see Boening 2000).

26.3.3  Some Unusual Phenomena Associated with Heavy-Metal Stress In the literature related to HM stress, there are some data that are not often discussed in metalstress related reviews. In some cases, the excess of one metal has a positive effect on the uptake of another one. On the other hand, low concentrations of HMs sometimes had a positive effect on plant metabolism. In this section, we briefly summarize these phenomena. 26.3.3.1  When the Excess of a Metal Alleviates the Stress Caused by Another Metal As stated and illustrated above (Table 26.1), the inhibitory effect of excess metals on plant metabolism are very often related to the fact that they compete for uptake and transportation and induce deficiency in essential nutrients. This is also true for unbalanced concentrations of essential HMs present in the soil, but it is more evident for toxic elements. When the amount of the toxic metal is low, its uptake into the cells or organs is probably rather weak because its chance to be taken up and to be transported is relatively low. The situation may dramatically change when the amount of toxic metal(s) increases in the environment. Additionally, changes in the concentration may also affect the amount of metals passively transported. Therefore, it is not surprising that in several of these cases, the toxic effects and symptoms induced by the metal in excess can be alleviated by the addition of the essential metal with which it is in competition (reviewed by Poirier et al. 2008). This way, the excess metal-induced deficiency, and the disturbed essential metal homeostasis can be recovered. In these cases, the presence of a beneficial element plays an antidote role against the non-beneficial element, even when it is given in excess.

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Several experiments have shown that the Fe nutrition status of plants may significantly modify nonessential metal uptake (reviewed by Fodor 2006). Experiments on the interaction of Fe supply with Cd uptake revealed that an overdose of Fe decreases Cd accumulation in chloroplasts of primary bean leaves treated with Cd via the nutrient solution (Siedlecka and Krupa 1996). Similarly, the addition of excess Cu decreased the Cd uptake of rice plants (Cui et al. 2008). A moderate excess of Fe results in increased growth and photosynthetic pigment content and more efficient light phase of photosynthesis in Cd-treated plants as a result of the recovery from Cd-induced physiological Fe-deficiency (Solti et al. 2008, for reviews, see Siedlecka and Krupa 1999, Fodor 2006). Cd-induced decreased photosynthetic rate, carotenoid and Chl contents, as well as disturbed plastid ultrastructure could be partially recovered by the supplementation of Mn in the growth media; at the same time the Cd content of the different plant organs (leaves, stems, and roots) decreased (Baszyński et al. 1980). Surprisingly, there are also results indicating the positive effect of toxic or nonessential HMs on the uptake of essential metals; however, the exact molecular background of these observations is not completely clear. Some experiments with lettuce have shown that increasing Cd concentrations increased the Mn uptake (Ramos et al. 2002). Pb contamination of cucumber leads to a slight increment in the transport of the essential nutrients, especially Mn (Varga et al. 1999, Cseh et al. 2000). Increasing Pb concentrations increased the Zn content in cabbage leaves (Sinha et al. 2006). Low concentrations of Cr3+ restored the chloroplast ultrastructure in Fe-deficient common bean plants (Poschenrieder et al. 1991). Silicon is known to effectively mitigate various abiotic stresses such as Cd, Mn, and Al pollution, and also salinity, drought, chilling, and freezing. However, mechanisms of Si-mediated alleviation of abiotic stresses remain poorly understood. The key mechanisms of Si-mediated alleviation of abiotic stresses in higher plants include (1) stimulation of antioxidant systems in plants, (2) complexation or coprecipitation of toxic metal ions with Si, (3) immobilization of toxic metal ions in growth media, (4) uptake processes, and (5) compartmentation of metal ions within plants (reviewed by Liang et al. 2007). In rice, the addition of Si to nutrient solutions had a positive effect on plant growth and could decrease the Cd accumulation in the shoots and had therefore an enhancing effect on shoot and root biomass under moderate Cd stress (Zhang et al. 2008). 26.3.3.2  When Nonessential Metals Added at Low Concentrations Have a Stimulating Effect The toxic and damaging effects of metals are usually observed when the stressors are applied at relatively high concentrations (10 −5–10 −3 M). Nevertheless, these harmful compounds used at low concentrations (defined as “low-concentration stressors,” 10 −8 –10 −6 M) may have a beneficial effect on plants. However, the effect depends on the developmental stage of the plant at the beginning of the treatment. The low-concentration stressors stimulate biosynthetic processes and growth in seedlings, and delay aging in detached leaves, which is reflected also by an increased metabolism (Nyitrai et al. 2007, 2009, Kovács et al. 2009). The stimulating effect is nonspecific, i.e., independent of the agent used (Nyitrai et al. 2003, 2004). The acceleration of plant growth is one of the stimulatory effects of nonessential metals observed in these studies. Short-term Cd treatments of maize roots result in small growth stimulation (Wójcik and Tukendorf 1999). Similarly, long-term treatments with Pb, Ni, and Ti induce a significant increase in root length, dry and fresh weights, and shoot growth in maize and bean (Nyitrai et al. 2003). Low-concentration stressors often stimulate the metabolic activity, i.e., respiration, Chl biosynthesis, and photosynthetic activity in seedlings (Prasad et  al. 2001, Nyitrai et  al. 2004, Nyitrai et  al. 2007). At the germination stage, Cd and Pb significantly increase the levels of Chls and carotenoids until the fourth day of development (Shaw and Rout 1998). Similarly, at relatively low concentrations, Pb has a slight stimulatory effect on the Chl content of cucumber leaves (Sárvári et al. 1999). Cd treatment of young maize seedlings increases the leaf Chl (Drazkiewicz et al. 2003,

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Drazkiewicz and Baszyński 2005) and carotenoid (Drazkiewicz and Baszyński 2005) content up to 100 μM, but causes chlorosis when applied above this concentration. Low concentrations of Cr3+ enhance the growth of bean plants and reduce the chlorosis in young leaves of Fe-deficient plants by increasing the concentration of Chls and carotenoids (Bonet et al. 1991, Poschenrieder et al. 1991). One of the most comprehensive surveys in this field is that of Nyitrai et al. (2003). In this study, low concentrations of different metals (Cd: 5 × 10 −8 and 10 −7 M, Pb and Ni: 10 −7 and 10 −6 M, Ti: 10 −6 and 10 −5 M) had a stimulating effect on Chl biosynthesis and photosynthetic activity of bean and maize. Treatments applied either in the nutrient solutions or by spraying the leaves were both effective. However, the extent of the stimulating effect depended on the species, the time course of treatment, the position of leaf, and the agent (Nyitrai et al. 2003). The stimulation of the photosynthetic activity was observed at different intervals during all treatments (Cd, Pb, Ni, Ti), while Chl a/b ratios of leaves or chloroplasts did not change considerably (Nyitrai et al. 2003). Low-concentration stressors increased the amount of PSI and LHCII. Electron microscopy of either maize or bean leaves did not show significant differences in the chloroplast lamellar systems between control and treated plants. The authors concluded that these low-concentration stressors generate nonspecific alarm reactions in plants, which may involve changes of the hormonal (e.g., cytokinin) balance (Nyitrai et  al. 2003, 2004). In a later study, the authors have demonstrated that in barley seedlings treated with low concentrations of Cd (5 × 10 −8 M), the amount of cytokinins increases in the roots, and it is transported to the leaves where it also causes stimulation (Kovács et al. 2009). The phosphatidylinositol-4,5-biphosphate-inositol-1,4,5-triphosphate/diacylglycerol and the mitogenactivated protein kinase signaling pathways, and not the stressor itself, were found to be responsible for the primary stimulation of cytokinin synthesis and/or activation in the roots (Kovács et al. 2009). Cd treatment at this low concentration did not induce any oxidative stress either in the roots or in the leaves, where Cd did not even accumulate to detectable amounts. In model systems (i.e., the detached old leaves of different plants), the beneficial effect of the moderate and weak stress that induces nonspecific alarm responses in plants could be studied and was shown to have a rejuvenating effect on plastids and their metabolism (Nyitrai et al. 2007, 2009). The physiological parameters of detached leaves are stimulated in a similar way to those observed in the case of seedlings (Nyitrai et al. 2003, Kovács et al. 2009). At the same time, the stressors stimulate starch accumulation in the chloroplasts, and a decrease of the large plastoglobuli typical for plastid senescence (Nyitrai et al. 2004, 2007). Under Pb (10−7 M) and Ti (10−6 M) treatment of detached, nonrooting barley leaves, the level of active cytokinins is not affected, indicating the direct effect of the stressors in this experimental system (Nyitrai et al. 2007). In both model systems (detached bean and barley leaves) the phosphatidylinositol-4,5-biphosphate-inositol-1,4,5-triphosphate/diacylglycerol signaling pathway is involved in the anti-senescence effect (Nyitrai et al. 2007, 2009). These interesting data provide further evidences for the complexity of interactions of plastids and metals.

26.4  Conclusion In case of metal pollution in the environment, an unbalanced metal content is often observed in plastids: potentially toxic HMs are present and essential elements are missing. Thanks to various strategies, the cells can cope with a moderate unbalance, but in case of strong unbalance, too many essential biomolecules are not functional any more, photosynthesis is altered and plant productivity is decreased. Although sufficient data are available on HM transporters, it appears that further studies on the mechanism of HM uptake and regulation in plastids are needed. Also, the metal translocation in the whole plant cell, and especially the transporters of the tonoplast that favor the elimination of toxic elements should be further studied. Moreover, the visualization of metal movement and transport at the cellular scale would greatly help. A possibility of preventing the entrance of the nonessential elements in plastids would be to produce mutants with more metal-specific transporters. The mechanisms allowing non-essential metals to have a stimulating effect on plant growth should be also further investigated.

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Abbreviations ATP Chl DGDG DNA HM HMA LHC MGDG NADP +/NADPH OEC PS RNA ROS RUBISCO SOD

Adenosine triphosphate Chlorophyll Digalactosyl-diacylglycerol Deoxyribonucleic acid Heavy metal HM ATPase Light-harvesting complex Monogalactosyl-diacylglycerol Nicotinamide adenine dinucleotide phosphate (oxidized/reduced form) Oxygen-evolving complex Photosystem Ribonucleic acid Reactive oxygen species Ribulose bisphosphate carboxylase oxygenase Superoxide dismutase

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Responses to Cadmium 27 Plant and Mercury Stress Elena Garmash, Svetlana Skugoreva, and Tamara Golovko Contents 27.1 Introduction........................................................................................................................... 713 27.2 Characteristics of Cadmium and Mercury............................................................................ 714 27.2.1 Cadmium................................................................................................................... 714 27.2.2 Mercury..................................................................................................................... 714 27.2.3 Toxicology of Cadmium and Mercury...................................................................... 715 27.3 Heavy Metal Environmental Pollution.................................................................................. 715 27.3.1 Heavy Metal Release into Environment.................................................................... 715 27.3.2 Assessment of Heavy Metal Environmental Pollution.............................................. 715 27.3.3 Heavy Metal Migration Processes............................................................................. 716 27.4 Heavy Metal Phytotoxicity.................................................................................................... 717 27.4.1 Growth....................................................................................................................... 718 27.4.2 Photosynthesis........................................................................................................... 718 27.4.3 Respiration................................................................................................................. 718 27.4.4 Metal-Induced Oxidative Stress................................................................................ 720 27.5 Heavy Metal Tolerance in Plants........................................................................................... 722 27.5.1 Roots as a Barrier on the Path of Metals Transport.................................................. 723 27.5.2 Antioxidant System of Plant Cells............................................................................. 723 27.5.3 Metal-Binding Complexes......................................................................................... 725 27.5.4 Metabolic Processes Changing.................................................................................. 726 27.6 Strategies in the Response of Plants to Heavy Metals.......................................................... 727 27.7 Phytoremediation................................................................................................................... 728 27.8 Conclusions............................................................................................................................ 728 References....................................................................................................................................... 729

27.1  Introduction In view of the increasing anthropogenic impact on the environment, the problem of emissions of heavy metals (HMs) gets more and more attention. Study on interaction effects between HM and biomolecules is of great theoretical and practical importance as it concerns economical and social life spheres, such as medicine, eco-toxicology, agriculture, recycling of wastes, etc. Despite the numerous reviews and reports on HM in literature, the study of phytotoxicity mechanisms is still incomplete (Masarovicˇová et al. 1999, Prasad and Hagemeyer 1999, Titov et al. 2007). HMs are a group of metals with relative atomic weight over 50 or density higher than 5 g cm−3. Pollutants mostly contain arsenic, cadmium, cobalt, chromium, copper, mercury, lead, nickel, and zinc. Several HMs such as iron, manganese, zinc, copper, nickel, and molybdenum are required by plants as micronutrients. Their concentrations in plant dry matter vary from 100 to 0.1 ppm (Taiz and Zeiger 2002). Mn is required for activity of some dehydrogenases, decarboxilases, oxidases, and peroxidases and is involved with other cation-activated enzymes and photosynthetic O2 evolution. 713

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Iron is a constituent of cytochromes and nonheme iron proteins in photosynthesis, N2 fixation, and respiration. Zinc and Cu are components of many enzymes involved in redox reactions as well. Molybdenum is a constituent of nitrogenase, nitrate reductase, and xanthine dehydrogenases. Nickel has been recently recognized as an important plant micronutrient as a component of urease. Other HMs such as cadmium, lead, mercury, arsenic, etc., are not essential for plants and are highly phytotoxic. HM concentration in soil is agreed to be toxic when it inhibits plant growth and development and decreases plant productivity by 10%–20% (Zyrin and Sadovnikova 1985). However, the identification of toxic levels of HM for plants is a very complicated problem. Knowledge of environmental conditions, HM forms and their stability in the environment, plant properties, etc., are of great importance for the assessment of HM toxicity. In this chapter, we have paid special attention to the phytotoxicity of two HMs—cadmium and mercury. By the resolution of the Environmental Protection Agency, these metals belong to the group of important environmental pollutants. They represent maximum potential danger for animals and people as they have a high cumulative effect and high rate of technogenic accumulation in the environment (Maistrenko et al. 1996, Chernykh and Ovcharenko 2002). According to the phytotoxicity classification, mercury is a very phytotoxic element as it inhibits the life activity of test organisms at concentrations in medium near (1 mg L−1); cadmium is a moderately toxic element as it inhibits the life activity of test organisms at concentrations 1–100 mg L−1 (Alexeev 1987). Cadmium and mercury are mobile in plants, and are accumulated in all plant organs, and seeds as well (Kabata-Pendias and Pendias 1984). Plants can volatilize mercury through leaves in the form of dimethylmercury, but dimethylmercury is synthesized 6000 times slower than methylmercury, which is very capable of penetrating through biological membranes (Gay et al. 1978).

27.2  Characteristics of Cadmium and Mercury Cadmium and mercury are chemical elements belonging to one triad group (Zn, Cd, Hg) (12) of the periodic table (IUPAC style). The standard atomic weight of Cd is 112.411 g mol−1 (atomic number 48), and that of Hg is 200.59 g mol−1 (atomic number 80).

27.2.1  Cadmium Cadmium is the soft, bluish-white bivalent metal, whose melting point is 321°C and whose density is 8.65 g cm−3. It is similar in many respects to zinc but forms more complex compounds. The most common oxidation state of the metal is +2. It has high affinity to sulfur, to SH groups in particular, which is responsible for a high accumulation of cadmium in living cells and its compounds’ toxicity. Cadmium is a relatively abundant element. The weight fraction of cadmium in the lithosphere varies around n · 10 −5%. Cadmium and zinc usually compose carbonate and sulfide ores, argillaceous sediments, and in shales. The role of cadmium in biology has been recently discovered (Lane et  al. 2005). Cadmiumdependent carbonic anhydrase, which is involved in supplying inorganic carbon for photosynthesis, has been found in the marine diatom Thalassiosira weissflogii. It is known that the diatoms live in environments with very low zinc concentration, and cadmium can substitute Zn in an active site of the enzyme in vivo. World cadmium production makes about 1.4 · 104 t year−1. Cadmium is produced as by-product when other metals are refined. Cadmium was for a long time used as pigment and for corrosionresistant plating on steel. Cadmium compounds were used to stabilize plastics (Scoullos et al. 2001).

27.2.2  Mercury Mercury is one of the six chemical elements that are liquid at or near room temperature and pressure, its melting point being −38.83°C and density (liquid) being 13.534 g cm−3. Mercury ions

Plant Responses to Cadmium and Mercury Stress

715

can form complex compounds with coordination numbers from 2 to 8. Mercury dissolves to form amalgams with gold, zinc, and many other metals. The weight fraction of mercury in the lithosphere equals n · 10-6%–10-5%. In the environment, mercury occurs as Hg0, Hg1+, and Hg2+. Native mercury, as inclusions in mountain rocks, is rare in occurrence. In nature, it exists in the form of bright red HgS, or cinnabar. This mineral is used for making red color (Trakhtenberg and Korshun 1990, Scoullos et al. 2001). World mercury production makes 8.4 · 103 t year−1. Mercury is used for producing thermometers, barometers, and manometers. Mercury vapor fills mercury-quartz and fluorescent lamps. Mercury contacts serve as position sensors. Metal mercury is applied for production of many important alloys, etc. (Trakhtenberg and Korshun 1990, Scoullos et al. 2001).

27.2.3  Toxicology of Cadmium and Mercury Owing to similar electronic structure, cadmium and mercury are thought to have similar effects on the biochemical systems of animals and humans. Both metals provoke calcium release from nephrocytes and hepatocytes and so cause the membrane potential decrease (Malis and Bonventre 1988). They are highly nephrotoxic as they accumulate in parenchymatous organs, in renal cortex especially, and remain in the organism for a long time (Ambrosi et al. 1991). Affected by cadmium and mercury, lipid peroxidation increases in human erythrocytes (Malis and Bonventre 1988). All cadmium and mercury compounds, as well as vapor, are toxic. Blood-absorbed HMs affect the nervous system, liver, and kidneys, and inhibit phosphorus-calcium exchange. When inhaled, mercury vapor affects respiratory tracts (mercury enters human organism usually as odorless vapor) (Chernykh and Ovcharenko 2002). In 1956, the so-called Minamata disease was reported that first appeared in South Japan because of methylmercury poisoning. This disease has symptoms as deformation of extremities, breathing difficulty, paralysis, and convulsions (Timothy 2001). Chronic cadmium poisoning causes anemia, decalcification, and bones’ destruction (Itai-itai disease) (Nogawa and Kido 1996).

27.3  Heavy Metal Environmental Pollution 27.3.1  Heavy Metal Release into Environment There are natural and technogenic sources of HM release into the environment (Kabata-Pendias and Pendias 1984, Alexeev 1987). Among natural sources, there are mountain rocks and minerals’ weathering, volcanic activity, and soil erosion. The other much stronger source of HM release is produced by anthropogenic factors (metal-working industries, power station, mining industry, cement factories, urban traffic, by-product of fertilizers, etc.). In areas with high anthropogenic pressure, HMs are 100 times more often involved in biochemical cycle as compared with natural HMs (Pacyna et al. 1984). World anthropogenic emissions of cadmium and mercury are equal to about 30 · 103 (Sanità di Toppi and Gabrielli 1999) and 8 · 103 t year−1 (Trakhtenberg and Korshun 1990), respectively. Land ecosystems are most technogenically impacted. Natural HM contents in soils have a great diapason of variation, depending on HM concentration in native rocks, on relief, and climate. For example, mercury concentration in surface soil layer varies within 0.01–0.9, cadmium 0.01–2.5, nickel 2–300, and lead 2–50 mg kg−1 (Kabata-Pendias and Pendias 1984, Chernykh and Ovcharenko 2002). HM contents in high-polluted areas exceed the above-cited values by tens of hundred times.

27.3.2  Assessment of Heavy Metal Environmental Pollution Under the steady increasing anthropogenic pressure on nature, it is important to assess the ecological situation with HM distribution in the environment. Assessment of soil pollution (for air and

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snow as well) is based on pollution comparison of city soils with background soils (in areas with low anthropogenic pressure). For this, concentration coefficient (Cc or clarke) as ratio between HM content in city and background soils is applied (Chernykh and Ovcharenko 2002). The Cc explains the pollution intensity but does not take into consideration a danger of the pollution for living organisms. Therefore, the value of maximum concentration limit (MCL) experimentally determined for every HM is applied for ecological and sanitary-hygienic assessment of soil pollution. The MCL of a pollutant means the maximum amount of the pollutant which does not have negative direct or indirect effect on human health, future generation, and sanitary life conditions (Chernykh and Ovcharenko 2002). The MCL values are not the same in different countries and are regularly revised. For example, MCL for cadmium and its organic compounds in drinking water in Russia is 0.001, in air 0.05 mg dm−3, and in soil 2 mg kg−1. MCL for mercury in drinking water is 0.0005, in air 0.005 mg dm−3, and in soil 2.1 mg kg−1 (Chernykh and Ovcharenko 2002). At present, the probabilistic approach proposed by the U.S. EPA (Environmental Protection Agency) in the early 1980s is widely used. This concept (“risk assessment”) considers the combined effect of pollutants and additional parameters (sex, age, genetic features of studied population). Based on the environmental quality monitoring analysis performed in areas of the industrial Kirov’s district (Kirov is a high industrially developed city), HM contents in soils and wild plants often exceed their Cc and MCL values. Mercury soil content was under MCL but 10–20 times as higher than mercury Cc (0.08), especially near the chemicals industrial complex. The content of mercury, cadmium, and other HM exceeded MCL values near the polymers plant (Skugoreva et al. 2008). It is well known that the main polluters and source of such HMs such as cadmium, lead, and copper in the Komi Republic (the Russian Arctic and subarctic) are discharge of coal mining and reprocessing, oil and gas industries (Walker et al. 2009).

27.3.3  Heavy Metal Migration Processes Knowledge on HM migration processes and on availability of different HM compounds in the environment is required for the HM toxicity assessment. Labile HM forms (water-soluble, exchangeable, acid-soluble HM compounds with organic and nonorganic ligands) are most dangerous because of their high biochemical activity and intensive accumulation in the environment and living organisms. According to the biogeochemical properties, cadmium, mercury, and lead simultaneously have high rates of mobility, aerosol enrichment, accumulation intensity, solubility, and bioconcentration capability (Roeva et al. 1996). Migration processes of HM largely depend on soil properties such as humus content, dispersion, and pH. Within the capability, soils absorb HM anions, and HMs acquire inactive state. Soil colloids have negative charge. They have hydroxyl groups and electron pairs of oxygen, as well as carboxylic and phenol groups of organic substances. Metal anions are attracted by soil colloids. The uptake of metals by plant roots increases with increasing pH in soil solution, which is due to competition between hydrogen ions (they have a higher affinity to negative-charged soil colloids) and metal ions at the bonding sites in colloids (Greger 1999). Soil organic matter lowers the mobility of metal ions by bonding metals to fulvic and humic acids. Clay and/or humus-enriched soils with high pH are generally known for bonding more metal ions and holding them for longer time, compared to other soil types. Cadmium in acid solution is more mobile than zinc (Chernykh and Ovcharenko 2002), and it can be highly toxic in acid soils, for example, podzolic soils in the North (the Komi Republic) (Zaboeva et al. 2002). It has been demonstrated that HM uptake rate by plants from hydroponic culture was higher than HM uptake from soil. Cd content in the hydroponic culture, where barley plants treated by 100 μM Cd were growing, in 6 days was 36% of initial concentration (Figure 27.1). Twelve percent of metal was taken up by plants, and the rest (52%) evaporated. On plots outside, where plants (pea, oat) were growing on fine-textured loamy arable podzolic soil (pH 5.7), soil Cd content did not essentially

717

Plant Responses to Cadmium and Mercury Stress 0.38% 0.67%

59%

20%

100% 36 mg Hg kg–1 (A)

79%

41%

15 daysa

40 daysa 12% 52%

100%

36%

100 μM Cd (B)

19 daysb

25 daysb

FIGURE 27.1  Dynamics of mercury in a soil-plant system (A) and cadmium in a nutrient solution-plant system (B) (with Pisum arvense L., cv. Nadezhda (A), and Hordeum distichum L., cv. Novichok (B) as examples). aDays after seedlings appearance, bdays after seeding. Mercury was added to a pot-filled ground in the form of Hg(NO3)2. (From Skugoreva, S.G. and Golovko, T.K., Agrokhimia (Agrochemistry), 2, 66, 2007b, in Russian.)

change, and annual Cd uptake by plants was less than 1% (Elkina 2007). Plants of Pisum arvense L. (cv. Nadezhda) were grown in soil substrate with initial mercury content in form of Hg(NO3)2 equal to 36 mg Hg kg−1. In 40 days of the experiment, the content of mercury in soil was 40% of the initial value. Only 0.4% of mercury was taken up by plants (Figure 27.1) (Skugoreva and Golovko 2007a). It is important that the soil and plants can volatilize Hg in form of dimethylmercury in significant quantities (Gay et al. 1978). Up to 6% of total mercury content per day can evaporate if soluble mercury salts were added to soil (Rogers and McFarlane 1979). Potentially human-dangerous food chains usually begin with polluted crop production. According to the Sanitary Rules and Norms in Russia, the MCL for Cd in plant green mass and grain is 0.03 and 0.05 mg kg−1 fresh weight, respectively. The MCL for mercury is 0.02 in vegetables and 0.03 mg kg−1 fresh weight in grain (Chernykh and Ovcharenko 2002). It was shown that plants accumulate human-dangerous concentration of mercury even under low Hg soil pollution (less than 18 mg Hg kg−1 soil).

27.4  Heavy Metal Phytotoxicity Excess HM, even of the necessary microelements, is toxic for plants. Knowledge of the mechanisms of metal toxicity in plants is still not well known. It is known that HM can bind to functionally important domains of biomolecules and thereby inactivate them. The result is the substitution of a micronutrient by toxic HM, deactivation and depolymerization of a macromolecule, improper protein synthesis and its replication, transcription, translation of genome, and production of the toxic substances underlies the HM toxicity (Bingham et al. 1986). Cadmium and mercury can cause cell death in plants by inactivating enzymes and structural proteins by acting mostly on sulphydril

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groups. About 100 enzymes are known to bind free metals through sulphydril groups and become inactive, which is the cause of disturbances in cell metabolism.

27.4.1  Growth Growth changes are often the first and most obvious reactions of plants under stress (Hagemeyer 1999). As a rule at high HM treatments, plant growth is significantly reduced due to inhibition of cell division and decrease of cell wall elasticity through accelerating the lignifications processes (Sanità di Toppi and Gabrielli 1999, Seregin and Ivanov 2001). Cd and other HMs induced mitotic disturbance, which resulted in increase in amount of fissionable cells in metaphase state (Seregin and Ivanov 2001). Different chromosomal aberrations and inhibiting spindle system in Allium cepa L root tips treated by mercuric fungicides were noted (Nandi, 1985). In our experiments, high cadmium concentration affected strongly growth processes: relative growth rate of barley plants decreased by 2–3 times (Table 27.1). Cool temperature regime enhanced the metal impact on plant growth evidently due to suppression of Cd immobilization and detoxification (Garmash and Golovko 2009). Lettuce plants were more sensitive to mercury impact (Table 27.2). Plants grown in soil substrate containing 36 mg Hg kg−1 decreased biomass twice compared to those under the control level; higher mercury concentration induced plant death. Up to 70% of growth reduction was induced by the high mercury concentration in garden radish and garden-cress plants.

27.4.2  Photosynthesis HMs are reported to have adverse effect on photosynthetic apparatus (Titov et  al. 2007, Greger and Ögren 1991, Krupa and Barzyński 1995, Prasad and Strzałka 1999, Mobin and Khan 2007). HMs disturb chlorophylls’ biosynthesis and chloroplasts’ structure, provoke the photosystems’ perturbance, inhibit the photosynthetic carbon reduction cycle enzymes, and interfere in stomatal and gas functions. The substitution of the central Mg in chlorophyll by HM (Hg, Cd, Cu, Ni, Zn, Pb) in vivo is an important type of damage in metal-stressed plants (Prasad and Strzałka 1999, Patra and Sharma 2000). According to our data (Figure 27.2), chlorophylls content in leaves under Cd and Hg impact decreases from 15% to 50%, depending on the external metal concentration due to likely inhibiting chlorophyll biosynthesis enzymes, in particular 5-aminolevulinic acid dehydrogenase and protochlorophyllide reductase (Stobart et al. 1985, Baryla et al. 2001, Mysliwa-Kurdziel and Strzałka 2004). High Cd concentration inhibited net photosynthesis (Figure 27.3). Carotenoids as pigments with high antioxidative capability were less affected by HM, which was observed in other studies (Krupa 1988, Khudsar et al. 2001, Lunačkova et al. 2003).

27.4.3  Respiration Mitochondria are thought to be more resistant to HM effects than chloroplasts. Moderate HM concentrations do not affect or even enhance respiration, whereas high concentrations reduce the intensity of this process due to enzyme inactivation (van Assche and Glijsters 1990, Chugh and Sawhney 1999) and disturbances in electron-transport chain (ETC) (Miller et al. 1973, Kesseler and Brand 1995). The effects of HM on individual enzymes of the citrate cycle are considered in detail and summarized (Lösch and Köhl 1999). In particular, HMs affect strongly the succinate dehydrogenase complex, malate dehydrogenase, and isocitrate dehydrogenase supplying reduced coenzymes (NADH, FADH2) into mitochondrial ETC. There is a little information about the effect of HM on the respiratory pathways ratio in the ETC. It is known that cytochrome oxidase activity is reduced by Cd and Hg (Vallee and Ulmer 1972). Engagement of alternative respiratory pathway (AP) as

21°C/17°C

13°C/8°C

Cd Concentration (μM) Parameter

Cd Concentration (μM)

Control

30

60

100

Control

30

60

100

0.110 ± 0.008 0.15 ± 0.04

0.096 ± 0.008 0.07 ± 0.03

0.090 ± 0.007 0.05 ± 0.03

0.098 ± 0.007 0.03 ± 0.06

0.087 ± 0.006 0.13 ± 0.05

0.058 ± 0.004 a 0.02 ± 0.02

0.062 ± 0.004 a 0.03 ± 0.02

0.057 ± 0.003 a 0.01 ± 0.04

Malonic dialdehyde, (MDA, nmol g−1 FW) Leaf 35.7 ± 0.6 Root 12.8 ± 1.6

36.2 ± 1.0 a 24.5 ± 0.5

32.3 ± 2.9 a 24.1 ± 0.3

27.5 ± 3.7 a 20.2 ± 0.9

24.0 ± 5.3 b 25.8 ± 1.1

33.8 ± 4.1 29.6 ± 4.4

21.6 ± 2.9 30.1 ± 2.2

32.3 ± 1.7 a 40.3 ± 2.4

Cd content, (mg kg−1 DW) Shoot Root

160 ± 50 4,100 ± 140

160 ± 50 5,600 ± 200

170 ± 20 15,240 ± 1,500

120 ± 40 3,800 ± 130

40 ± 50 5,050 ± 170

506 ± 65 10,597 ± 106

Plant dry weight (W, g) Relative growth rate (RG, g g−1 day−1)

— —

— —

Plant Responses to Cadmium and Mercury Stress

Table 27.1 Morphophysiological Parameters and Cadmium Accumulation in the Organs of 25-Day-Old Barley Plants (Hordeum distichum L., cv. Novichok) Grown under Two Temperature Regimes (Day/Night) and Different CdSO4 Concentrations in Nutrient Solution

Source: Garmash, E.V. and Golovko, T.K., Russ. J. Plant Physiol., 56, 343, 2009. a Notes: Significant differences between the control and Cd-treated plants are indicated by (p ≤ 0.05), for W, RG = 30, for MDA, n = 6, Cd content, n = 3. Significant b differences from plants grown at temperature regime 21°C/17°C are indicated by (p ≤ 0.05).

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Table 27.2 Effect of Hg(NO3)2 Soil Pollution on Plant Organs Biomass Accumulation (g DW Plant−1) Hg Treatments (mg kg−1)d Species

Control

36

Garden-cress (Lepidium sativum L.) 30 day-old plants Root 0.066 ± 0.011 0.028 ± 0.004a Shoot 0.22 ± 0.03 0.22 ± 0.02

90 0.018 ± 0.002a 0.18 ± 0.02

Lettuce (Lactuca sativa L., cv. Moskovskii parnikovyi) 50-day-old plants Root Plant death 0.060 ± 0.011 0.032 ± 0.008a Shoot 0.85 ± 0.13 0.21 ± 0.06c Garden radish (Raphanus sativus var. radicula, cv. 18 days) 40-day-old plants Root 0.045 ± 0.012 0.034 ± 0.007 0.014 ± 0.004 Shoot 0.56 ± 0.14 0.40 ± 0.07b 0.29 ± 0.06c Garden radish (Raphanus sativus var. radicula, cv. Sofit) 40-day-old plants Root 0.022 ± 0.005 0.021 ± 0.004 0.027 ± 0.004a Shoot 1.08 ± 0.20 0.74 ± 0.19 0.72 ± 0.14 Sources: Skugoreva, S.G. and Golovko, T.K., Agrokhimia (Agrochemistry), 5, 85, 2007a, in Russian; Skugoreva, S.G. and Golovko, T.K., Agrokhimia (Agrochemistry), 2, 66, 2007b, in Russian. Notes: Significant differences between the control and Hg-treated plants are indicated by a (p ≤ 0.05), b (p ≤ 0.01), or c (p ≤ 0.001), respectively, n = 4. d Hg(NO3)2 pollution levels (36 and 90 mg Hg kg−1 DW) correspond to 10 and 25 of MCLs of Hg in soil, respectively.

alleviating the formation of reactive oxygen species (ROS) way under Cd effects was observed (Garmash and Golovko 2009). AP activation is believed to be one of the mechanisms for maintenance of root cell homeostasis under cadmium-induced stress.

27.4.4  Metal-Induced Oxidative Stress HMs stimulate the formation of ROS, either by direct electron transfer involving metal cations or as a consequence of metal-mediated inhibition of metabolic reactions (Dietz et al. 1999). These ROS generated by aerobic metabolism include singlet oxygen (1O2), superoxide ions (O2−), and peroxides, the most widely distributed being hydrogen peroxide (H2O2). Plants in their own life use ROS as signaling molecules, especially in response to various stresses or threats to the plant integrity, as pathogen attacks, or nonoptimal growth conditions (Foyer and Noctor 2003). These molecules can thus act as messengers to trigger protein deactivation or induce the transcription of specific sets of genes in plant cells (Gadjev et al. 2006). On the other hand, plants need to control the levels of these oxidants because of their harmful nature. Therefore, plants have evolved a complex antioxidant defense system (antioxidative enzymes and nonenzymatic antioxidants), which can be regulated according to the environmental conditions. Under non-stress or moderate stress conditions, the antioxidant system is sufficient to ensure redox homeostasis and thus prevent oxidative damage (Dietz et al. 1999). If the antioxidative machinery cannot manage the increased rate of ROS formation, uncontrolled oxidation and radical chain reactions will result in “oxidative blast” (Minibaeva and Gordon 2003).

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Plant Responses to Cadmium and Mercury Stress 14 12

a

Pigment concentration, mg g–1 DW

10

*

8

*

b

6 4 2 (A)

0 8 7 6 5 4 3 2 1 0

0

30

60

100 μM Cd

a *

*

b

Control

(B)

90 mg Hg kg–1

36

External metal concentration

FIGURE 27.2  Effects of cadmium (A) and mercury (B) on pigments concentration in plant leaves. a—chlorophylls, b—carotenoids. A—25-day-old barley plants (Hordeum distichum L., cv. Novichok) after 6 days of CdSO4 treatment; B—40-day-old garden radish plants (Raphanus sativus var. radicula, cv. 18 days). Mercury was added to a soil in the form of Hg(NO3)2. Significant differences between the control and metal-treated plants are indicated by * (p ≤ 0.05), n = 10. (From Skugoreva, S.G. and Golovko, T.K., Agrokhimia (Agrochemistry), 5, 85, 2007a, in Russian.)

Pn , mg CO2 g–1 DWh–1

30

a b

25 20 15

*

*

10 5 0

0

100 Cd concentration, μM

FIGURE 27.3  Effect of cadmium on net photosynthesis rate (Pn ) in leaves of 25-day-old barley plants (Hordeum distichum L., cv. Novichok) after 6 days of CdSO4 treatment under two temperature regimes (day/night): 13°C/8°C (a) and 21°C/17°C (b). Significant differences between the control and Cd-treated plants are indicated by * (p ≤ 0.05), n = 6.

ROS attack all important biomolecules, including nucleic acids, proteins, lipids, and amino acids. As a consequence of oxidative damage, the lipid peroxidation, plasmalemma and cytoskeleton destruction, chloroplasts and mitochondria degradation, and inhibition of photosynthesis increase in the presence of toxic metal concentrations (Dietz et al. 1999). The possible result is the cell disorganization and death. In our experiments, the increased accumulation of malondialdehyde (MDA)

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Table 27.3 Malonic Dialdehyde Content in the Organs of Some Plant Species (nmol g−1 FW) under Different Hg(NO3)2 Soil Pollution Hg Treatments (mg kg−1)d Species

Control

36

Garden-cress (Lepidium sativum L.) 30-day-old plants Leaf 95.2 ± 3.3 101.9 ± 3.3 Root 40.9 ± 2.8 48.8 ± 1.0a

90 103.5 ± 1.7 61.2 ± 3.3a

Lettuce (Lactuca sativa L., cv. Moskovskii parnikovyi) 50-day-old plants Leaf 116.9 ± 8.4 145.3 ± 1.7b Plant death Root 84.0 ± 3.7 147.5 ± 3.5c Garden radish (Raphanus sativus var. radicula, cv. 18 days) 40-day-old plants Leaf 128.6 ± 15.0 130.3 ± 11.7 133.6 ± 1.7 Root 84.3 ± 2.7 104.2 ± 5.8b 104.7 ± 1.3c Edible root 75.2 ± 6.7 80.2 ± 1.7 78.5 ± 1.7 Garden radish (Raphanus sativus var. radicula, cv. Sofit) 40-day-old plants Leaf 130.3 ± 8.4 120.2 ± 3.3 150.3 ± 3.3a Root 99.9 ± 1.3 102.0 ± 2.7 121.1 ± 2.8c Edible root 73.5 ± 3.3 73.5 ± 1.7 86.8 ± 1.7a Sources: Skugoreva, S.G. and Golovko, T.K., Agrokhimia (Agrochemistry), 5, 85, 2007a, in Russian; Skugoreva, S.G. and Golovko, T.K., Agrokhimia (Agrochemistry), 2, 66, 2007b, in Russian. Notes: Significant differences between the control and Hg-treated plants are indicated by a (p ≤ 0.05), b (p ≤  0.01), or c (p ≤ 0.001), respectively, n = 4. d Hg(NO3)2 pollution levels (36 and 90 mg Hg kg−1 DW) correspond to 10 and 25 of MCLs of Hg in soil, respectively.

as a metabolite indicator of lipid peroxidation of membranes after HM treatment was found (Tables 27.1 and 27.3). The maximal concentration of MDA in roots of lettuce grown on mercury-polluted soil (36 mg Hg kg−1) and in barley roots treated with 100 μM Cd under cool temperature conditions was observed.

27.5  Heavy Metal Tolerance in Plants Plants are able to control the intracellular concentration of HM. Many of them are adapted to elevated HM concentrations due to evolution of tolerance mechanisms. Among the metal tolerance mechanisms in plants, selective metal ions uptake, binding and sequestration of metal cations, metabolic processes changing and engagement of alternative metabolic pathways, metal removal through root secretion, and leaves abscission have been extensively documented (Prasad and Hagemeyer 1999, Hall 2002, Titov et al. 2007). Overall, there are three groups of the tolerance mechanisms: (1) prevention of metal ions inflow to the cell, (2) intracellular tolerance mechanisms, and (3) formation of metal-binding complexes.

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27.5.1  Roots as a Barrier on the Path of Metals Transport A first barrier against HM stress operates at the level of root-soil relationship. Root exudates can affect HM, and in particular Cd absorption, by plants through changing the physical and chemical characteristics of rhizosphere. The influence of root exudates on HM bioavailability and toxicity may include modifying the rhizosphere pH, chelating/complexing and deposition with metal ions, and altering the community construction, and the numbers and the activities of rhizospheric microbes (Seregin and Ivanov 2001, Dong et al. 2007). There is circumstantial evidence that plants can benefit from mycorrhiza colonization on contaminated soils (Colpaert and Vandenkoornhuyse 2001). Mycorrhiza is thought to improve soil nutrition of the plant-host and protect the plant from exposure to metal contaminants by reduced assimilation and/or transfer of metals to the host. The prevention of excess metal uptake might be realized by fungi excretion of metal-immobilizing substances, extracellular sequestration, or wellregulated uptake system coupled to stable plasmalemma. Roots as the main barrier on the path of metals transport to shoots are able to immobilize HM ions (including Cd and Hg) by means of the cell wall and extracellular carbohydrates (Sanità di Toppi and Gabrielli 1999, Seregin and Ivanov 2001). HM ions seem to be mostly bound by pectic sites and hystidyl groups of the cell wall. The importance of Cd binding to cell walls and the limitation of its subsequent translocation to shoots has been demonstrated for root cells of hyperaccumulators and non-hyperaccumulating plants (Shevyakova et al. 2003, Cosio et al. 2005). Under low concentrations of HM in the medium, the metal ions enter the roots and reach the xylem through an apoplastic pathway where they can be complexed by several ligands (Sanità di Toppi and Gabrielli 1999, Seregin and Ivanov 2001). Casparian band and plasma membrane of endodermal cells can hold the ions to a certain metal concentration in tissue. Roots transport HMs across their plasma membrane either by diffusion (mostly by exchange absorption) or by active transport via cation channels or carrier proteins (Costa and Morel 1994, Hall and Williams 2003). These transport mechanisms are believed to be typical for all metal ions, including Cd, Hg, Zn, Cu, Pb, etc. However, Hg2+ is thought to enter the root cells only after disturbance of plasma membrane sites by binding sulfhydryl groups of membrane proteins. Organic lipophilic mercury compounds move across lipidic phase of plasma membrane readily (Trakhtenberg and Korshun 1990). In our experiments, the HM content in plant roots was higher than that in shoots and soil. Under high mercury soil pollution, the metal content in aboveground part and in roots increased (Table 27.4). It has been also demonstrated that at cool temperature regime and high Cd concentration (100 μM) in nutrient solution, the root barriers lost their function, and cadmium was translocated to shoots: the Cd content in the shoots sharply increased (by four times) (Table 27.1).

27.5.2  Antioxidant System of Plant Cells At high metal concentration in medium, the barriers and sites of metal ions binding in roots are not sufficient to prevent transport of HM to the shoots. Therefore, oxidative stress in cells can be developed. Antioxidant defense system plays an important role in detoxifying ROS. The antioxidative machinery is antioxidative enzymes and nonenzymatic antioxidants (Dietz et al. 1999). The most prominent antioxidative enzymes are the ROS-detoxifying superoxide dismutases (SOD), catalases, and a large number of H2O2-reducing peroxidases. The important nonenzymatic antioxidants are glutathione, ascorbate, carotenoids, flavonoids, tocopherol, aromatic hydroxy-acids, anthocyanins, polyamines, polyphenols, and thiols; some of them are regenerated by antioxidative enzymes. Enhanced activities of ascorbate peroxidase, catalase, and glutathione reductase in two mustard (Brassica juncea L.) cultivars alleviated Cd stress and protected the photosynthetic activity (Mobin and Khan 2007). Cd2+ in concentrations of 0.01, 0.1, and 1 mM stimulated SOD activity and glutathione reductase in pea leaves, but ascorbate peroxidase activity increased only

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Table 27.4 Hg Content in the Organs of Some Plant Species under Different Hg(NO3)2 Soil Pollution Hg Treatments (mg kg−1)d Species

Control

36

90

Field pea (Pisum arvense L., cv. Nadezhda) 15-day-old plants Root 3.4 ± 0.3 399 ± 41b Shoot 6.6 ± 0.6 45 ± 0.1c

1116 ± 89b 108 ± 9c

40-day-old plants Root Shoot Leaf Stem

1180 ± 110b 21 ± 3a 7.3 ± 0.1c 11 ± 1b

4.6 ± 0.2 4.0 ± 0.6 2.4 ± 0.1 1.8 ± 0.1

289 ± 4c 9.2 ± 1.3a 2.8 ± 0.2 3.4 ± 0.1b

Garden-cress (Lepidium sativum L.) 20-day-old plants Root 2.2 ± 0.3 43 ± 1.8c Shoot 1.6 ± 0.1 3.8 ± 0.1a Stem 1.9 ± 0.3 2.1 ± 0.6

257 ± 8c 16 ± 3a 5.2 ± 0.2b

30-day-old plants Root Leaf Stem

1670 ± 36c 62 ± 7a 24 ± 1a

17 ± 1 25 ± 1 20 ± 1

245 ± 5c 15 ± 2a 14 ± 2a

Lettuce (Lactuca sativa L., cv. Moskovskii parnikovyi) 50-day-old plants 65 ± 3b Plant death Root 14 ± 1 Shoot 4.4 ± 0.1 10.1 ± 0.5c Garden radish (Raphanus sativus var. radicula, cv. 18 days) 40-day-old plants Root 5.2 ± 0.7 48 ± 1c 312 ± 17b Edible root 1.9 ± 0.1 7 ± 0.1c 18 ± 2b b Leaf 2.4 ± 0.1 16 ± 1 28 ± 2b a Stem 2.1 ± 0.4 4.5 ± 0.7 4.8 ± 0.1b Garden radish (Raphanus sativus var. radicula, cv. Sofit) 40-day-old plants Root 5.0 ± 0.1 10.7 ± 0.2c 498 ± 30b c Edible root 1.62 ± 0.03 6.50 ± 0.03 26 ± 4b b Leaf 2.4 ± 0.1 8.1 ± 0.6 33 ± 3b Stem 1.5 ± 0.1 4.90 ± 0.02c 26 ± 2b Sources: Skugoreva, S.G. and Golovko, T.K., Agrokhimia (Agrochemistry), 5, 85, 2007a, in Russian; Skugoreva, S.G. and Golovko, T.K., Agrokhimia (Agrochemistry), 2, 66, 2007b, in Russian. Notes: Significant differences between the control and Hg-treated plants are indicated by a (p ≤ 0.05), b (p ≤ 0.01), or c (p ≤ 0.001), respectively, n = 4. d Hg(NO3)2 pollution levels (36 and 90 mg Hg kg−1 DW) correspond to 10 and 25 of MCLs of Hg in soil, respectively.

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Table 27.5 Peroxidase Activity in the Organs of Some Plant Species (mL 0.01 n I2 g−1 FW) under Different Hg(NO3)2 Soil Pollution Hg Treatments (mg kg−1)d Species

Control

36

Garden-cress (Lepidium sativum L.) 30-day-old plants Leaf 6.58 ± 0.41 6.67 ± 0.43 Root 18.3 ± 0.5 33.8 ± 0.6c

90 7.10 ± 0.43 19.40 ± 0.86

Lettuce (Lactuca sativa L., cv. Moskovskii parnikovyi) 50-day-old plants Leaf 4.19 ± 0.40 147.5 ± 3.5 Plant death Root 5.59 ± 0.50 7.48 ± 0.10a Garden radish (Raphanus satuvus var. radicula, cv. 18 days) 40-day-old plants Leaf 8.60 ± 0.30 8.31 ± 0.10 16.4 ± 0.60b Root + edible root 7.68 ± 0.10 7.22 ± 0.34 16.2 ± 0.41c Source: Skugoreva, S.G. and Golovko, T.K., Agrokhimia (Agrochemistry), 5, 85, 2007a, in Russian. Notes: Significant differences between the control and Hg-treated plants are indicated by a (p ≤ 0.05), b (p ≤ 0.01), or c (p ≤  0.001), respectively, n = 4. d Hg(NO3)2 pollution levels (36 and 90 mg Hg kg−1 dry weight) correspond to 10 and 25 of MCLs of Hg in soil, respectively.

under high cadmium concentration, peroxidase activity—under low Cd concentration in medium (Balakhnina et al. 2005). Peroxidase activity in many plants is found to increase under mercury stress (Siegel et al., 1987). We also found peroxidase activation in roots of the plants grown in soil substrate containing 36 mg Hg kg−1 (Table 27.5). Peroxidases are known to catalyze the reduction of H2O2 to H2O by oxidizing reduced substances (ascorbate, glutathione, etc.) High Hg concentration inhibited the enzyme activity.

27.5.3  Metal-Binding Complexes The production of metallothioneins (MTs) and metal-binding complexes is a widespread mechanism of HM detoxification in higher plants. MTs are a family of cycteine-rich, low molecular weight (3.5–14 kDa) proteins. MTs have the capacity to bind such metals as Zn, Cu, Cd, Hg, etc., through the thiol group of its cysteine residues, which represents nearly 30% of its amino acidic residues (Prasad 1999). Phytochelatins (PCs) are γ-glutamyl peptides containing glutamate, cysteine, and glycine. PCs are synthesized from glutathione in the presence of HM by the enzyme phytochelatin synthase (Grill et al. 1985, Prasad 1999). It is known that a very significant role in HM detoxification and tolerance is played by vacuolar compartmentalization, which prevents the free circulation of metal ions in the cytosol and forces them into a limited area. And HM-phytochelatin complexes can transport into vacuole. In the vacuole, because of the acidic pH, these complexes dissociate and HM can be complexed by vacuolar organic acids (citrate, oxalate, malate), and, possibly, by amino acids (Prasad 1999, Sanità di Toppi and Gabrielli 1999, Seregin and Ivanov 2001). Apo-phytochelatins may return to the cytosol, where they can continue to carry out their shuttle role (Sanità di Toppi

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mg SH g–1 protein

300

*

250 200 150 100 50 0

b a 0

* 30 60 Cd concentration, μM

* 100

FIGURE 27.4  Content of SH group in soluble protein of 25-day-old barley plants (Hordeum distichum L., cv. Novichok) after 6 days of CdSO4 treatment. a—leaves, b—roots. Significant differences between the control and Cd-treated plants are indicated by * (p ≤ 0.05), n = 6.

and Gabrielli 1999). Phytoferritin is an iron-protein complex. The protein moiety of ferritin is composed of 24 subunits, which form channels with the deposition of Fe as ferric hydroxyl phosphate (Prasad 1999). The functions of ferritin in plants are storage of Fe and protection of the cell against the toxic effects of free ionic iron. Phytoferritins are usually abundant in roots, root nodules, senescing cells, and seeds. This Fe-binding complex readily meets the demand for iron of the developing chloroplasts. Overall, metal-binding complexes reduce cytoplasmic toxicity of certain concentrations of HM. They are less toxic to cellular plant metabolism than free metal ions. We found a change of soluble protein structure in barley roots under cadmium impact (Figure 27.4). The amount of the SH groups reacting with HM and detoxifying it was significantly increased compared to the control.

27.5.4  Metabolic Processes Changing The activation of a reparation system and/or changing of metabolic processes can be one of the HM tolerance mechanisms in plants. Respiration is an important plant function, because it is source of energy and intermediates. As process connecting with all sides of plant activity, respiration reflects metabolism state. We found that HMs in moderate concentrations do not change or even increase respiratory capacity. Though, the plant growth was inhibited in mercury stress conditions, respiration rate in plant roots was 20%–40% higher under Cd stress compared to the control (Figure 27.5). It is likely related to the activation of metabolic processes associated with functional cell integrity maintenance and reparation of damages (Golovko 1999). In other experiment, high cadmium concentrations suppressed respiration rate of barley plants, especially in roots, and induced engagement of alternative pathway (AP) respiration (Garmash and Golovko 2009). The contribution of AP increased to 40% of the total respiration (Figure 27.6). At the same time, the lipid peroxidation is intensified, which is an indication of increased oxidative stress (Table 27.1). AP is known to maintain redox balance during mitochondrial electron transport (Millenaar and Lambers 2003) and to alleviate the formation of ROS (Maxwell et al. 1999). A significant AP involving in the roots at low Cd concentration (30 μM) under warm temperature regime, when the respiration rate is relatively high, is evidently required for continuation and active functioning of the citric acid cycle. Organic acids of the cycle are able to bind Cd in vacuoles (Sanità di Toppi and Gabrielli 1999, Seregin and Ivanov 2001). Under high Cd stress (100 μM) and low temperature regime, plant growth was ceased (Table 27.2) and partial death of plants was observed. It is known that during the development of programmed cell death in plants, APs may maintain mitochondrial function after insult with a death-inducing compound or may prevent chronic oxidative stress within the mitochondrion (Robson and Vanlerberghe 2002).

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**

160 140 120

*

**

*

* *

*

*

**

* * Control

100 80 60 40 20 0

Sofitb

20 daysa 30 daysa Garden-cress 36

Lettuce 54

18 daysb

15 daysa 40 daysa

Garden radish

90

108

Field pea

180 mg Hg kg–1 DW

FIGURE 27.5  Effect of mercury on respiration rate of underground organs in some plant species, % from control values. aDays after seedlings appearance, bcultivars of garden radish (40 days after seedlings appearance). Mercury was added to a soil in the form of Hg(NO3)2. Significant differences between the control and Hg-treated plants are indicated by * (p ≤ 0.05), n = 5. (From Skugoreva, S.G. and Golovko, T.K., Agrokhimia (Agrochemistry), 5, 85, 2007a, in Russian.) 3500

O2 uptake, μL g–1 DW h–1

2500

Leaves a

21/17°C

Roots

b

1500

*

*

500 0 3500

13/8°C

2500

*

1500

*

500 0

0

30

60

100

0

30

60

100

Cd concentration, μM

FIGURE 27.6  Effect of SHAM, the inhibitor of AOX, on the rate of O2 uptake by the organs of 25-day-old barley plants after 6 days of CdSO4 treatment under two temperature regimes (day/night). a—control (no inhibitor); b—25 mM SHAM. Significant differences between the control and SHAM-treated samples are indicated by * (p ≤ 0.05), n = 12. (From Garmash, E.V. and Golovko, T.K., Russ. J. Plant Physiol., 56, 343, 2009.)

27.6  Strategies in the Response of Plants to Heavy Metals Plants have developed different tolerance strategies to grow on soils rich in HMs (Baker 1981). A large number of tolerant plants are called excluders: they are able to restrict root uptake and, in particular, root-to-shoot translocation of HM. These plants have a barrier to avoid uptake of metals. Under high HM levels, the barriers lose their function, and the uptake increases. In other plants, called indicators, passive metal uptake occurs so that the internal concentration reflects the external

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level. Indicators have been widely used in the detection of HM impact on the environment. The best phytoindicators are mosses and lichens, but they are not effective for soil biomonitoring. It was found that suitable phytoindicator among angiosperms is dandelion (Taraxacum officinale L.) (Bezel’ and Zhuikova 2007). Roots and leaves of this plant are able to uptake metals from polluted soil and air, respectively. Dandelion and other species of Asteraceae, which are the most advanced evolutionary plant species and have a wide reaction norm, accumulated more HM such as Cu, Cd, and Co than other plants (Fabaceae, Poaceae) (Bezel’ and Zhuikova 2007). The third type of uptake characteristic is plants accumulators. These plants have high accumulation of metals at low external metal concentrations. Accumulators have certain internal mechanisms of HM detoxification, which allow the plant to accumulate high amounts of metals. And some plant species called hyperaccumulators accumulate HMs to exceedingly high levels in the aboveground parts. The term “hyperaccumulator” was introduced by Brooks et al. (1977) for plants that accumulate more than 1 mg Ni per gram of dry weight in their shoots in their natural habitats. To date, more than 400 species of hyperaccumulators of Ni, Co, Mn, Cd, and Zn belonging to 45 families have been identified (Baker et al. 2000). Many of the hyperaccumulators belong to Brassicaceae (Sanità di Toppi et al. 2001).

27.7  Phytoremediation Plants concentrate the contaminants within their tissues, thereby reducing the amount of hazardous waste in the environment. This plant property underlies phytoremediation technology (Saxena et al. 1999, Mukherjee 2001). Generally, plant hyperaccumulators are used for phytoremediation, and most of the commonly known hyperaccumulators belong to the Brassicaceae family (Sanità di Toppi et al. 2001). The volume of contaminated plant biomass can be reduced by ashing or composting; the residue should be treated as a hazardous waste or can be recycled for the production of valued metals (Mukherjee 2001). Phytoremediation is cost-effective in comparison with current expensive engineering methods, such as washing or incineration of soils. Phytoremediation is used for remediation of soils and decontamination of air and water. Nowadays, transgenic plants might represent a strong tool for developing effective HM phytoremediation strategies. Pilon-Smits et al. (1999) overexpressed the E.coli γ-glutamylcystein synthetase and glutathione synthetase enzymes in Brassica juncea. These transgenic plants concentrated more Cd than normal plants in their shoots. It has been reported that a modified bacterial mercuric ion reductase has been introduced into Arabodopsis thaliana, which converts Hg2+ into Hg0 and volatilize Hg in significant quantities (Rugh et al. 1996). Phytovolatilization is a perspective method for removing Hg as well as As and Se from the contaminated soils (Mukherjee 2001).

27.8  Conclusions The last 10–15 years were marked by considerable progress in studying the problem of plants tolerance and adaptation to HM environment pollution. However, the problem is not completely solved yet. Further production development, the earth population upsurge and resource consuming, contamination of the environment, and accelerating imbalance in nature compel us to take strong measures regarding defense and maintenance of biodiversity. Therefore, extension of knowledge on ecological adaptations of plants is of great importance. Plant responses to an HM depend on genotype tolerance, its actual age, phase of development, life form, climatic and edaphic factors, and finally concentration and exposure time of the metal. In nature, the effects of pollutants on plants are often modified by other existing factors. Plants in areas with high anthropogenic pressure are more sensitive to other abiotic and biotic effects. Different plants tolerances to cadmium and mercury effects caused by the HM uptake and accumulation peculiarities, allocation of the metals to the plant organs, responses of the plant metabolism, detoxification mechanisms, and the plant growth conditions were observed. Mercury in two concentrations in soil (36 and 90 mg Hg kg−1)

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729

suppressed growth of plants. The most adverse effect of the metal was found on lettuce (Lactuca sativa L., cv. Moskovskii parnikovyi) growth. This cultivar can be recommended as phytoindicator for biomonitoring mercury-polluted soil. It was also shown that the cool temperature regime impaired the growth of the barley plants treated by cadmium. Alternative respiratory pathway is believed to be one of the mechanisms for maintenance of the root cell homeostasis under cadmiuminduced stress. Knowledge on plant responses to HM effects is attractive to the further development of phytoremediation, development of genotypes and phytocenoses tolerant to HM, and efficient use of natural resources.

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Mysliwa-Kurdziel, B. and K. Strzałka. 2004. Influence of Cd(II), Cr(VI) and Fe(III) on early steps of deetiolation process in wheat: Fluorescence spectral changes of protochlorophyllide and newly formed chlorophyllide. Agriculture, Ecosystems & Environment 106:199–207. Nandi, S. 1985. Studies on the cytogenetic effect of some mercuric fungicides. Cytologia 50:921–926. Nogawa, K. and T. Kido. 1996. Itai-itai disease and health effects of cadmium. In Toxicology of Metals, ed. L.W. Chang, pp. 353–369. New York: CRC Press. Pacyna, J.M., A. Semb, and D.E. Hanssen. 1984. Emission and long-range transport of trace elements in Europe. Tellus 36B:163–178. Patra, M. and A. Sharma. 2000. Mercury toxicity in plants. Botanical Review 66:379–422. Pilon-Smits, E.A.H., Y.L. Zhu, M. Pillon, and J.N. Terry. 1999. Overexpression of glutathione synthesizing enzymes enhances cadmium accumulation in Brassica junceae. In Proceedings of Extended Abstracts of the fifth International Conference on the Biochemistry of Trace Elements, Vienna, Austria, July 11–15, 1999, eds. W.W. Wenzel et al., pp. 890–891. Vienna, Austria: University of Agricultural Sciences. Prasad, M.N.V. 1999. Metallothioneins and metal binding complexes in plants. In Heavy Metal Stress in Plants: From Molecules to Ecosystems, eds. M.N.V. Prasad and J. Hagemeyer, pp. 51–72. Berlin, Heidelberg, Germany, New York: Springer Verlag. Prasad, M.N.V. and J. Hagemeyer. 1999. Heavy Metal Stress in Plants: From Molecules to Ecosystems. Berlin, Heidelberg, Germany, New York: Springer Verlag. Prasad, M.N.V. and K. Strzałka. 1999. Impact of heavy metals on photosynthesis. In Heavy Metal Stress in Plants: From Molecules to Ecosystems, eds. M.N.V. Prasad and J. Hagemeyer, pp. 117–138. Berlin, Heidelberg, Germany, New York: Springer Verlag. Robson, C.A. and G.C. Vanlerberghe. 2002. Transgenic plant cells lacking mitochondrial alternative oxidase have increased susceptibility to mitochondria-dependent and -independent pathways of programmed cell death. Plant Physiology 129:1908–1920. Roeva, N.N., F.Ya. Rovinskii, and E.Ya. Kononov. 1996. Special features of the behavior of heavy metals in various natural environments. Journal of Analytical Chemistry 51:352–364. Rogers, R.D. and C.M.C. McFarlane. 1979. Factors influencing the volatilization of mercury from soil. Journal of Environmental Quality 8:255–260. Rugh, C.L., H.D. Wilde, N.M. Stack et al. 1996. Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial MerA gene. The Proceedings of the National Academy of Sciences of the United States of America 93:3182–3187. Sanità di Toppi, L., and R. Gabrielli. 1999. Response to cadmium in higher plants. Environmental and Experimental Botany 41:105–130. Sanità di Toppi, L., M.A. Favali, R. Gabbrielli, and P. Gremigni. 2001. Brassicacea. In Metals in the Environment, ed. M.N.V. Prasad, pp. 219–257, Chap. 8. New York: Marcel Dekker. Saxena, P.K., S. KrishnaRaj, T. Dan, M.R. Perras, and N.N. Vettakkorumakankav. 1999. Phytoremediation of metal contaminated and polluted soils. In Heavy Metal Stress in Plants: From Molecules to Ecosystems, eds. M.N.V. Prasad and J. Hagemeyer, pp. 305–329. Berlin, Heidelberg, Germany, New York: Springer Verlag. Scoullos, M.J., G.H. Vonkeman, I. Thornton, and Z. Makuch. 2001. Mercury–Cadmium–Lead Handbook for Sustainable Heavy Metals Policy and Regulation. Dordrecht, the Netherlands: Kluwer Academic Publishers. Seregin, I.V. and V.B. Ivanov. 2001. Physiological aspects and lead toxic effects on higher plants. Russian Journal of Plant Physiology 48:523–524. Shevyakova, N.I., I.A. Netronina, E.E. Aronova, and Vl.V. Kuznetsov. 2003. Compartmentation of cadmium and iron in Mesembryanthemum crystallinum plants during the adaptation to cadmium stress. Russian Journal of Plant Physiology 50:678–685. Skugoreva, S.G. and T.K. Golovko. 2007a. Dynamics of mercury in a soil–plant system (with Pisum arvense L. as an example) (in Russian). Agrokhimia (Agrochemistry) 5:85–88. Skugoreva, S.G. and T.K. Golovko. 2007b. Effect of mercury (II) nitrate on the growth and metabolism of lettuce and garden radish (in Russian). Agrokhimia (Agrochemistry) 2:66–71. Skugoreva, S.G., S.Yu. Ogorodnikova, T.K. Golovko, and T.Ya. Ashikhmina. 2008. Phytotoxicity of Phosphororganic Substances and Mercury (in Russian). Ekaterinburg, Russia: Ural Division RAS. Stobart, A.K., W.F. Griffiths, I. Amen-Bukhari, and R.P. Sherwood. 1985. The effect of Cd2+on the photosynthesis of chlorophyll in leaves of barley. Physiologia Plantarum 63:293–298. Taiz, L. and E. Zeiger (Ed.). 2002. Plant Physiology, 3rd edn. Sunderland, MA: Sinauer Associates, Inc. Publishers. Timothy, S.G. 2001. Minamata: Pollution and the Struggle for Democracy in Postwar Japan. Cambridge, MA and London, U.K.: Harvard University Press.

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Titov, A.F., V.V. Talanova, N.M. Kaznina, and G.F. Laidinen. 2007. Plant Tolerance to Heavy Metal Impact (in Russian). Petrozavodsk, Russia: Karelian Scientific Centre RAS. Trakhtenberg, I.M. and M.N. Korshun. 1990. Mercury and Its Compounds in the Environment (Sanitary and Ecological Aspects) (in Russian). Kiev, Russia, Vysha Shkola. Vallee, B.L. and D.D. Ulmer. 1972. Biochemical effects of mercury, cadmium and lead. Annual Review of Biochemistry 41:91–128. Van Assche, F. and H. Glijsters. 1990. Effects of metals on enzyme activity in plants. Plant, Cell & Environment 13:195–206. Walker, T.R., P.D. Crittenden, and V.A. Dauvalter et al. 2009. Multiple indicators of human impacts on the environment in the Pechora Basin, north-eastern European Russia. Ecological Indicators 9:765–779. Zaboeva, I.V., E.M. Lapteva, V.A. Beznosicov, G.M. Vtiurin, and G.A. Simonov. 2002. Guide to Scientific Soil Excursion (Forest Zone) (in Russian). Syktyvkar, Russia: Komi Scientific Centre Ural Division RAS. Zyrin, N.G. and L.K. Sadovnikova. 1985. Chemistry of Heavy Metals, Arsenic and Molybdenum in Soils (in Russian). Moscow, Russia: Moscow State University.

Part V Plant and Crop Responses to Weeds, Pests, Pathogens, and Agrichemical Stress Conditions

in Plants and Crops 28 Stress Induced by Parasitic Weeds Andrea Cavalieri and Asghar Heydari Contents 28.1 Introduction........................................................................................................................... 735 28.2 Types of Parasitic Plants........................................................................................................ 736 28.2.1 Important Parasitic Weed Species............................................................................. 737 28.2.1.1 Orobanchaceae............................................................................................ 737 28.2.1.2 Scrophulariaceae......................................................................................... 738 28.2.1.3 Cuscutaceae................................................................................................ 738 28.3 Metabolic Pathway................................................................................................................. 739 28.4 Damage.................................................................................................................................. 741 28.5 Conclusion............................................................................................................................. 742 References....................................................................................................................................... 744

28.1  Introduction The story of agriculture is mainly the story of weed interference. Weeds are considered one of the main problems in agriculture and are responsible for large yield losses in various crops every year (Lampkin, 1990; Bhuler, 1996; Anaya, 1999). Their effect on the agricultural production can be quantifiable to a loss of one-third of the total production, due to competitive factors (Oerke et al., 1994). Plants undergo continuous exposure to various biotic and abiotic stresses in their natural environment. Stress is often defined as any factor that decreases plant growth and reproduction below the potential of the genotype (Osmond et al., 1987). However, biotic stress remains a broadly defined and poorly understood form of plant stress, partly because its application is difficult to control, and partly because its physiological consequences are often highly variable (Ayres, 1992). To survive under such conditions, plants have evolved complex mechanisms to perceive external signals, allowing optimal response to environmental conditions (Fujita et al., 2006). For example, plants compete with each other for light, water, and nutrient elements; therefore, the potential inherent ability to directly inhibit competitors is their strategy to survive. Competition takes place in optimal habitats; therefore, plants or even groups of plants have to move to less favorable and sparsely populated habitats. Movement will only be successful if nutrition can be obtained elsewhere. While the vast majority of plants and weeds are autotrophic where nutrition is guaranteed by the formation of leaves and roots, there are heterotrophic plants, such as parasitic ones, where nutrition is guaranteed by their host. Parasitic weeds are serious pests in agricultural fields and pose a tremendous threat to world agriculture, mainly because they are at present almost uncontrollable. Parasitic plants have lost their autotrophic way of life during their development process. The system is made up of the coexistence of two different organisms, which supports one of them (parasite) on the expense of the other (host). The host through leaves can synthesize organic substance and photosynthesize (chlorophyll + solar energy), while through roots, it can synthesize inorganic substance and the uptake of water and minerals. On the other hand, the parasite obtains carbohydrates from another organism. Hence, they are directly or indirectly dependent on autotrophic plants for carbohydrates, nitrogen, 735

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water, etc. Usually, the host differs from the total number of different species that can be parasitized, and is called “host range,” while it becomes “host preference” when it develops into the most desirable host for optimal growth. Parasites acquired crops as their food resource, enabling them to survive in large numbers even on nutrient-poor sites, since parasitism allows plants to access a rich hydroponic nutrient source. Evolution is opportunistic and parasites are the greatest opportunists. Parasitic weeds are serious pests in agricultural fields; they are a taxonomically diverse group of angiosperms that rely partially or completely on host plants for carbon, nutrients, and water. Root-parasitizing weeds are of economic importance because they reduce crop yield and quality. The parasites remove water, minerals, and photosynthates from the crop, reducing the host’s ability to grow and develop. Crops that are parasitized usually grow more slowly, and, depending on the severity of infestation and the parasite, biomass production is lowered or the host is almost killed. For decades it was assumed that improved breeding and agronomy could overcome the parasites, and little effort was invested in understanding the nature of parasitism. Because of the lack of control methods at the time of attachment, the main means of controlling parasitic weeds had focused on the varying levels of host tolerance and on reducing the soil seed bank, preventing seed set and inhibiting spread from infested to noninfested areas (Boari and Vurro, 2004; Vurro et al., 2009).

28.2  Types of Parasitic Plants Parasitic weeds are generally divided into two major categories (as root and stem parasites, according to the plant organ they attach to), but can be classified in a broader way as follows: • Parasite: a plant depending on another plant for part or all of its nutrition • Hemi-parasite or semi-parasite: a plant that is only partially parasitic, possessing its own chlorophyll and photosynthetic ability (may be facultative or obligate) • Holoparasite: a plant that is totally parasitic, lacking chlorophyll, and thus unable to synthesize organic carbon • Obligate parasite: a plant that cannot establish and develop independently • Facultative parasite: a plant that can establish and grow independently, but normally behaves as a parasite to obtain some of its nutrition Parasitic plants are defined by the production of specialized feeding structures, described as haustorium. The haustorium is the key organ in all parasitic plants, physically and physiologically bridging the parasite and the host. Nutrients and water are transferred through the haustorium from the conductive system of the host into that of the parasite, and all hormonal interactions between the two organisms are facilitated by this organ. The haustorium develops when intrusive cells of the parasite penetrate host tissues, reaching the vascular system of the host (Smith et al., 2001). Parasites become established via germination. Seeds land on the host tissue and germinate after reaching a chemical stimulus from the host. A modified lateral root becomes a haustorium; this root is chemotrophic, that is, responding to a chemical gradient, and contacts the host epidermis. The root then attaches by pushing against the plant and forming a disc, called a hapteron, and secretes a polysaccharide adhesive. The root tip then mechanically penetrates the host, apparently without enzymatic digestion, and establishes a vascular connection by attaching vessels and positioning phloem next to the leaky host phloem. Mistletoes were formerly alleged to receive no host carbohydrates, but a substantial heterotrophic carbon gain has been measured in mistletoes, even without phloem connections. A direct xylem-to-xylem continuity between host and parasite is not easy to demonstrate. Mistletoes often exhibit high transpiration rates during the day, through stomates and cracks in the epidermis. Losing substantial water from the leaves and stems of the parasite results in a steep water potential gradient, favorable to drawing water into the mistletoe plant. Nitrogen is supplied to the parasite in the xylem stream, and the high transpiration rates; hence, high water demands appear instead to represent a nitrogen-gathering mechanism for the mistletoe. Typical thick, fleshy root parasites

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generally lack any adaptations to restrict water loss from their achlorophyllous stems and leaves because they tend to lack the waxy coating, cuticle. Dodder and mistletoes are serious problems for plants. Dodder is weedy and can cover woody plants and damage certain economically important crop plants. Mistletoe can become so abundant on a tree that most of the foliage is of the parasite, not of the host. In general, experts generally state that parasitic plants rarely, perhaps never, kill the host plant, so that the host and parasite live unhappily together in some balance.

28.2.1  Important Parasitic Weed Species The economically important parasitic weed species derive from three families: • Orobanchaceae: Orobanche • Scrophulariaceae: Striga, Alectra • Cuscutaceae: Cuscuta 28.2.1.1  Orobanchaceae According to the occurrence of Orobanche spp., the major problems with these weeds are found around the Mediterranean Sea that cause major yield reductions in crops. Yield reduction is dependent on the coincidence and the severity of attack, with yield losses from 5% to 100%. Orobanche spp. are essentially restricted to dicotyledonous hosts. Species of the family Asteraceae are preferred by Orobanche, which, in fact, provide about 150 hosts parasitized by 59 Orobanche species. The genus Orobanche, as currently classified, contains over 100 species of obligate root holoparasites in both the Old and New Worlds. These plants are known by the English name “broomrape” because they were thought to grow as tubers (“rapum”) from brooms (the common name for the legume Cytisus). The genus reaches its greatest diversity in the Mediterranean climates and in Western Asia. Most of the economically important pathogens are species of the Old World. The major crop hosts for Orobanche are legumes, solanaceous crops (eggplant, tomato, tobacco, and potato, but not Capsicum peppers), umbels (carrot, parsley, and celery), cole crops (cabbage and cauliflower), lettuce, and sunflower. Control is difficult due to seed longevity in the soil (more than five decades), small seed size (less than the width of a human hair), fecundity (thousands of seeds per plant), and a subterranean phase (seeds germinate beneath the soil and parasitize the host before they emerge and appear). Broomrapes have their greatest impact in the Balkans, the Nile Valley, Central Asia, southern India, and Nepal. Damage varies with the level of infestation, and total crop failures have occurred in some cases. There have been numerous studies of the host range of Orobanche species. It has been shown that Orobanche ramosa can parasitize plants from 11 different dicot families, in fact, more different hosts than any other broomrape. Major agronomically important hosts include solanaceous crops, cabbage, cauliflower, hemp, carrots, lettuce, and some legumes. The related species O. aegyptiaca causes especially severe damage to melons in Central Asia, where broomrape not only reduces the yield and weakens the melons, but also induces the production of a toxin within the melons that renders them unmarketable. O. aegyptiaca and O. ramosa attack especially Solanaceae, like eggplant, potato, tobacco, tomato, etc. Grain and fodder legumes are parasitized by O. crenata and O. minor, while O. cumana threatens sunflower. The most damaging species in legume crops is O. crenata, occurring mainly in the Mediterranean region. The most important legume hosts are faba bean, lentil, pea, chickpea, and common vetch. Serious damage in faba bean occurs, for example, in Egypt, Morocco, and Spain. Orobanche aegyptiaca, although more widespread than O. crenata, is of less importance in legumes due to the different temperature requirements. This species requires higher temperatures for germination and development. Therefore, it is more often found parasitizing summer crops, like tobacco or tomato, instead of the legumes grown during winter.

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Although Orobanche is spread all over the world, losses occur mainly in South and East Europe, North Africa, and West Asia. Parasitism can cause huge or limited yield reduction depending on the number of parasite attachments per host plant and the time of infestation. In some cases, farmers have to abandon their fields. During the period 1968–1978, the faba bean production area in Egypt dropped down by 29% because of heavy infestation with O. crenata. In Yogoslavia, the production area for sunflowers decreased by 37% in the 1950s (Nickrent and Musselman, 2004). The parasite could be not forced back until resistant varieties were grown. Damage by the parasite is dependent on the infestation level in the field, that is, the probability of the host root to meet the seeds of a parasitic weed. Yield loss is also dependent on the time of infestation and, therefore, on the duration of infection. Most of the attached parasites are not visible aboveground, since usually only about 1%–30% of the attached plants emerge. 28.2.1.2  Scrophulariaceae Striga is at the moment the biggest biological hindrance in grain and corn production in Africa. Three species are seriously damaging: Striga asiatica and Striga hermonthica on cereals, and Striga gesnerioides on legumes like cowpea, groundnut, and bambaranut. Striga asiatica and Striga hermonthica are almost entirely specific to grasses, while Striga gesnerioides prefers dicot host. The most important species from the economical viewpoint is Striga hermonthica, attacking sorghum and millet crops in the Sahelian area. Crop losses due to Striga were estimated to more than $7 billion. The occurrence of the economic importance of Striga species is reported in 59 countries, especially in east and west Africa and Asia. Witchweeds (Striga spp.) have a greater impact on humans worldwide than any other parasitic plants, because their hosts are subsistence crops grown widely in Africa and Asia. Such crops include maize, sorghum, pearl millet, finger millet, rice, as well as sugarcane and legume crops, such as cowpea and groundnut. The name “witchweed” derives from the effect these parasites have on their host, in which most damage occurs before the parasite is visible aboveground. This “bewitching” behavior is also reflected in the Latin name, which means “hag” or “witch.” Striga is an obligate hemiparasite that reaches its greatest diversity in the grasslands of Africa, although it also occurs in India, the Far East, and Australia. Two species, S. asiatica and S. hermonthica cause the most damage to crops worldwide. Most of the Striga species have complex life cycles. Several discrete steps can be recognized: diaspore, after ripening, conditioning, haustorial induction, attachment, penetration, seedling development, emergence, and flowering. The genus Alectra spp. (hemiparasitic) comprises about 30 species. Four are reported to be significant pests. The most important is Alectra vogelii, parasitizing mainly cowpea, bambara, and groundnut. Excessive damage is caused by Alectra picta, attacking predominantly Vigna unguiculata (cowpea), a food legume of the savannah areas in Africa. Alectra orobanchoides can be a problem in Nicotiana tabacum (tobacco) in South Africa, while Alectra fluminensis has been described as a pest in Saccharum officinarum (sugarcane) in Latin America. 28.2.1.3  Cuscutaceae Cuscuta spp., or dodder species, are among the best known of all parasitic plants. The biology and control of dodders has been reviewed in Dawson et al. (1994). Dodders have a broad host range, although monocots are less preferred. The genus Cuscuta contains three subgenera. The first subgenus, Monogyna, are robust vines that may attack and kill fruit trees, the second subgenus, Cuscuta, are more delicate in structure and favor herbaceous hosts, the third subgenus, Grammica, have very low host specificity and are easily found parasitizing different host species simultaneously (Nickrent and Musselman, 2004). Although dicots are preferred, attack on monocots has been observed. Dodders may be the most important parasitic weeds of legumes in temperate regions. Of particular importance is Cuscuta campestris on alfalfa (Medicago sativa). The alfalfa and dodder seeds are similar in size, and so the parasite is spread with the host. The wide range of hosts attacked by dodders is reviewed in Dawson et al. (1994). The most effective means of control is seed sanitation.

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Because the surface of dodder seeds is minutely roughened, dodder seeds stick to felt rollers, while alfalfa seeds pass over. Dawson et al. (1994) also reviewed several herbicide treatments that are directed at the newly germinated seeds of dodder.

28.3  Metabolic Pathway The germination of parasitic plants represents a pivotal role in their metabolic pathways. This process is unique because it depends on the reception of chemical stimulus. A typical life cycle is shown in Figure 28.1. The seeds of parasitic weeds buried in the soil become sensitive to germination-stimulating signals from host roots. Usually, as soon as a crop is planted, the roots of crop plants produce germination stimulants and induce the germination of part of the parasitic weed seed population (Bouwmeester et al., 2003). In addition, it is widely reported that, following imbibitions of the seeds, and before chemical stimulation, a wet environment is required for several days in suitable temperatures to render the imbibed seeds responsive to germination stimulants and to allow germination (Joel, 2000). Seeds of most parasitic plants will readily germinate only if the appropriate environmental conditions with respect to water, oxygen, temperature, and light are met (Boone et al., 1995; Estabrook and Yoder, 1998, Sauberon et al., 2007). This preparatory phase is termed “conditioning” or “preconditioning.” After this stage, the radicle of the parasite grows to the host root. Haustorium-inducing factors are produced by the host root and a haustorium is formed. A xylem connection is established and the parasitic plant emerges. A large part of the damage to the host is inflicted before the emergence of the parasite. The parasitic plant reaches maturity and produces flowers and seeds. The seeds end up in the soil seed bank where they gradually become sensitive to germination signals. Seeds of parasitic weeds are buried in the soil and are subsequently “preconditioned” (Boone et al., 1995; Joel, 2000; Bouwmeester et al., 2003). Many studies on seed conditioning have tried to explain such a pathway, but no single research explains whether any of these molecular and metabolic developments correspond to the receptivity of germination stimulation. It has been speculated that conditioning may allow the leaching of germination inhibitors from the seed, or that conditioning may increase the permeability of the seed coat or other seed structures, or that structural modifications occurring in the seeds allow the stimulant to access its putative cellular target (Godwin et al., 1998). Parasitic plants use host molecules to trigger developmental

Dormancy

Colonization

Conditioning

Seed dispersion

Germination

Flowering

Emergence Parasitization

Host Seeds

FIGURE 28.1  Life cycle of a parasitic plant.

Parasite

Penetration

Attachment

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programs essential for parasitism. One such program rules the development, growth, and role of haustoria. The haustoria is a parasite-specific organ responsible for the attachment and invasion of host tissues that attach, attack, and act as physiological conduits for robbing the host plant of water, minerals, and carbohydrates (Kuijt, 1969; Musselman and Dickison, 1975). All of the information necessary to define plant ontogeny is established within the meristem, a space of just a few millimeters. Haustoria develop from changes in the growth and development of the meristem in response to external stimuli. Plant apical meristems consist of an incessant embryonic tissue maintaining root or shoot elongation. In some parasitic species, primary haustoria develop as a transformation of root apexes (Kuijt, 1969). In others, secondary haustoria develop from parenchymal cortical cells near the root tip as well as at more proximal positions along the root (Baird and Riopel, 1983; Estabrook and Yoder, 1998). The attachment of the parasite to the host is facilitated by mucilaginous substances produced by haustorial hairs. Attachment is not discriminatory and can occur on plastic or string as readily as host roots. The epidermal cells that overlay the swollen root cortex develop long haustorial hairs that function in host attachment (Baird and Riopel, 1983). After the haustorium initiation, cortical cells differentiate into vessel elements that form a xylem bridge between the host and the parasite. Haustorium development is induced in the roots of parasitic plants by molecules exuded from host roots. In contrast, the development of vessel members requires a direct contact with the host tissues. Therefore, it is likely that at least two deferent types of host signals are necessary for haustoria to mature. Even though a large number of host species can be parasitized, only a subset is selected when the parasite is presented with a choice (Atsatt and Strong, 1970; Werth and Riopel, 1979; Gibson and Watkinson, 1991). Host selection presumably allows the parasite to maximize the number of beneficial associations, such as those with nitrogen-rich legumes. Legumes, in fact, are often favored because the parasite’s mineral requirements are met in part through connections with the host (Visser et al., 1990; Seel et al., 1993; Press, 1995). On the other hand, host selection allows the parasite to minimize nonproductive associations, such as those between roots of the same individual or between roots of closely related individuals (Yoder, 1997). A clear example of this is the avoidance of self-parasitism by most parasitic plants. Some parasites, like Striga, completely lack haustoria in the absence of host plants. Haustoria may be produced by mono- or dikaryotic mycelium of rust fungi. Monokaryotic haustoria (M-haustoria) merely appear as intracellular extensions of intercellular hyphae with no significant morphological specialization (Voegele and Mendgen, 2003). Dikaryotic haustoria develop from external haustorial mother cells (HMC) with a slim neck that penetrates into the host cell and a haustorial body that forms distally to the neck (Voegele and Mendgen, 2003). Penetration generally is mediated by a combination of intrusive growth and enzymatic digestion (Kuijt, 1969). The evidence for intrusive, mechanical penetration comes from the appearance of crushed host cells at the site of haustoria entry. Upon infection, increased turgor pressure at the tip of the appressorium allows it to mechanically penetrate the cuticle and host cell walls (Estabrook and Yoder, 1998). The HMC, therefore, functionally resemble an appressorium. However, it remains to be elucidated if the functional similarity extends to the molecular level. The expanding haustorium invaginates the host plasma membrane and a new membrane is probably synthesized. Therefore, haustoria are not truly intracellular. They remain outside the physiological barrier of the host cell. With the formation of the haustorial body, a zone of separation between the plasma membranes of the host and the parasite is established. It consists of the fungal cell wall and the extrahaustorial matrix. The extrahaustorial matrix resembles an amorphous mixture of components, mainly carbohydrates and proteins, partly of fungal, but primarily of plant origin (Harder and Chong, 1991). The initial biotrophic phase is characterized by the presence of an interfacial matrix separating host and parasite plasma membranes (Perfect and Green, 2001). Upon the switch to necrotrophic growth, the host plasma membrane surrounding the hyphae disintegrates and parasitic growth continues with narrower unsheathed hyphae. It, therefore, seems likely that this zone of separation plays an important role in the

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maintenance of the biotrophic lifestyle. Undoubtedly, the extrahaustorial matrix represents a formidable trading place for the exchange of nutrients and information between the host and the parasite (Heath and Skalamera, 1997).

28.4  Damage The effects that parasitic plants have on host plants are diverse. The acquisition of host resources can exert strong effects on host growth, allometry, reproduction, and physiology (Press et al., 1999). They can lower host growth rate and reproductive output, change host architecture, and even influence host interactions with other organisms, such as mycorrhizae, pathogens, herbivores, and pollinators. Generally, parasitism reduces host productivity and/or reproductive effort, either in root parasites (Matthies, 1995, 1996, 1997; Seel and Press, 1996; Davies and Graves, 1998; Matthies and Egli, 1999) or shoot parasites (Jeschke et al., 1994a,b; Silva and Martínez del Rio, 1996; Tennakoon and Pate, 1996; Howell and Mathiasen, 2004). The presence of a parasite usually alters drastically the host metabolic functions, such as photosynthesis, respiration, and uptake of water and solutions. The damage generated by the parasite in such circumstances is more important than direct competition for resources. For example, host productivity can be brutally reduced by parasite-induced change in CO2 exchange, or loss of water from the host may lead to a decrease in the host stomatal conductance and, consequently, a fall in the rate of host photosynthesis. Basically, a parasite affects host performance by abstracting resources that are essential for the subsistence of the host. Host-derived materials may be transferred from source (crop) to sink (parasite) through straw-like penetrations, called oscula, into the host vascular system (Dörr, 1997). Hence, competition between host and parasite takes place for water, inorganic nutrients, and organic compounds. The level of injury generated by the parasite depends not only on how much the resources are removed but also on the supply available to the host, which may be constrained by the environmental conditions. In most cases, the reduction in host performance is significant, and in the most extreme cases, parasitism may result in host death (Aukema, 2003). Significantly, for community-level impacts, effects on the host are often disproportionately great in comparison to the size of the parasite. This can result from both inefficient use of the resources by the parasite, such that reduction in host biomass is generally greater than the increase in parasite biomass (Matthies, 1995, 1996, 1997; Marvier, 1998; Matthies and Egli, 1999), and from impacts on host physiology that further impair host performance (Ehleringer et al., 1986; Watling and Press, 2001). Further impacts can occur through effects on the host allometry and architecture (Ehleringer et al., 1986; Wanner and Tinnin, 1986; Parker and Riches, 1993; Sala et al., 2001; Meinzer et al., 2004). Parasites can affect host photosynthesis in a variety of ways and at a range of scales, acting at either the leaf and/or the whole-plant level. The magnitude of the impact varies with species, both host and parasite, and ranges from tiny to cruel. Any mechanism that lowers the host photosynthetic rate will also increase the chance of photoinhibition occurring in infected plants. The reduction in host biomass is not explained simply by source–sink relationships, but by phytotoxic effects that change the partitioning of the host photosynthate from shoot to root and an overall reduction in the photosynthetic rate (Ransom et al., 1996). Many species also cause a shortening of the internodes in infected hosts, which causes increased self-shading and, thus, a reduction in light interception (Press et al., 1999). In addition to the injurious effects that some parasitic plants have on host photosynthesis at the leaf level, some are also able to impact negatively on whole-plant carbon fixation through the effect they have on host architecture. Graves et al. (1989), in a carbon budget model, estimated that over 80% of the reduction in host growth could be attributed to the reduction in photosynthesis, while Cechin and Press (1993) demonstrated that the effect on photosynthesis was generated critically by the deficiency in nitrogen. Anyway, the degree of pathogenicity exhibited by parasitic plants is great, from those that exert little impact on their hosts to those that dramatically affect the host physiology and fecundity. Among factors affecting pathogenicity are the biomass ratio of parasite to host, the number of parasites attached to an individual host plant, and the length of time required for the parasite to complete its life cycle.

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Even though there can be dissimilarity in pathogenicity and life cycle dynamics, all parasitic plant species have evolved under the condition that they do not kill their hosts prior to successful reproduction.

28.5  Conclusion The changing agroclimatic conditions have boosted the infestation and proliferation of the parasitic weeds at an increasing rate. As a result, a war has been waged against these parasitic plants. Unfortunately, the results are not as estimated. The reason is mainly due to the close affiliation of parasitic plants to their host, their complex life cycle and the longevity of seeds (Joel et al., 2007). All these characteristics have not allowed researches to develop a feasible “parasitic weed control”! For decades it has been assumed that crop breeding and agranomy production could overcome parasitism. This belief have generated little interest in understanding the real nature and physiology of parasitism. Their origin, the evolution from nonparasitic plants, their population structures, their evolutionary pathways toward becoming crop parasites, and their ecology are still unclear (Balázs et al., 2009). In fact, due to the lack of knowledge about parasite weeds, it is still impossible to predict whether the infestation levels will be high or low, showing how much needs to be learnt. What has still to be realized and metabolized by a majority of the people is that parasitic plants are not normal weeds. Parasitic plants are a diverse group of organisms that have evolved in a number of ways. This evolution relies on an incessant parasite dependency on the host, ranging from complete dependency upon host nutrients and carbohydrates to an ability to survive and reproduce in the absence of the host. Hence parasitic plants, which are not in direct contact with the soil and soil factors for their growth, have evolved survival means different from normal green plants. For this reason, contrary to general belief, parasitic plants have all the benefit by not billing their hosts, or at least, prior to successful reproduction. Therefore, it is much more difficult to manage or control parasitic weeds. As native components of the ecosystem that have coevolved with their hosts, management rather than eradication is the only rational approach. Understanding the complex and fascinating biology of parasitic plants will require continued research by those engaged in both basic and applied scientific disciplines. Preventing early growth stages, such as seed germination, host attachment, and tubercle development, could be a strategy to deal successfully with the parasite, bringing about its management. Above all, control methods should focus on reducing the soil seed bank and regulate the parasite’s early developmental stages, as most of the damage to the host is inflicted before parasitic plants emerge above the soil. In this context, natural compounds that inhibit or stimulate seed germination could be attractive and environmentally friendly tools to reach that objective (Boari and Vurro, 2004). Most of the damage to the host is done before the parasitic weed emerges above the soil. To overcome this phase, the approach should be the accomplishment of different methods with more emphasis on those that are particularly addressed to neutralize key processes in the parasite. Such methods should lead to a more rational management strategy able to reduce crop damage and suppress the expansion of a virulent seed bank in the fields. As already highlighted, no conventional control can be exerted due to the intimate relation between the parasite and the host. In fact, when the parasite is observable, injury to the crop has already been accomplished. The ineffectiveness of conventional control methods is due to numerous factors, such as the complex nature of the parasites, that reproduce prolifically, have tiny but long-lived seeds, and are difficult to detect until irremediable injury to the crop has been done. So, the only alternative could be the application of the main principles of parasitic weeds management by means of reducing the soil seed bank, preventing seed set, and constraining the spread from infested to noninfested areas. The close relation between the host and the parasite hinders control such as through the application of herbicides. To be effective, the herbicides should be applied at a stage of the crop when the parasite is still underground. At this stage, the herbicides concentrate in the parasites by translocation through the host plant or through the soil solution until they totally defeat

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and die. In point of fact, identifying the timing and the rate of herbicide application is critical because a proper concentration proportional to the parasite biomass has to be achieved without causing damage to the crop. Due to the scalar growth of the parasite and by the presupposition that when the parasite is detectable it is already too late, the herbicide application is not economically effective. Although numerous control measures have been developed in agriculture, only a few are effective due to environmental conditions. One of the most suitable control options could be the development of resistant crop varieties. Unfortunately, in many crops, no resistant varieties have been discovered to date. In fact, as native components of the same ecosystem, parasites and hosts have coevolved in a certain environment through a successful survivor strategy. It looks like the only approach in parasitic weed management could consist of the integration of direct different cultural practices, such as crop and cultivar choice, together with biological control. As in an integrated weed management system (IWMS), a solution to this problem could be combining agronomic choice with biological control. The common practice to tackle weed infestation is to use products obtained by chemical synthesis, although herbicide resistance in weeds is rapidly expanding throughout the world with higher costs of production and greater environmental impact (Anaya, 1999; Lemerle et al. 2001). Inevitably, this practice has caused both ecological and environmental problems and risk to human health (Wyse, 1994; Buhler, 1996; Caporali, 2004). For this reason, in the last years, much emphasis has been put on finding out new and alternative solutions to weed management (Dudai et al., 1999; Cavalieri and Caporali, 2010) and pest control. Research on traditional management of crop pests has mainly focused on a more rational use of natural components and processes of the agroecosystem, providing a chance for more appropriate technologies to be used for a sustainable production without damaging the environment (Anaya, 1999). As a consequence, considerable interest has increased in the development of natural products, plant extracts, and essential oils. Biological control by means of essential oils has been already tested for weed control in contraposition to conventional management, such as synthetic herbicides. Results permitted to assert that essential oils are effective in reducing seed germination in controlled conditions, while environmental factors and enzymatic and microbiological transformations make their application in the open field more difficult (Cavalieri, 2009). Biological control is particularly attractive in suppressing root parasitic weeds in annual crops, because the intimate physiological relationship with their host plants makes it difficult to apply conventional weed control measures. Besides the use of essential oils, a novel approach to increase the level of control is to use a “multiple-pathogen strategy” (Charudattan, 2001). In this strategy, two or more pathogens are combined and applied before or after parasite emergence. The possibility of using fungal toxins as natural herbicides is another alternative (Boari and Vurro, 2004; Vurro et al., 2009). Unfortunately, as already observed for essential oils, many toxins are not selective, as they are able to cause the same toxic effects both on host and on nonhost plants. Further research is necessary to develop an appropriate technology for biological control applications. If properly and successfully developed, the advantages of such tools should lie in their application at a very low rate and concentration. The application could be made when the host plants are already fully developed and vigor, so that they inhibit seed germination of the parasitic plant avoiding any toxic effect to the crop. Biological control, such as the application of toxins or essential oils, is a feasible tool even if it apparently does not seem to be persistent. The low persistence due to their biodegradability and easy catabolization in the environment has to be reconsidered in the scenario of natural products used in some kind of sustainable agriculture, such as organic farming. Vurro et al. (2009), have already demonstrate as the application of fungal toxins as inhibitor of seed germination affects the size and shape of the germ tube, while Zanellato et al. (2010) have demonstrate the genotoxic and phytotoxic effects of essential oils as preemergence bioherbicides. Both the application have the advantage of not being persistent in the environment, and dangerous for the human health. The shortening effect could further protect the development of the host plant from the parasite, reducing and/or defeating the seed germination enough to not be able to reach the root of the host or compete with it.

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References Anaya, A.L. 1999. Allelopathy as a tool in the management of biotic resources in agroecosystems. Critical Reviews in Plant Sciences 18: 697–739. Atsatt, P.R. and D.R. Strong. 1970. The population Biology of Annual grassland hemiparasites, I. The Host Environment Evolution 24(2): 278–291. Aukema, J.E. 2003. Vectors, viscin, and viscaceae: Mistletoes as parasites, mutualists, and resources. Frontiers in Ecology and Environment 1: 212–219. Ayres, P.G. 1992. Plants versus pests and pathogens: An old story but the same story? In Ayres, P.G., ed., Pests and Pathogens: Plant Responses to Foliar Attack. Bios Scientific Publishers, Oxford, U.K. Baird, Wm.V. and J.L. Riopel. 1983. Experimental studies of the attachment of the parasitic angiosperm Agalinis purpurea to a host. Protoplasma 118: 206–218. Balázs, E., Vurro, M., and J. Gressel. 2009. Managing parasitic weeds: Integrating science and practice. Pest Management Science 65: 451–614. Boari, A. and M. Vurro. 2004. Evaluation of Fusarium spp. and other fungi as biological control agents of broomrape (Orobanche ramosa). Biological Control 30: 212–219. Boone, L.S., Fate, G., Chang, M., and D.G. Lynn. 1995. Seed germination. In Press, M. and J. Graves, eds., Parasitic Plants. Chapman and Hall, London, U.K., pp. 14–38. Bouwmeester, H.J., Matusova, R., Zhongkui, S., and M.H. Beale. 2003. Secondary metabolite signaling in host-parasitic plant interactions. Current Opinion in Plant Biology 6: 358–364. Buhler, D.D. 1996. Development of alternative weed management strategies. Journal of Production Agriculture 9: 501–505. Caporali, F. 2004. Agriculture and Health – The Challenge of Organic Agriculture. Editeam, Cento, Italy, 97pp. Cavalieri, A. 2009. From construction to diffusion of knowledge in Agroecology. PhD dissertation, University of Tuscia, Viterbo, Italy. Cavalieri, A. and F. Caporali. 2010. Effect of essential oils of cinnamon, lavender and peppermint on weed germination. Allelopathy Journal 25(2): 441–452. Cechin, I. and M.C. Press. 1993. Nitrogen relation of the sorghum-Striga hermonthica host–parasite association, germination, attachment and early growth. New Phytologist 124: 681–687. Charudattan, R. 2001. Biological control of weeds by means of plant pathogens: Significance for integrated weed management in modern agro-ecology. Bio Control 46: 229–260. Davies, D.M. and J.D. Graves. 1998. Interactions between arbuscular mycorrhizal fungi and the hemiparasitic angiosperm Rhinanthus minor during coinfection of a host. New Phytologist 139: 555–563. Dawson, J.H., Musselman, L.J., Wolswinkel, P., and I. Dörr. 1994. Biology and control of Cuscuta. Review of Weed Science 6: 265–317. Dörr, I. 1997. How Striga parasitizes its host: A TEM and SEM study. Annals of Botany 79: 463–472. Dudai, N., Poljakoff-Mayber, A., Mayber, A.M., Putievsky, E., and H.R. Lerner. 1999. Essential oils as allelochemicals and their potential use as bioherbicides. Journal of Chemical Ecology 25: 1079–1089. Ehleringer, J.R., Ullmann, J., Lange, O.L., Farquhar, G.D., Cowan, I.R., Schulze, E.D., and H. Ziegler. 1986. Mistletoes—A hypothesis concerning morphological and chemical avoidance of herbivory. Oecologia 70: 234–237. Estabrook, E.M. and J.I. Yoder. 1998. Plant–plant communications: Rhizosphere signaling between parasitic angiosperms and their hosts. Plant Physiology 116: 1–7. Fujita, M., Fujita, Y., Noutoshi, Y., Takahashi, F., Narusaka, Y., Yamaguchi-Shinozaki, K., and K. Shinozaki. 2006. Crosstalk between abiotic and biotic stress responses: A current view from the points of convergence in the stress signaling networks. Current Opinion in Plant Biology 9: 436–442. Gibson, C.C. and A.R. Watkinson. 1991. Host selectivity and the mediation of competition by the root hemiparasite Rhinanthus minor. Oecologia 86: 81–87. Godwin, K.S., Aflakpui, P.J., Gregory, and R.J. Froud-Williams. 1998. Effect of temperature on seed germination rate of Striga hermonthica (Del.) Benth. Crop Protection 17: 129–133. Graves J.D., Press, M.C., and G.R. Stewart. 1989. A carbon balance model of the sorghum-Striga hermonthica host-parasite association. Plant, Cell & Environment 12: 101–107. Harder, D.E. and J. Chong. 1991. Rust haustoria. In Mendgen, K. and D.-E. Lesemann, eds., Electron Microscopy of Plant Pathogens. Springer, Berlin, Germany, pp. 235–250. Heath, M.C. and D. Skalamera. 1997. Cellular interactions between plants and biotrophic fungal parasites. Advances in Botanical Research 24: 195–225. Howell, B.E. and R.L. Mathiasen. 2004. Growth impacts of Psittacanthus angustifolius Kuijt on Pinus oocarpa Schiede in Honduras. Forest Ecology and Management 198: 75–88.

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Jeschke, W.D., Rath, N., Baumel, P., Czygan, F.C., and P. Proksch. 1994a. Modeling the flow and partitioning of carbon and nitrogen in the holoparasite Cuscuta reflexa roxb and its host Lupinus albus L.1. Methods for estimating net flows. Journal of Experimental Botany 45: 791–800. Jeschke W.D., Baumel P., Rath N., Czygan F.C., and P. Proksch. 1994b. Modeling of the flows and partitioning of carbon and nitrogen in the holoparasite Cuscuta reflexa roxb and its host Lupinus albus L.2. Flows between host and parasite and within the parasitized host. Journal of Experimental Botany 45: 801–812. Joel, D.M. 2000. The long-term approach to parasitic weeds control: Manipulation of specific developmental mechanisms of the parasite. Crop Protection 19: 753–758. Joel, D.M., Hershenhorn, J., Eizenberg, H., Aly, R., Ejeta, G., Rich, P.J., Ransom, J.K., Sauerborn, J., and D. Rubiales. 2007. Biology and management of weedy root parasites. In Janick, J., ed., Horticultural Reviews. John Wiley, New York, pp. 267–350. Kuijt, J. 1969. The Biology of Parasitic Flowering Plants. University of California Press, Berkeley, CA. Lampkin, N. 1990. Organic Farming. Farming Press, Ipswich, U.K., 715pp. Lemerle, D., Verbeek, B., and B. Orchard. 2001. Ranking the ability of wheat varieties to compete with lolium rigidum. Weed Research 41: 197–209. Marvier, M.A. 1998. Parasite impacts on host communities: Plant parasitism in a California coastal prairie. Ecology 79: 2616–2623. Matthies, D. 1995. Parasitic and competitive interactions between the hemiparasites Rhinanthus serotinus and Odontites rubra and their host Medicago sativa. Journal of Ecology 83: 245–251. Matthies, D. 1996. Interactions between the root hemiparasite Melampyrum arvense and mixtures of host plants: Heterotrophic benefit and parasite-mediated competition. Oikos 75: 118–124. Matthies, D. 1997. Parasite–host interaction in Castilleja and Orthocarpus. Canadian Journal of Botany 75: 1252–1260. Matthies, D. and P. Egli. 1999. Response of a root hemiparasite to elevated CO2 depends on host type and soil nutrients. Oecologia 120: 156–161. Meinzer, F.C., Woodruff, D.R., and D.C. Shaw. 2004. Integrated responses of hydraulic architecture, water and carbon relations of western hemlock to dwarf mistletoe infection. Plant, Cell & Environment 27: 937–946. Musselman, L.J. and W.C. Dickison, 1975. The structure and development of the haustorium in parasitic Scrophulariaceae. Botanical Journal of the Linnear Society, 70(3): 183–212. Nickrent, D.I. and L.J. Musselman. 2004. Introduction to Parasitic Flowering Plants. The Plant Health Instructor. Oerke, E.C., Dehne, H.W., Schönbeck, F., and A. Weber. 1994. Crop Production and Crop Protection— Estimated Losses in Major Food and Cash Crops. Elsevier Science, Amsterdam, The Netherlands, 808pp. Osmond, C.B., Austin, M.P., Berry, J.A., Billings, W.D., Boyer, J.S., Dacey, J.W.H., Nobel, R.S., Smith, S.D., and W.E. Winner. 1987. Stress physiology and the distribution of plants. Bioscience 37: 38–48. Parker, C. and C.R. Riches. 1993. Parasitic Weeds of the World: Biology and Control. CAB International, Wallingford, U.K. Perfect, S.E. and J.R. Green. 2001. Infection structures of biotrophic and hemibiotrophic fungal plant pathogens. Molecular Plant Pathology 2: 101–108. Press, M.C. 1995. Carbon and nitrogen relations. In Press, M.C. and J.D. Graves eds., Parasitic Plants. Chapman and Hall, London, U.K., pp. 102–123. Press M.C., Scholes J.D., and J.R. Watling. 1999. Parasitic plants: Physiological and ecological interactions with their hosts. In Press, M.C., Scholes, J.D., and M.G. Barker, eds., Physiological Plant Ecology. Blackwell Science, Oxford, U.K., pp. 175–197. Ransom, J.K., Odhiambo, G.D., Eplee, R.E., and A.O. Diallo. 1996. Estimates from field studies of the ­phytotoxic effects of Striga spp. on maize. In Moreno, M.T., Cubero, J.I., Berner, D., Joel, D., Musselman, L.J., and C.  Parker, eds., Advances in Parasitic Plant Research. Junta de Andalucia, Dirección General de Investigación Agraria, Cordoba, Spain, pp. 327–333. Sala A., Carey E.V., and R.M. Callaway. 2001. Dwarf mistletoe affects whole-tree water relations of Douglas fir and western larch primarily through changes in leaf to sapwood ratios. Oecologia 126: 42–45. Sauberon, J., Müller-Stöver, D., and J. Hershenorn. 2007. The role of biological control in managing parasitic weeds. Crop Protection 26: 246–254. Seel, W.E. and M.C. Press. 1996. Effects of repeated parasitism by Rhinanthus minor on the growth and photosynthesis of a perennial grass, Poa alpina. New Phytologist 134: 495–502. Seel, W.E., Parsons, A.N., and M.C. Press. 1993. Do inorganic solutes limit growth of the facultative hemiparasite Rhinanthus minor in the absence of host? New Phytologist 123: 283–289.

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Silva, A. and C. Martínez del Rio. 1996. Effects of the mistletoe Tristerix aphyllus (Loranthaceae) on the reproduction of its cactus host Echinopsis chilensis. Oikos 75: 437–442. Smith, D., Barkman, T.J., and C.W. de Pamphilis. 2001. Hemiparasitism. Encyclopedia of Biodiversity, vol. 3. Academic Press, San Diego, CA, pp. 317–328. Tennakoon K.U. and J.S. Pate. 1996. Effects of parasitism by a mistletoe on the structure and functioning of branches of its host. Plant, Cell & Environment 19: 517–528. Visser, J.H., Dörr, I., and R. Kollmann. 1990. Compatibility of Alectra vogelii with different leguminous host species. Journal of Plant Physiology 135: 737–745. Voegele, R.T. and K. Mendgen. 2003. Rust haustoria: Nutrient uptake and beyond. New Phytologist 159: 93–100. Vurro, M., Boari, A., Evidente, A., Andolfi, A., and N. Zermane. 2009. Natural metabolites for parasitic weed management. Pest Management Science 65: 566–571. Wanner J. and R.O. Tinnin. 1986. Respiration in lodgepole pine parasitized by American dwarf mistletoe. Canadian Journal of Forest Research 16: 1375–1378. Watling J.R. and M.C. Press. 2001. Impacts of infection by parasitic angiosperms on host photosynthesis. Plant Biology 3: 244–250. Werth, C. and J.L. Riopel. 1979. A study of the host range of Aureolaria pedicularia (L.) Raf. (Scrophulariaceae). American Midland Naturalist Journal 102: 300–306. Wyse, D.L. 1994. New technologies and approaches for weed management in sustainable agriculture system. Weed Technologies 8: 403–407. Yoder, J.I. 1997. A species-specific recognition system directs haustorium development in the parasitic plant Triphysaria (Scrophulariaceae). Planta 202: 407–413. Zanellato, M., Masciarelli, E., Casorri, L., Boccia, P., Sturchio, E., Pezzella, M., Cavalieri, A., and F. Caporali. 2009. The essential oils in agriculture as an alternative strategy to herbicides: A case study. International Journal of Environmental and Health 3: 198–213.

of Insect Pests 29 Involvement in Plant and Crop Stress Stefano Speranza, Angelo Mazzaglia, Antoine Harfouche, and Asghar Heydari Contents 29.1 Introduction........................................................................................................................... 747 29.2 Plant Interactions with Insect Herbivores.............................................................................. 749 29.2.1 How Insect Herbivores Find a Host Plant.................................................................. 749 29.2.2 Damage to Plants Caused by Insect Herbivores........................................................ 750 29.2.2.1 Herbivory in Aboveground and Belowground Plant Tissues...................... 750 29.2.2.2 Plant Compensation to Insect Herbivores Damage.................................... 751 29.3 Plant Responses to Insect Herbivores Attack........................................................................ 752 29.3.1 Early Events in Plant–Insect Interactions.................................................................. 753 29.3.2 Plant Defense Signaling Pathways............................................................................. 754 29.3.3 Direct Defense Responses......................................................................................... 755 29.3.4 Chemical Communication during Herbivory............................................................ 756 29.4 Detrimental Effects of Insect Herbivores to Plants............................................................... 757 29.4.1 Effects on Photosynthesis.......................................................................................... 757 29.4.1.1 Direct Reduction of Photosynthetic Capacity............................................. 757 29.4.1.2 Indirect Reduction of Photosynthetic Capacity.......................................... 758 29.4.2 Effects on Yield......................................................................................................... 759 29.4.2.1 The Yield Loss............................................................................................ 759 29.4.2.2 Yield Losses Assessment............................................................................ 760 29.4.2.3 Economic Injury Level and Economic Threshold...................................... 760 29.4.2.4 Damage in Natural and Agricultural Environments................................... 760 29.5 Molecular Approaches to Insect Resistance.......................................................................... 761 29.6 Conclusions............................................................................................................................ 762 References....................................................................................................................................... 763

29.1  Introduction Several stresses can be involved during the life of plants. One of the major stresses is caused by tissue damage. Tissue damage in plants is most often associated with insect herbivore infestation. The insect uses the plant as a source of food but the plant tissue consists of dilute nutrients in a matrix of indigestible structural compounds, such as cellulose and lignin, and a variety of allelochemicals. The insect differs from other animals in that it lacks the capacity to synthesize sterols. The insect, in fact, must extract sterol together with several other essential nutrients (amino acids, carbohydrates, lipids, fatty acid, vitamins, trace element) from their food (Behmer and Nes, 2003). Optimal insect growth, survival, and fecundity require certain proteins: carbohydrate ratios, which may vary considerably among species and developmental stages (Schoonhoven et  al., 2005). The food is not totally converted to insect biomass but follow the decrease line from food ingested to growth and 747

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Table 29.1 Average Value of Performance and Indices of Nutritional Utilization by Mandibulate and Haustellate Herbivorous Insects (Range in Parentheses) Mandibulate (i.e., Lepidoptera) Herbs Grasses Tree Haustellate (i.e., Homoptera) Herbs

AD (%)

ECD (%)

RCR (mg per mg per Day)

RGR (mg per mg per Day)

53 43 39

41 45 37

2.0 (0.27–6.0) 2.0 (0.07–4.8) 1.5 (0.31–5.0)

0.37 (0.03–1.5) 0.29 (0.06–0.62) 0.17 (0.03–0.51)

60

65

1.0 (0.90–1.6)

0.39 (0.11–0.67)

Sources: Slansky, F. and Scriber, J.M., Food consumption and utilization, in Comprehensive Insect Physiology, Biochemistry, and Pharmacology, vol. 4, eds. G.A. Kerkut and L.I. Gilbert, pp. 87–163, Pergamon, New York, 1985; Waldbauer, G.P., Adv. Insect Physiol., 5, 229, 1968. Notes: AD, approximate digestibility (also termed absorption efficiency); ECD, efficiency of conversion of digested food to body substance (also termed metabolic efficiency); RCR, relative consumption rate; RGR, relative growth rate (product of RCR × AD × ECD) expressed as the amount of growth attained (mg dry matter) per unit of body weight (mg dry matter) per unit of time.

reproduction. The performance (the extent to which an insect herbivore is able to realize maximum growth and reproduction) is preferentially expressed in rate parameter, and when looking at performance and utilization value, large differences appear to exist between different feeding guilds, such a mandibulate feeders of herbs and forbs versus woody plants or mandibulate versus haustellate (piercing–sucking) species (Table 29.1). Estimates of crop production removed by foliage-feeding insects typically ranges from 5% to 30% (Mattson and Addy, 1975), and insect outbreaks can reduce net primary productivity by more than 70% in some terrestrial ecosystems (Cyr and Pace, 1993; Agarwal et al., 2006). The loss of productivity to herbivory traditionally has been estimated as the amount of leaf tissue removed (Ohmart et al., 1983; Lowman, 1985). It is estimated that insects (mandibulates) consume in the order of 10% of all annually produced plant biomass, these data varies considerably with vegetation type, time, and locality (Barbosa and Schultz, 1987; Damman, 1993; Coupe and Cahill, 2003). Losses to sap-feeding insect (haustellate mouth parts) are more difficult to measure, but are estimated to be around 5% of the net primary production (Schoonhoven et al., Table 29.2 Numbers of Herbivorous Species in Some Insect Order Herbivorous Species Insect Order

No. of Species (Estimate)

No.

%

Coleoptera Lepidoptera Diptera Hymenoptera Hemiptera Orthoptera Thysanoptera

349.000 119.000 119.000 95.000 59.000 20.000 5.000

122.000 119.000 35.700 10.500 53.000 19.900 4.500

35 100 30 11 90 99 90

Source: Schoonhoven, L.M. et  al., Insect–Plant Biology, 2nd edn., Oxford University Press, Oxford, New York, 2005.

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2005). Measuring the intensity of insects herbivory is often difficult and estimate of losses can differ two- to fivefold among the methods used (Coley and Barone, 1996; Schowalter, 2000; Peterson and Higley, 2001). Insects often inflict much more damage in agro-ecosystems than in natural settings (Peterson and Higley, 2001). Conspicuous among herbivores are the Lepidoptera (butterfly and moth), Hemiptera (bugs, leafhoppers, aphids, etc), and Orthoptera (grasshoppers and locust), but large parts of Coleoptera, Hymenoptera, and Diptera (Schoonhoven et al., 2005) (Table 29.2) are also present. Tissue damage usually induces local osmotic stress responses that are often found to be a key component in the response to mechanical wounding (Reymond et al., 2000; Deneckamp and Smeekens, 2003). In this chapter, we analyze in depth how the insect herbivores find host plants and arrive in a patch, how damaging the plant tissue causes stress, how the plant responds to damage, and the effects of this stress on growth and reproduction of the plant.

29.2  Plant Interactions with Insect Herbivores 29.2.1  How Insect Herbivores Find a Host Plant Insects are often said to show programmed behavior and stereotyped, predictable sequence of behavioral act called reaction chains (Atkins, 1980) and that more or less distinct behavioral elements follow one another in a fixed order after succession of stimuli, as demonstrated by Zohren et  al. (1968). The study shows the behavior of Delia radicum (cabbage root fly), where a gravid female fly lands on the cabbage leaf in response to yellow-green wavelengths (500–600 nm). Then she walks along the leaf throughout the latent phase, pausing now and then to groom or to make short flights. At the leaf-blade run phase, she walks continuously, repeatedly along the leaf edge and often changing direction. She assesses the suitability of the plant with taste hairs on her tarsi. Then, she moves on to a midrib of a leaf or a stem, once she contacts the appropriate chemical stimuli, followed quickly by stem run. Next, she moves around the stem base, entering the stem circling phase, keeping her head downward, and then climbing up the stem few centimeters. Afterward, she starts probing the soil with her ovipositor waiting for the chemical stimuli that when perceived, she finally lays her eggs in the soil close to the stem (Zohren et al., 1968). When the outcome of a sensory evaluation is the rejection of a particular plant or plant tissue as food or oviposition site, the insect jumps back to earlier steps in the reaction sequence. In the process of host-plant selection, two main consecutive phases may be distinguished: searching and contact-testing. A standardized host-plant selection sequence can be described as follows (Schoonhoven et al., 2005): • The insect herbivore has no physical contact with a plant and either rests or moves about randomly, walking or flying. • It perceives plant-derived cues, optical and/or olfactory. • It responds to these cues in such a way that the distance between its body and the plant decreases. • The plant is found (contacted by either touching or climbing, or by landing). • The plant surface is examined by contact-testing (palpation of surface). • The plant may be damaged and the content of tissue released by nibbling or test-biting (for biting–chewing species), probing (piercing–sucking species), or puncturing with the ovipositor. • The plant is accepted (one or more eggs being laid or continued feeding) or is rejected, resulting in the insect’s departure. When it arrives in a patch of potential host plants, it may exhibit repetition of the same sequence with respect to different plant individuals of the same or other species. Once an insect has established contact with a potential host plant, elaborate evaluation behavior ensues, during which the insect uses both mechanosensory and chemosensory stimuli offered by the plant. At the behavioral

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level, it has been ample documents that acceptance is determined by the balance between stimulatory and inhibitory compounds (Schoonhoven et al., 2005). The crucial decision to accept or to reject a plant is based not only on sensory information of plant cues but also on the insect’s physiological status (satiety, sexual maturity, eggs maturation, etc.; Barton Browne, 1993).

29.2.2  Damage to Plants Caused by Insect Herbivores The herbivorous are distinguishable based on the degree of food-plant specialization in monophagous, oligophagous, and in polyphagous. The first occurs only on one or a few closely related plant species; many lepidopterans, hemypterans, and coleopterans fit into this category; the second feed on a number of plant species, all belonging to the same plant family; the latter category are herbivorous that seem to exercise little choice and accept many plants belonging to different plant families. The classification of an insect species into this three categories are difficult to sustain, and it is often more convenient to distinguish the insect herbivorous only in specialist (monophagous and oligophagous) from generalist (polyphagous species) (Pashley, 1986; Bryne and Bellows, 1991; Howard et al., 1994). 29.2.2.1  Herbivory in Aboveground and Belowground Plant Tissues For the first category, the insects may consume every anatomical part of plant and also show specialization with regard to feeding site they occupy on their hosts. Many caterpillars, beetles, and grasshoppers are leaf foragers (folivores), ingesting relatively large chunks of leaf material. These extensive tissue damage caused by these insects activate JA-dependent and JA-independent wound response and herbivore responsive genes (Walling, 2000). Other insects such as hemipterans and homopterans show more specific needs. Plant bugs penetrate epidermal cells and ingest cell contents, aphids suck mainly from the sap flow in phloem sieve elements. Spittlebugs and cicadellinine leafhoppers often tap the xylem (Thompson, 1994). Phloem-feeding whitefly and aphid cause small wounds in plant foliage that are perceived as pathogens by the plant’s defense system and activate the SA-dependent and JA/ET-dependent signaling pathways (Walling, 2000). Another insects of other order are the leaf-mining insects that live and feed during their larval stage between the upper and lower epidermis of leaf blade and devour the parenchymal tissues. In this category, there are some insects that may excavate different layers of the leaf parenchyma. The Betula papyrifera leaves, for example, are infested by two hymenopterous leaf miners: the Fenusa pusilla feed on the entire mesophyll, whereas the Messa nana larvae feed only on palisade parenchyma (DeClerck and Shorthouse, 1985; Scheirs et al., 2001). The different leaf part taste affects not only the leaf miners but also insect ingesting leaf pieces. The larvae of the caterpillars Lymantria dispar and Catocala spp. can discriminate between the basal, lateral, and terminal leaflets of their compound-leaved food plant and show dislike to basal leaflets (Gall, 1987). Stems of plant may harbor stem-borers as some species of Lepidoptera, Coleoptera, and Hymenoptera, and the bark of woody plants are often infested by the Coleoptera bark beetles (Scolytidae and others). Wood may be used as food by some Lepidoptera, Coleoptera, and Hymenoptera, which are adapted to these unbalanced diets. Some insect species are specialist feeders on flowers, fruits, or seeds, and others are more specialized to induce the formation of galls in various plant parts (Williams, 1994). The variation in adaptations to certain plant tissue is principally due to nutritional factor. The different plant parts show different dietary value, and the smaller the size of the body of herbivore, the finer is the scale of heterogeneity of the plant tissue it meets. For example, for the Lepidoptera polyphagous Mamestra configurat, when larvae feeding on the pods of rape, one of their host plants, remain smaller and show a 30% increase mortality rate compared with conspecific larvae feeding on foliage (Bracken, 1984). Another different example is the larvae of Dasineura brassicae that are specialized feeders on the pod of rape and survive only on these plant parts (Åhman, 1985). Nutritional factors are not the only determinant of feeding site specialization, several other physiological and ecological factors must also be involved in insects that live belowground. Recent data have shown that the total biomass

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of the life belowground is much vaster than all that we observe aboveground. A considerable subterranean herbivore feeds on plant roots, and intimate interactions between insect and plant are likely to mirror the aboveground relationship (Schoonhoven et al., 2005). Root damage may result in inadequate uptake of water, nutrient, mineral, and thereby reduce the growth of aboveground plant part and cause severe yield losses (Maron, 2001). The subterranean herbivory root feeders may also affect their aboveground counterpart and vice versa via changes in their hosts’ chemistry or physiology (Van der Putten et al., 2001). For example, the infested root by rice water weevils (Lissorhoptrus oryzophilus) markedly reduce the growth rate of fall armyworms (Spodoptera frugiperda) feeding on the leaves of the attacked plants, and, reciprocally, severe defoliation by the S. frugiperda had a negative effect on performance of rice water weevils (Tindall and Stout, 2001). Herbivores root damage may also affect indirect plant defense in cotton plants (Gossypium herbaceum) infested by root feeding wireworms (Agriotes lineatus) which increase their extrafloral nectar production in comparison to control plant with their roots intact. Extrafloral nectar production allows plant to recruit predator, which in turn protect the plant against aboveground insect herbivores (Wäckers and Bezemer, 2003). Crop production removed by foliage-feeding insects typically ranges from 5% to 30% (Mattson and Addy, 1975), with maximum to 70% net primary productivity in peculiar ecosystems (Cyr and Pace, 1993; Agarwal et al., 2006). It is widely believed that herbivore–plant interactions and plant defenses vary with latitude. Herbivory is suggested as being more intense and plant defenses are better developed at lower latitudes (Dobzhansky, 1950; MacArthur, 1969; Lowman, 1985; Coley and Aide, 1991; Coley and Barone, 1996; Grime, 2001; Pennings and Silliman, 2005). An increasing foliar damage trend on Betula pubescens and B. pendula along decreasing latitude was detected in Fennoscandia, while no geographical or climatic pattern was detected on B. pendula in its more southern distribution region (e.g., Central Europe) (Kozlov, 2008). Several studies that have estimated rates of folivory have often used quite different methods from one region to another, complicating the comparison (Pennings and Silliman, 2005). This problem appeared to be solved using a closely standardized methodology to compare folivory (not including sap suckers) in selected temperate forest species along a latitudinal gradient using an extensive network of sites (Adams and Zhang, 2009). 29.2.2.2  Plant Compensation to Insect Herbivores Damage Plants are equipped with various mechanisms to reduce the deleterious effects of herbivory. Structurally, plants are modular organisms, which consists of repetitive multicellular units, each with its own meristem and none of these units is vital for the plant as a whole. This particular modularity reduces the adverse effects of herbivory considerably and allows for easy recovery from tissue removal. How a plant will be affected by herbivory is influenced by variation in environmental conditions (Maschinski and Whitham, 1989); genetic variation in plant response to environmental stress (Rosenthal and Kotanen, 1994; Paige, 1999); phenotypic plasticity (Alward and Joern, 1993); developmental plant stage (Chiarello and Gulmon, 1991; Coley and Barone, 1996; Mercader and Isaacs, 2003; Pedigo and Rice, 2006); the timing (Higley, 1992; Marquis, 1992; Mercader and Isaacs, 2003) or pattern (Meyer, 1998) of herbivory, and type of herbivory (Welter, 1989; Meyer and Whitlow, 1992; Delaney and Macedo, 2001); bottom or up defoliation (Damascos, et al., 2005); plant anatomical characteristic, mode of plant reproduction, stored reserves; and availability of water, nutrient, and light (Whitham et al., 1991; Delaney and Macedo, 2001; Utsumi et al., 2009). These factors influence the effectiveness of plant compensatory responses after herbivory (Nowak and Caldwell, 1984; Maschinski and Whitman, 1989; Herms and Mattson, 1992; Trumble et al., 1993; Rosenthal and Kotanen, 1994). The recovery from herbivory results from the presence of meristem (often dormant) and the ability to redirect resources as nutrient and photosynthetic products to regrowing tissue (Haukioja, 1991). Under good resource conditions, plants can partially or wholly compensate or overcompensate (production of more biomass than has been lost to herbivory) for losses in feeding (Belsky, 1986).

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Table 29.3 Environmental Factors and Plant Traits Involved in Plant’s Compensatory Response to Herbivory Undercompensation Herbivory late in season Low water, nutrients, and/or light High competition Meristem limitation Slow growth Nonintegrated plant modules Woody perennials

Equal or Overcompensations Herbivory early in season Low water, nutrients, and/or light Low competition No meristem limitation Fast growth Integrated plant modules Annuals and biannuals

Source: Whitham, T.G. et al., Plant response to herbivory, the continuum from negative to positive and underlying physiological mechanism, in Plant–Animal Interactions. Evolutionary Ecology in Tropical and Temperate Regions, ed. P.W. Price, T.M. Lewinsohn, G.W. Fernandes, and W.W. Benson, pp. 227–256, Wiley, New York, 1991.

In fact, Huttunen and colleagues (2007) show that climatic change will have a positive impact on the compensatory ability of defoliated plants when these grow in fertile soil. Herbivore-induced modifications in plants often have negative impact on the survival and reproduction of insect herbivores because of decrease of morphological resistance (Masters and Brown, 1992; Inbar et al., 1995; Denno et al., 2000; Tindall and Stout, 2001; Wise and Weinberg, 2002; Denno and Kaplan, 2007). It is difficult in the present state of the art to make generalization regarding the ecological importance of compensatory responses because of many factors involved (Table 29.3). The compensatory regrowth response can have positive effects on abundance and performance of herbivorous insects via increasing food quality and/or quantity (Danell and Huss-Danell, 1985; Damman, 1989; Strauss, 1991; Martinsen, et al., 1998; Nakamura, et al., 2003). Herbivore-induced plant response also indirectly affects predator abundance and/or predation pressure through bottom-up trophic cascade (Masters et al., 2001; Bailey and Whitham 2003; Nakamura et al., 2005, 2006). Several authors have argued that increased abundance or species richness at lower trophic levels may result in increased abundance or species richness at higher trophic levels (Hunter and Price, 1992; Abrams, 1995; Siemann, 1998). Hence, the plant regrowth response following herbivory may have a subsequent influence on abundance and species richness of predaceous arthropods by altering both species composition and abundance of prey herbivores (Utsumi et al., 2009). There are differences, moreover, between monocot and dicot plants with respect to optimal conditions for overcompensation. The difference is caused, probably, by a difference in meristem location in the two groups of plants, which entails important physiological consequences. Monocot herbs, in fact, grew more after herbivory in high resource conditions, whereas recovery from herbivory in dicot herbs and woody plants was significantly better in low resource conditions (Hawkes and Sullivan, 2001). Compensation responses to insect that do not destroy the photosynthetic machinery, such as phloem sap-feeding species, are more difficult to measure.

29.3  Plant Responses to Insect Herbivores Attack Here, we review the latest information on the plant immunity to herbivorous insects based on biochemical and molecular research advances. Insect herbivores use various feeding strategies to get nutrients from their host plants. Being sessile within their local environment and in order to protect

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themselves from herbivorous insects, plants have to respond in a rapid and effective way. Therefore, they have evolved an array of sophisticated and highly adaptive responses to herbivory, which include constitutive and inducible defense strategies. Inducible defenses are exhibited in two forms, direct and indirect, and appear to be widespread in the plant kingdom. Inducible direct defenses have been exclusively studied since the 1970s (Green and Ryan, 1972; Farmer and Ryan, 1992; McCloud and Baldwin, 1997; Baldwin and Preston, 1999), whereas the inducible indirect defenses have been studied since the 1980s (Sabelis and van de Baan, 1983; Sabelis and Dicke, 1985; Dicke and Sabelis, 1988; Turlings et al., 1990). Inducible direct defenses include the production of compounds that exert repellent, anti-nutritive, or toxic effects on herbivores as well as physical barriers such as leaf hardiness and trichomes that increase plant fitness in the presence of herbivores. Plants respond to insect attack by producing toxins and defensive proteins that target physiological processes in the insect instead of acting as passive victims in these interactions. Major known defense chemicals comprise plant secondary metabolites, protease inhibitors, lectins, and amino acid deaminases and oxidases. Indirect defenses include plant volatiles organic compounds (VOCs) and nectar rewards. Both are induced by insects and play a role in attracting natural enemies of the herbivore (Kessler and Baldwin, 2004). Moreover, herbivorous insects trigger induced defenses both locally and systematically through signaling pathways involving jasmonate, ethylene (ET), and salicylates. The plant immunity to herbivore insects is highly dynamic and is initiated by the recognition of insect oral secretions and signals from injured plant cells. These initial cues are transmitted within the plant by signal transduction pathways that include calcium ion fluxes, phosphorylation cascades, and, in particular, the jasmonate pathway, which plays a central and conserved role in promoting resistance to a broad spectrum of insects (Howe and Jander, 2008). The joint effects of plant direct and indirect defense provide a robust resistance to a broad spectrum of insect herbivores in natural ecosystems (Gatehouse, 2002; Kessler et al., 2004). In addition to induced defensive traits, plants can minimize the fitness consequences of tissue loss by activating physiological processes, such as sequestration of sugars in belowground tissues, which allow the plant to tolerate herbivory better (Schwachtje et al., 2006).

29.3.1  Early Events in Plant–Insect Interactions Plants that are swiftly and accurately able to recognize, decipher the incoming signal, and effectively respond to a wide array of attacking insects are the ones with greater success to withstand insect stress. These events that take place the first seconds to minutes are responsible for recognition and triggering of signal transduction pathways, but they are still poorly understood. They start from damage-induced ion imbalances, variations in membrane potentials, calcium flux, production of reactive oxygen species (ROS), and mitogen-activated protein kinases (MAPK) activities. Additionally, insect elicitors have been identified by many studies to allow plants to differentiate between herbivory and mechanical wounding. Moreover, there is evidence that R genes are involved in the control of host plant resistance to insect attack. Although all herbivory results in plant tissue damage, tissue disruption per se is not always a reliable indicator of insect attack. Therefore, to avoid wasting defensive resources, plants must differentiate insect feeding and simple mechanical damage, such as that caused by hail or wind in natural settings. Some responses, including the up-regulation of genes required for cell repair and response to osmotic stress, would likely occur as a result of either herbivory or mechanical wounding. However, the production of toxic secondary metabolites and other defensive responses would presumably benefit only herbivore-challenged plants (Howe and Jander, 2008). Plants may differentiate mechanical wounding from herbivory through the recognition of compounds in insect oral secretion as well as through the use of as yet unknown mechanisms that gauge the quantity and quality of tissue damage (Howe and Jander, 2008). Oral secretions from herbivorous insects elicit volatile release in their host plants. The emission of volatiles is one of the well-studied plant defense responses to insect attack. Numerous plant–insect

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interaction studies have shown that insect feeding or application of oral secretions to wound sites elicits a different or more intense volatile response than mechanical damage alone (Turlings et al., 1990; De Moraes et al., 2001; Arimura et al., 2004). However, it is not the case for aphids, as aphids-derived elicitors are yet to be identified. A study by Miles (1999) showed that aphid salivary enzymes such as peroxidases and pectinase may be elicitors of plant defense responses, yet this hypothesis needs to be further tested. Genetic proof coming from various monocot and dicot crop species supports the idea that R gene products mediate resistance to phloem-feeding insects (Smith and Boyko, 2007). Two plant NBS-LRR (nucleotide binding site–leucine rich repeat) proteins that contribute to the recognition of hemipteran herbivores have been isolated: the tomato Mi-1 gene and the melon vat gene. The former provides resistance to some isolates of Macrosiphum euphorbiae (potato aphid) and Bemisia tabaci (silverleaf whitefly), although not to Myzus persicae (green peach aphid) (Rossi et al., 1998; Nombela et al., 2003). The latter confers increased resistance to both Aphis gossypii (cotton aphid) and the transmission of plant viruses by this aphid species (Dogimont, 2007). By analogy to plant defense against pathogens, these findings suggest a gene-for-gene interaction between the plant and the aphid. However, the presumed avirulence proteins in aphid saliva have not yet been identified (Howe and Jander, 2008). Furthermore, Ca2+ has been implicated as a second messenger in many plant signaling pathways, including responses to herbivory (Maffei et al., 2007). Experiments involving Ca2+ chelator treatment in Phaseolus lunatus (lima bean) resulted in the prevention of defense gene induction in response to feeding by Tetranychus urticae (two-spotted spider mite) and volatiles from miteinfested neighboring plants (Arimura et al., 2000). Despite the fact that MAPK pathway leading to insect resistance has not been identified yet, evidence that such pathway is involved in some plant–insect interactions is shown. Mi-1 mediated resistance was attenuated when the expression of certain MAPKs and MAPK kinases was reduced by VIGS (virus-induced gene silencing) in tomato (Solanum lycopersicum; Li et al., 2006).

29.3.2  Plant Defense Signaling Pathways Salicylic acid (SA), jasmonic acid (JA), and ET are recognized as key players in the regulation of defense responses to insect herbivores that inflict various types of tissue damage in the plant (Howe, 2004; Lorenzo et al., 2004; Pozo et al., 2004; Grant and Lamb, 2006; Van Loon et al., 2006; Harfouche et al., 2006; Von Dahl and Baldwin, 2007). Other plant hormones, including abscisic acid (ABA; Mauch-Mani and Mauch, 2005), brassinosteroids (Nakashita et al., 2003), and auxin (Navarro et al., 2006; Wang et al., 2007), have also been involved in plant defense to insect herbivores, yet their significance needs further studies. By means of synergistic and antagonistic interactions (cross talk), which depend on the herbivorous insect and the attacked plant, a fine-tuned response is achieved that regulates gene expression and, thus, controls the production of protective metabolites (Maffei et al., 2007). Silencing of the octadecanoid pathway in N. attenuata resulted in plants that were more vulnerable to insect attack, demonstrating the importance of this pathway for plant defense against insects (Kessler et al., 2004). What is more, the level of SA was raised only moderately in corn (Zea mays) (Schmelz et al., 2003) and Arabidopsis (Stotz et al., 2002; Traw et al., 2003) after insect attack, knowing that microbial infections generally caused much higher levels of SA than insect feeding. Also, increase in ET production is another early and active response of plants to insect attack. ET synergizes volatile emission in corn (Ruther and Kleier, 2005) and modulates many other defense responses to insect herbivory (Baldwin et al., 2006; Harfouche et al., 2006). The antagonistic roles played by the JA pathway in affecting plant resistance to chewing and phloemfeeding herbivores in monocotyledonous plants reported by Zhou et al. (2009) question the general assumption that JA-dependent defenses enhance plant resistance against both chewing and phloemfeeding herbivores. The silencing OsHI-LOX (a chloroplast-localized type 2 13-lipoxygenase gene of rice) makes rice more susceptible to chewing herbivores, but enhances resistance to a phloem feeder (Zhou et al., 2009).

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29.3.3  Direct Defense Responses In order to protect themselves against insect attack, plants are equipped with an array of defense mechanisms, one of which is the direct defense. Direct plant defenses can be generally divided into two categories: antinutrition and toxicity. It is likely that all plants exhibit constitutive or induced accumulation of toxic secondary metabolites as part of their defense against herbivory (Howe and Jander, 2008). Research on plant defenses to insect pests using numerous plant species has revealed a large variety of small molecules that have toxic and antinutritional effects. Terpenoids that are the most metabolically diverse class of plant secondary metabolites, with more than 40,000 known structures, play a role in plant defense (Aharoni et al., 2005). Also, alkaloids such as nicotine, caffeine, morphine, cocaine, and strychnine are likely evolved as direct defense against insect pests. Tanins, saponins, glucosinolates, and furnaocoumarins are other well-studied classes of plant secondary metabolites that exhibit defensive properties to insect attack. Advances in plant molecular biology research have led to the identification of defensive toxins biosynthetic pathways. Genes encoding all five enzymes involved in the biosynthesis of 2,4-dihydroxy-1, 4-benzoxazin-3-one (DIBOA), a toxin found in maize, wheat, and other Gramineae have been discovered (Frey et al., 1997). In addition, most of the genes required for the production of glucosinolates, a diverse class of metabolites found in the model plant Arabidopsis thaliana and other Cruciferae, have been identified (Halkier and Gershenzon, 2006). Using this information and to better understand how it can be applied to improve plant immunity to insect pests, Tattersal et al., (2001) have engineered A. thaliana with three enzymes from grain sorghum (Sorghum bicolor) to produce the cyanogenic glycoside dhurrin, thereby enhancing resistance to yellow-striped flea beetle (Phyllotreta nemorum). Many defensive compounds are potentially toxic to the plants that produce them. Therefore, the storage of relatively benign precursors that are activated by herbivory is a recurring theme in plant biology. For instance, all three of the defensive systems mentioned in the above paragraph include compounds that are sequestered in plants, but not activated until the onset of herbivory. DIBOA is stored as inactive DIBOA-glucoside, glucosinolates are enzymatically activated to produce toxic breakdown products, and the respiratory inhibitor hydrogen cyanide is released from cyanogenic glycosides during herbivory (Howe and Jander, 2008). Two aspects should be clarified here, one pertains to the synergistic effects in defense against herbivory that may be provided by the complex mixture of toxins found in many plants, and the other to the metabolic diversity in toxin production by individual plants that can also provide defense against multiple insects with different feeding styles and resistance mechanisms. Hummelbrunner and Isman (2001) demonstrated that combining two monoterpenoids almost increased the toxicity 10 times more to tobacco cutworm (Spodoptera litura) than would have been predicted from a simple additive effect. Results of research experiments involving four insect herbivores demonstrated that tryptophan-derived indole and methionine-derived aliphatic glucosinolates have differing effects on Hemiptera and Lepidoptera (Mewis et al., 2005). What’s more, a better defense against M. persicae has been gained by Indole glucosinolates, which break down in the absence of the activating enzyme myrosinase (Barth and Jander, 2006) than do the more stable aliphatic glucosinolates (Kim and Jander, 2007). Besides, plants challenged with insect express defensive proteins that exert direct effects on the attackers. The plant’s insect defense protein arsenal includes proteinase inhibitors (PIs), cysteine proteases, lectins, chitinases, polyphenol oxidases (PPOs), lipoxigenases (LOXs), arginases, and threonine deaminase (TD), the discovery of which has been eased by proteomic and functional genomics analyses. PIs play an active role in the active defense response by impairing various mechanistic classes of digestive proteases in the insect midgut (Green and Ryan, 1972; Ryan, 1990). PIs inhibit insect gut proteases, which results in amino acid deficiencies that negatively affect the insect growth and development (Zavala et al., 2004; Lison et al., 2006). However, PIs defense efficacy is often foiled by the insect’s adaptive ability to express digestive proteases that are insensitive to the host plant complement of PIs or that inactivate PIs (Jongsma et al., 1995; Giri et al., 1998; Rivard et al., 2004;

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Bayes et al., 2005). This evolutionary arms race between two distinct defense mechanisms may be reflected by the diversity and rapid evolution of certain PI gene families (Talyzina and Ingvarsson, 2006). Members of the cysteine protease family of enzymes play another role in defending the plant against insect attack by disrupting the chitin-rich peritrophic membrane that protects the gut epithelium (Konno et al., 2004; Mohan et al., 2006). Besides, plant lectins and chitinases may also target carbohydrate-containing components of the insect gut (Peumans and Vandamme, 1995; Lawrence and Novak, 2006). What’s more, oxidative enzymes such as PPO and LOX covalently modify dietary protein through the production of reactive o-quinones and lipid peroxides, respectively (Felton et al., 1994; Constabel et al., 1995; Wang and Constabel, 2004). Based on the premise that defensive proteins are relatively resistant to gut proteases and, as a consequence, are highly enriched during passage of the food bolus through the insect (Howe and Jander, 2008), proteomic analysis of gut content and feces (frass) of insect herbivores in tomato-reared Manduca sexta (tobacco hornworm) larvae has led to the identification of isoforms of arginase and TD, which degrade the essential amino acids arginine and threonine, respectively, in the lepidopteran midgut (Chen et al., 2005).

29.3.4  Chemical Communication during Herbivory The emission of volatiles is another important plant response affecting insect attackers either directly and indirectly. In response to herbivory, they can provide a direct defensive benefit by deterring further conspecific oviposition (De Moraes et al., 2001) or an indirect benefit by attracting predators (Kessler and Baldwin, 2001). This phenomenon is well studied in many plant species. The specificity of this interaction has revealed with the expression of a Z. mays herbivore-induced terpene synthase (TPS10), that forms (E)-β-farnesene, (E)-α-bergamotene, and other sesquiterpenes, in A. thaliana (Schnee et al., 2006). Females of the parasitoid Cotesia marginiventris were subsequently attracted to TPS10-producing A. thaliana by associating this odor with their prey, Spodoptera littoralis (Egyptian cotton worm). Due to the fact that wild-type A. thaliana does not produce significant amounts of volatile terpenes, this experiment shows that a single herbivoreinduced gene from Z. mays is sufficient to elicit this indirect defense. More work with Z. mays demonstrates that the emission of volatiles provides an indirect defense against underground herbivory. Challenged by western corn rootworm (Diabrotica virgifera) larvae, maize roots release (E)-β-caryophyllene, which attracts Heterorhabditis megidis nematodes that feed on the beetle larvae (Rasmann et al., 2005). Treatment of nonproducing plants with (E)-β-caryophyllene attracted H. megidis and resulted in herbivory reduction. Additionally, volatiles emitted by insect damage communicate at inter- and intra-plant level by providing a signal that allows nearby plants to get ready for an imminent insect attack occurs. This process results in a rapid and robust response to subsequent herbivory and it is called priming (Arimura et al., 2000; Karban et al., 2000; Engelberth et al., 2004). Some of the good examples of inter-plant signaling via endogenous volatiles emission are summarized here. Nicotiana attenuata planted adjacent to clipped Artemisia tridentata (sagebrush) received a blend of VOCs that affected gene expression and caused more rapid induction of PI production upon subsequent feeding by M. sexta (Kessler et al., 2004). Likewise, green leafy volatiles (primarily degradation products of linoleic and linolenic acids) released by Z. mays primed plants in close proximity to respond more robustly following mechanical damage and application of caterpillar oral secretions (Engelberth et al., 2004). At first glance, it would appear that eavesdropping on volatile signals should provide a defensive benefit only to the receiving plant. However, in a tree or other large plant, volatiles transferred between branches or leaves of the same individual would potentially allow faster communication of imminent threats than would phloem-mediated propagation of a systemic signal (Howe and Jander, 2008). Such intra-plant chemical-mediated priming has been demonstrated in field experiments, where clipped A. tridentata showed that defense priming depends on the movement of an airborne signal between damaged and undamaged branches (Karban et al., 2006).

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29.4  Detrimental Effects of Insect Herbivores to Plants 29.4.1  Effects on Photosynthesis Insect infestation and damage are one of the most important crop stresses. Insect herbivore damage is assessed by surveying the amount of tissue removed from foliage. This approach, however, assumes that the remaining leaf tissue functions normally. Plants can replace leaf area loss by herbivores through new leaf production but change the photosynthetic rate of the remaining leaves (Chabot and Hicks, 1982) directly and indirectly, as efficiently showed by Nabity and colleague (2009) who discussed how photosynthesis is affected by arthropod herbivores, and the importance of indirect suppression of the surviving mechanisms compared to the loss of photosynthetic capacity from reduced leaf area (Núñez-Farfán et  al., 2007). Application of thermal and fluorescent imaging shows that surviving tissue is adversely affected. Photosynthesis is one aspect of primary physiology common in vast majority of higher plants, and leaf chlorophyll fluorescence is increasingly being used as a sensitive tool to assay for the degree of stress that a plant experiences from chronic or acute form of stress (Roháček, 2002). In the wider sense, photosynthesis is central to the performance of autotrophic plants, not in isolation or unique but combined with the processes determining growth and development as part of the whole organism’s function and reproductive performance and survival. Photosynthetic organisms, therefore, have developed many varied mechanisms to avoid or minimize imbalance and to maintain homeostasis. All mechanisms are based ultimately on gene expression: adaptation to adverse conditions requires changes in expression to alter the amounts and activities of system components to maintain or readjust photosynthetic efficiency under adverse conditions and to counteract abiotic and biotic factors (Lawlor, 2009). Thus, from an ecological (and crop production) perspective, ignoring indirect suppression of photosynthesis by arthropods may underestimate its importance (Peterson and Higley, 1993, 2001; Neves et al., 2006; Lawlor, 2009). These “indirect” effects on photosynthesis may be considerably greater than the direct removal of leaf area (Welter, 1989; Zangler et al., 2002; Aldea et. al., 2005). Since 1989, Welter examined an extensive body of literature across multiple guilds and found over 50% of all plant–insect interactions resulted in a loss of photosynthetic capacity. Defoliation generally increases photosynthesis, whereas specialized cell-content feeding decreases photosynthesis. These results were partially revised in a recent literature. Feeding on specialized tissues typically reduces photosynthesis, regardless of whether the infested component is the phloem or xylem (Haile et al., 1999; Macedo et al., 2003a,b; Heng-Moss et al., 2006), the stem (Macedo et al., 2005), or general leaf fluids (Haile and Higley, 2003). There are some evidence indicating that increased photosynthesis occurs in the presence of phloem feeding, particularly when the annual photosynthesis rate is estimated (Dungan et al., 2007). The contrast between the results of Welter following the literature where, defoliation injury often does not alter photosynthetic capacity, within plant families (e.g., legumes and some species of Asclepiadaceae) or between hardwoods and crops (Peterson et al., 1992, 1996, 2004; Delaney, 2008); however, there are examples where defoliation reduced (Delaney and Higley, 2006; Retuerto et al., 2006) or increased photosynthesis (Coley and Barone, 1996; Aldea et al., 2005; Dungan et al., 2007; Turnbull et al., 2007). Insect herbivory, whether defoliation or by feeding on specific tissues (e.g., phloem or xylem), triggers a complex and interacting array of molecular and physiological responses in plants. These responses potentially reduce the photosynthetic capacity in remaining leaf tissues to a greater extent than the direct removal of photosynthetic surface area. For example, the removal of only 5% of the area of an individual wild parsnip leaf by caterpillars reduced photosynthesis by 20% in the remaining foliage (Zangler et al., 2002). 29.4.1.1  Direct Reduction of Photosynthetic Capacity The removal of leaf tissue by herbivores represents a “direct” reduction of photosynthetic capacity (Nabity et al., 2009).

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29.4.1.2  Indirect Reduction of Photosynthetic Capacity In addition to directly damaging photosynthetic tissue, herbivores may indirectly affect remaining leaf tissue by diverting resources to defense or disrupt the transport of nutrient and water (Welter, 1989; Sack et al., 2003; Lawlor, 2009). The insect attack to xylem or phloem may alter water transport, stomatal aperture, and sucrose transport and loading, thereby reducing photosynthesis in remaining leaf tissue (Welter, 1989). Severing tissue vasculature alters leaf hydraulics and, subsequently, nutrient or osmotica transport (Aldea et al., 2005; Sack and Holbrook, 2006). If insect feeding is subtle enough to avoid outright cell rupture, modulation of nutrients sequestered by feeding will alter plant osmotica or sink/source relationships (Girousse et al., 2005; Dorchin et al., 2006). These effects also may be mediated by the plant’s response. Insect infestation, or even the perception of attack, can induce a myriad of defense-related responses while concomitantly reducing the expression of photosynthesis-related genes (Kessler and Baldwin, 2002). Where plant defenses are constitutively expressed, the release of biocidal compounds against infesters may damage photosynthetic or homeostatic mechanisms vital for plant function (e.g., Zangler et al., 2002). Indirect effects of herbivory were assigned to four classes: severed vasculature, altered sink demand, defense-related autotoxicity, and defense-induced down-regulation of photosynthesis (Nabity et al., 2009). 29.4.1.2.1  Alteration of Photosynthesis and Water Balance Damage to leaf venation provoked by insect infestation alters leaf hydraulic conductance thereby reducing stomatal conductance and photosynthesis (Nabity et al., 2009). If there are not alternative pathways for water transport, the consequences of damage to venation can persist for weeks after the initial injury and lead to leaf desiccation (Sack and Holbrook, 2006). The foliage damage injury, which severs venation indiscriminately or feeding on specific tissues, may physically obstruct fluid flow with insect mouthparts (stylets) or cell fragments and alter photosynthesis and water balance in remaining leaf tissue (Reddal et al., 2004; Delaney and Higley, 2006). Skeletonizing of soybean leaves by Japanese beetles increased water loss from the cut edges. Damaging the interveinal tissue increased transpiration by 150% for up to 4 days post-injury, and this uncontrolled water loss had no detectable effect on CO2 exchange, severed vasculature induced for 2 days increase in photosynthetic efficiency in undamaged tissue of damaged leaves (Aldea et al., 2005; Nabity et al., 2009). The effects of defoliation on photosynthesis seem to be less predictable than damage caused by other feeding guilds. In hardwoods, leaf gall and fungal damage consistently reduced photosynthetic efficiency at distances ≥1 cm from the point of direct damage, whereas defoliation resulted in only highly local reductions ( 0.05) different from that in the control. In postemergence seedling death experiment in the microcosm, the disease incidence in soils treated with pendimethalin and prometryn increased significantly (P < 0.05) by 64%, 60%, 50% and by 64%, 59%, 57%, relative to the control 1, 2, and 3 weeks after inoculation, respectively. The disease incidence in the soil treated with trifluralin was not significantly (P > 0.05) different from the control (Heydari and Misaghi, 1998). Results of field studies corresponded with those of microcosm experiments (Tables 30.1 and 30.2). The stand count in plots treated with pendimethalin and prometryn at Safford significantly (P < 0.05) decreased by 30%, 28%; 39%, 30%; and 47%, 39%, for 15, 25, and 50 days after sowing, respectively (Table 30.1). The difference in the amount of disease in plots treated with trifluralin and in nontreated plots at Safford was not significant (P > 0.05), relative to the control. The stand count in plots treated with prometryn at Tucson significantly (P < 0.05) decreased by, 41%, 49% and 54% relative to the control, 15, 25 and 50 days after sowing, respectively (Table 30.2). Pendimethalin and trifluralin did not cause significant changes in the stand count at Tucson (Table 30.2).

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Table 30.1 Plant Stand for Soils Treated with Each Test Herbicide and Infested with Rhizoctonia solani Inoculum for the Safford Field Experiment Time (Days after Sowing) Treatment R. solani only R. solani + pendimethalin R. solani + prometryn R. solani + trifluralin

15

25

50

166(14) a 117(19) b 121(18) b 163(16) a

126(12) a 77(19) b 88(16) b 132(13) a

105(12) a 56(11) b 64(15) b 112(8) a

Notes: Stand is represented as mean (the average number of emerged seedlings in one plot or one replicate sown with 400 seeds). Each mean is an average of four values. Means values followed by the same letter in each column are not significantly different at the 0.05 probability level (P > 0.05) according to the Duncan multiple range test. Values in parentheses are standard deviations.

Table 30.2 Plant Stand for Soils Treated with Each Test Herbicide and Infested with Rhizoctonia solani Inoculum for the Tucson Field Experiment Time (Days after Sowing) Treatment R. solani only R. solani + pendimethalin R. solani + prometryn R. solani + trifluralin

15

25

50

101(13) a 91(56) a 60(28) b 104(24) a

87(11) a 75(43) ab 44(21) b 97(21) a

79(18) a 55(12) ab 36(15) b 70(11) a

Notes: Stand is represented as mean (the average number of emerged seedlings in one plot or one replicate sown with 400 seeds). Each mean is an average of four values. Means values followed by the same letter in each column are not significantly different at the 0.05 probability level (P > 0.05) according to the Duncan multiple range test. Values in parentheses are standard deviations.

The reported impact of herbicides on the intensity of pathogen-induced stress has not been always the same in different studies. For example, prometryn, which increased cotton seedling mortality in our studies, did not do so in a previous study (El-Khadem et al., 1984). Moreover, trifluralin that has been reported to increase R. solani-induced cotton seedling death (Moustafa-Mahmoud et al., 1993) did not affect the disease in our study. Differential responses may be due to the differences in soil moisture, soil temperature, herbicide concentration, races of pathogens, plant varieties, the composition of rhizosphere microflora, and the rate of herbicide inactivation in various experiments. The development of tolerance to herbicides by pathogens as a result of a long-term herbicide use may also be a contributing factor.

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30.6  Effect of Herbicides on Population of Biocontrol Bacteria in the Rhizosphere To assess the impact of herbicides on the activity of biocontrol bacteria, it is necessary to examine the impact of herbicides on rhizosphere populations of biocontrol bacteria. This is because the ability of these bacteria to develop in the rhizosphere of target plants is a prerequisite for their biocontrol activity (Filippi et al., 1995). Any soil factors which can potentially interfere with the ability of biocontrol bacteria to develop in the rhizosphere is expected to affect their biocontrol activity as well. However, despite its importance, as far as we have been able to determine, except for our study (to be discussed below), the impact of herbicides on population of biocontrol bacteria in the rhizosphere has not been studied. We studied the potential impact of three widely used herbicides, pendimethalin, prometryn, and trifluralin, on populations of five plant disease-suppressing bacterial isolates (three isolates of Pseudomonas fluorescens and two isolates of Burkholderia cepacia) in the rhizosphere of cotton seedlings (Heydari et al., 1997). All isolates have been efficient cotton root colonizers and have been capable of suppressing pathogen-induced stress. All five isolates were used in microcosm experiments and one isolate (D1) was tested in the field. In microcosm experiments, population sizes of most of the bacterial isolates in the rhizosphere of cotton seedlings in soils treated with each of the three herbicides were significantly (P < 0.05) lower than those in the untreated soils 2 weeks after sowing. The ability of all three test herbicides to reduce isolate D1 population in the rhizosphere declined with time over a 4 week period of monitoring. The population of the bacterium recovered from roots in the herbicide-treated soils was significantly (P < 0.05) lower than those recovered from controls after 1 and 2 weeks but were equivalent to the controls 3 and 4 weeks after sowing (Heydari et al., 1997). Results of the field experiments were similar to those of the microcosm experiments (Tables 30.3 and 30.4). Pendimethalin and prometryn caused significant (P < 0.05) decrease in the D1 population in the rhizosphere 15 and 25 days after sowing at Safford (Table 30.3). Trifluralin had no significant effect on the D1 population at this location (Table 30.3). Pendimethalin and prometryn

Table 30.3 Population Sizes (×106 cfu g−1 Root) of Burkholderia cepacia (Isolate D1) in the Rhizosphere of Cotton Seedlings Grown in Soils Treated or Not Treated with Pendimethalin, Prometryn or Trifluralin, 15, 25, and 50 Days after Sowing in Safford Field Experiment Time (Days after Sowing) Treatment Control (no herbicide) Pendimethalin Prometryn Trifluralin

15

25

50

5.3 (2.5) a 2.6 (1.8) b 2.4 (1.1) b 4.1 (2.2) a

3.0 (2.2) a 1.4 (1.3) b 1.8 (1.3) b 2.4 (1.9) ab

2.2 (1.8) a 1.7 (1.1) a 1.8 (1.4) a 1.9 (1.3) a

Notes: Each mean is an average of four values obtained in one experiment with four replicates. Means values followed by the same letter in each column are not significantly different at the 0.05 probability level (P > 0.05) according to the Duncan multiple range test. Values in parentheses are standard deviations.

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Table 30.4 Population Sizes (×106 cfu g−1 Root) of Burkholderia cepacia (Isolate D1) in the Rhizosphere of Cotton Seedlings Grown in Soils Treated or Not Treated with Pendimethalin, Prometryn or Trifluralin, 15, 25, and 50 Days after Sowing in Tucson Field Experiment Time (Days after Sowing) Treatment Control (no herbicide) Pendimethalin Prometryn Trifluralin

15

25

50

7.2 (4.2) a 3.0 (0.9) b 2.0 (1.5) b 5.6 (2.9) a

2.8 (2.0) a 1.3 (0.8) b 1.1 (0.9) b 1.6 (1.0) b

1.0 (0.8) a 0.8 (0.5) a 0.7 (0.5) a 1.0 (0.6) a

Notes: Each figure (mean value) is an average of four values obtained in one experiment with four replicates. Means values followed by the same letter in each column are not significantly different at the 0.05 probability level (P > 0.05) according to the Duncan multiple range test. Values in parentheses are standard deviations.

caused significant (P < 0.05) decrease in the D1 population in the rhizosphere 15 and 25, but not 50 days after sowing at Tucson (Table 30.4). The trifluralin-induced decline in the D1 population was significant only 25 days after sowing (Table 30.4). Isolate D1, which was originally recovered from cotton plants in the field, may have developed tolerance to trifluralin as a result of the continuous exposure to this herbicide in the field.

30.7  Effect of Herbicides on Biocontrol Activity of Bacteria in the Rhizosphere Our earlier finding that soil atmospheric composition can modulate biocontrol activity of selected bacteria (Kim and Misaghi, 1996) spurred us to examine the impact of other soil factors on the activity of biocontrol bacteria in the rhizosphere. We, therefore, examined the impact of three widely used herbicides, pendimethalin, prometryn, and trifluralin on the efficacy of isolate D1 (a biocontrol bacterium) to reduce the severity of R. solani-induced seedling death. Isolate D1 has been capable of reducing the incidence of R. solani-induced seedling death in the field (Zaki et al., 1998). In both field and microcosm experiment the efficacy of isolate D1 was reduced in the presence of two of the three test herbicides. In Safford field experiment isolate D1 reduced seedling death severity significantly (P < 0.05), compared to the control (not treated with D1), 15, 25, and 50 days after sowing only in nonherbicide treated plots and in plots treated with trifluralin, and not in plots treated with pendimethalin and prometryn (Table 30.5). In Tucson field experiment, biocontrol bacterium (isolate D1) reduced cotton seedling death in plots not treated with herbicides and in those treated with trifluralin significantly, compared to the control (not treated with D1) 15, 25, and 50 days after sowing (Table 30.6). Pendimethalin and prometryn both decreased the efficacy of isolate D1 in reducing cotton seedling death significantly (P < 0.05), 25 and 50 days after sowing in Tucson experiment (Table 30.6). In contrast to our findings, the herbicides pendimethalin and metribuzin have been reported to enhance biocontrol activity of Streptomyces corchorusii and S. mutabilis in greenhouse tests (Elshanshoury et al., 1996).

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Table 30.5 Stand Count (The Number of Emerged Seedlings) in Soils Treated with Each Test Herbicide, Inoculated with Rhizoctonia solani and/or Biocontrol Bacterium (Burkholderia cepacia, Isolate D1) 15, 25, and 50 Days after Sowing in Safford Field Experiment Time (Days after Sowing) Treatment R. solani alone R. solani + D1 R. solani + D1 + pendimethalin R. solani + D1 + prometryn R. solani + D1 + trifluralin

15

25

50

166(15) b 217(23) a 153(53) b 163(13) b 202(23) a

126(12) b 193(19) a 121(50) b 127(15) b 186(20) a

105(12) b 184(15) a 98(40) b 85(26) b 161(23) a

Notes: Each figure (mean value) represents the average number of emerged seedlings in one plot (one replicate) sowed with 400 seeds. Each figure is average of four values obtained in one experiment with four replicates. Means values followed by the same letter in each column are not significantly different at the 0.05 probability level (P > 0.05) according to the Duncan multiple range test. Values in parentheses are standard deviations.

Table 30.6 Stand Count (The Number of Emerged Seedlings) in Soils Treated with Each Test Herbicide, Inoculated with Rhizoctonia solani and/or Biocontrol Bacterium (Burkholderia cepacia, Isolate D1) 15, 25, and 50 Days after Sowing in Tucson Field Experiment Time (Days after Sowing) Treatment R. solani alone R. solani + D1 R. solani + D1 + pendimethalin R. solani + D1 + prometryn R. solani + D1 + trifluralin

15

25

50

101(13) b 171(29) a 117(33) b 168(51) a 180(17) a

87(11) b 164(29) a 107(32) b 121(25) ab 157(19) a

79(18) b 158(25) a 102(32) b 110(31) b 151(21) a

Notes: Each figure (mean value) represents the average number of emerged seedlings in one plot (one replicate) sowed with 400 seeds. Each figure is average of four values obtained in one experiment with four replicates. Means values followed by the same letter in each column are not significantly different at the 0.05 probability level (P > 0.05) according to the Duncan multiple range test. Values in parentheses are standard deviations.

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30.8  M  echanism of Herbicide-Mediated Alterations in Pathogen-Induced Stress The mechanism of the observed herbicide-mediated change in the intensity of pathogen-induced stress is not known. The phenomenon may be due to the effect of herbicides on the plant, on the pathogen, on the activity of indigenous microbial competitors and/or on the interactions among these entities. Herbicides may cause changes in plant root physiology such as root exudation (Brown and McCarter, 1976; Elshanshoury et al., 1996). These changes, in turn, may alter microbial community structures in the rhizosphere in ways which may encourage or discourage the development of competitors of an introduced biocontrol bacterium. Such changes may enhance or depress the activity of biocontrol bacteria. Herbicides may alter the intensity of pathogen-induced stress by changing plant resistance levels to pathogens. Starratt and Lazarovits (1996) showed that the application of dinitroaniline herbicides induced resistance in tomato seedlings to pathogen, Fusarium oxysporum f. sp., lycopersici. Herbicides also may cause changes in crop plants, which may influence the outcome of plant–pathogen interactions (Heydari et al., 1997). Herbicides have been reported to cause alteration in growth, lignin-containing substances, B-glucoside, wax layer on leaves, and in the release of glucose from roots (Starratt and Lazarovits, 1996; Wyse et al., 1976). While the height, biomass, and root densities of cotton seedlings grown in soils treated with pendimethalin or prometryn were generally lower than those of control (untreated soil) in our studies, the differences were not statistically significant (P > 0.05), indicating that physical characteristics of cotton seedlings were not affected by test herbicides. The observed herbicide-mediated changes in the intensity of pathogen-induced stress may be due to the effect of herbicides on the pathogen (Altman and Campbell, 1977; Black et al., 1996). Such effect may be stimulatory or inhibitory. For example, in the R. solani–sugar beet combination, herbicide cycolate may have interfered with the growth of the fungus and at the same time may enhance root exudates (Altman and Campbell, 1977). In such cases, the impact of the herbicide on the intensity of pathogen-induced stress is determined by the balance of stimulatory and inhibitory effects. In our study, the growth of R. solani in in vitro condition was not significantly affected by pendimethalin, prometryn, or trifluralin. Herbicide-mediated alterations in pathogen-induced stress may also be due to the effect of herbicides on indigenous microbial antagonists of pathogens (Elshanshoury et al., 1996; Heydari et al., 1997). The observed absence of soil-borne diseases in some fields in the presence of susceptible hosts and virulent pathogens is most likely due to the presence of indigenous microbial antagonists of the pathogen (Heydari et  al., 1997). As pointed out earlier, we found that herbicides, pendimethalin, prometryn, and trifluralin decreased the populations of some biocontrol bacteria in the rhizosphere of cotton. Finally, herbicides may change the intensity of pathogen-induced stress by interfering with the activity of fungicides used to curb the pathogenic activity of pathogens (Awadalla and El-Refaie, 1994; Hans and Dodan, 1982; Moustafa-Mahmoud et al., 1993; Youssef et al., 1987). Application of fluchlovalin and alachlor to the soil altered the effectiveness of fungicides to control the cowpea damping-off (Hans and Dodan, 1982). Application of nurflurazon, pendimethalin, fluometuron, prometryn, fomesafen, and oxyfluorfen to the field soil significantly reduced the efficacy of fungicides, tolclofos-methyl, pencycuron, carboxin, flutonalit, metalaxyl, and chloroneb against cotton seedling diseases (Youssef et al., 1987). In contrast the antifungal activity of fungicides, captan, and mounsrin was shown to be increased in the presence of herbicides, paraquat, and simazine (Awadalla and El-Refaie, 1994). While plants, pathogens, and antagonistic microorganisms are perhaps the primary target of herbicides, the possibility of other herbicide-mediated changes cannot be overlooked. One such possibility is an alteration of the microclimate as a consequence of removal of weeds as suggested by Awadalla and El-Refaie (1994), We agree with Altman and Campbell (1977) that no single factor may be solely responsible for the observed herbicide effect on the outcome of plant–pathogen interactions.

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30.9  M  echanism of Herbicide-Mediated Changes in Biocontrol Activity of Bacteria The observed herbicides interference with the biocontrol activity of isolate D1 (Heydari et al., 1997) and Streptomyces sp. (Elshanshoury et al., 1996) is most likely due to the effect of herbicides on the biocontrol agents. The sensitivity of microorganisms to herbicides has been demonstrated (Atlas et al., 1978; Forlani et al., 1995; Heydari et al., 1997). Results of our preliminary studies also have shown that the growth of isolate D1 in a liquid medium was reduced by 48%, 44%, and 32%, 24 h after exposure to pendimethalin, prometryn, or trifluralin. Herbicide-mediated change in the performance of biocontrol agents may also be a consequence of herbicide-induced change in pathogen and plant, which was discussed earlier. For example, increased activity of a pathogen (in terms of growth and aggressiveness) in the presence of a herbicide may tip the balance in favor of the pathogen, reducing the effectiveness of the biocontrol agent. Herbicides may also provide favorable environment for some indigenous competitors of the biocontrol agent. Herbicideinduced changes in plant may cause alterations in microbial community structures, encouraging or discouraging biocontrol activity. Another possibility is the herbicide-mediated shift in the quality and the quantity of root exudates and/or border cells in plants (Wilcox, 1996), which can alter microbial community structure. Finally, herbicides may affect cross-communication among microorganisms and plant roots, causing drastic changes in the activity of the introduced biocontrol agents and their indigenous competitors. Cross-communication among microorganisms and between plants and microorganisms has been demonstrated (McKenny et al., 1995; Pierson and Pierson, 1996).

30.10  Conclusions Results of studies presented here clearly show that herbicides, which are being used extensively throughout the world, (a) may alter severity of pathogen-induced stress, (b) may affect the efficacy of biocontrol agents used to curb pathogen-induced stress, and (c) microorganisms are differentially sensitive to herbicides. In our study trifluralin did not cause any significant change in the incidence of cotton seedling death both in the microcosm and in the field experiments. Rhizosphere-associated microorganisms including R. solani, may have developed some levels of tolerance to this herbicide due to its wide-spread and long-term use in cotton fields. The results presented here have important implications for disease management. This is particularly true for seedling diseases in which plants are vulnerable to attack by pathogens when most herbicides may still be present in the soil at biologically active levels. Selection of herbicide must be done cautiously in areas where plant diseases are important. Ideally, available herbicides for a particular crop need to be screened for their effect on pathogen-induced stress and on the biocontrol activity of selected biocontrol agents. Since bacterial isolates are differentially sensitive to herbicide, it may also be possible to first select a herbicide and then to choose a biocontrol agent whose biocontrol activity is not adversely affected by the presence of the selected herbicide. It may also be possible to genetically construct bacterial isolates with increased tolerance to herbicides. The sensitivity of some microorganisms, including biocontrol-active B. cepacia to herbicides as was reported by several workers cited in this chapter and herbicides ability to increase the incidence of some diseases as was reported by several workers cited in this chapter. Provide additional support in favor of the concept of integrated pest management (IPM) strategy. This strategy encourages crop specialists to base their decision regarding the use of a pesticide not only on the effectiveness of the pesticide against the target pest but also on its potential impact on all crop pests in the region. The selection of an ideal pesticide (one which is not harmful to the crop, to the nontarget microorganisms, and to the beneficial insects) is difficult because of the number and diversity of pests involved in any one crop in any region. For example, in addition to insects and weeds, cotton seedlings are damaged by a number of soilborne pathogens besides R. solani. Despite these problems,

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IPM strategies need to be developed for specific crops in specific regions. The development of IPM requires knowledge of the impact of a selected pesticide not only on its intended target but also on plants as well as on beneficial and harmful microorganisms and insects. Application of IPM strategy also requires major changes in agricultural development policies and institutions as was reported by some workers cited in this chapter. While many herbicides are readily biodegraded within a week, others may remain active for up to 2 months following application to the soil. We found that the ability of all three test herbicides to reduce biocontrol bacterium (isolate D1) populations in the rhizosphere decreased with time, perhaps due to degradation of the herbicides. However, all three herbicides tested in Safford field experiment were biologically active up to 25 days after sowing. The persistence of herbicides in the soil depends on several factors including soil moisture, temperature, pH, organic matter content, clay content, and chemical structure of the herbicides as was reported by some workers cited in this chapter. With the increasing world population, providing food for fast-growing populations is becoming a critical issue. A sustainable agricultural system as the most important food source is therefore extremely important and should be taken very seriously. Harmful pests (insects, pathogens, weeds) are among the major stress-inducing agents for the plants and the most important limiting factors for the agricultural yield and production. Nowadays, biological control is considered as one of the most important strategies for management of plant pests, including harmful pathogens. To achieve a successful biological control in the field, studying the interactions among environmental factors and biocontrol active microorganisms is important and critical. Such studies can potentially help scientists and farmers to promote and increase the efficiency of biological control agents in the field, increase the yield and productivity of crops and plants, and protect the environment and biological resources.

References Agrios, G.N. 1988. Plant Pathology, 3rd edn. Academic Press, San Diego, CA, 803pp. Ahmad, I., J. Bissett, and D. Malloch. 1995. Influence of the bioherbicide phosphinothricin on interactions between phytopathogens and their antagonists. Canadian Journal of Botany, 73: 1750–1760. Altman, J. and C.L. Campbell. 1977. Pesticide-plant disease interactions. Effect of cycolate on sugar beet damping-off induced by Rhizoctonia solani. Phytopathology, 67: 1163–1165. Altman, J. and A.D. Rovira. 1989. Herbicide–pathogen interactions in soil-born root diseases. Canadian Journal of Plant Pathology, 11: 166–172. Atlas, R.M., D. Parmer, and R. Bartha. 1978. Assessment of pesticide effect on non-target soil microorganisms. Journal of Soil Biology and Biochemistry, 10: 231–239. Awadalla, O.A. and M. El-Refaie. 1994. Effect of herbicides on the toxicity of fungicides against Rhizoctonia solani causing damping-off of cotton. Journal of Phytopathology, 140: 187–192. Black, B.D., J.S. Russin, J.L. Griffin, and J.P. Snow. 1996. Herbicides effects on Rhizoctonia solani foliar blight of soybean (Glycine max). Journal of Weed Science, 44: 711–716. Brown, E. and S.M. McCarter. 1976. Effect of a seedling disease caused by Rhizoctonia solani on subsequent growth and yield of cotton. Phytopathology, 66: 111–115. Campbell, C.L. and J. Altman. 1977. Pesticide–plant disease interactions: Effect of cycolate on growth of Rhizoctonia solani. Journal of Phytopathology, 67: 557–560. Cook, R.J. and K.F. Baker. 1983. The Nature and Practice of Biological Control of Plant Pathogens. APS Press, St. Paul, MN, 539pp. Deacon, J.W. and L.A. Berry. 1993. Biocontrol of soil-born plant pathogens: Concepts and their applications. Journal of Pesticide Sciences, 37: 417–426. El-Khadem, M. and G.C. Papavizas. 1984. Effect of the herbicides EPTC and linuron on cotton diseases caused by Rhizoctonia solani and Fusarium oxysporum f. sp. vasinfectum. Journal of Plant Pathology, 33: 411–416. El-Khadem, M., M. Zahran, and M.K. El-Kazzaz. 1979. Effect of the herbicides trifluralin, dinitramine and fluometuron on Rhizoctonia disease in cotton. Plant and Soil, 51: 463–470. El-Khadem, M., M.K. El-Kazzaz, and M.A. Hassan. 1984. Influence of different pre-emergence herbicides on cotton diseases caused by Rhizoctonia solani and Fusarium oxysporum f. sp. vasinfectum. Plant and Soil, 79: 29–36.

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Elshanshoury, A.R.E., S.M. Abuelsououd, O.A. Awadalla, and N.B. Elbandy. 1996. Effects of Streptomyces corchorusii, Streptomyces mutabilis, pendimethalin, and metribuzin on the control of bacterial and Fusarium wilt of tomato. Canadian Journal of Botany, 74(7): 1016–1022. Filippi, C., G. Bagnoli, M. Volterrani, and G. Picci. 1987. Antagonistic effects of soil bacteria on Fusarium oxysporum f. sp. dianthi (Prill and Dell). Synd. And Hans. III. Relation between protection against Fusarium wilt in carnation and bacterial antagonists colonization on roots. Plant and Soil, 98: 161–167. Forlani, G., M. Mantelli, M. Branzoni, E. Nielsen, and F. Favilli. 1995. Differential sensitivity of plant-associated bacteria to sulfonylurea and imidazolinone herbicides. Plant and Soil, 176: 243–253. Gentili, E., A. Mariotti, A. Vincenzi, A. Mazzaglia, A. Heydari, N.W. Schaad, L. Varvaro, and G.M. Balestra. 2008. Dieback of hazelnut: Isolation and characterization of two potential biocontrol agents. Journal of Plant Pathology, 90(2): 381–384. Hans, P.K. and D.S. Dodan. 1982. The influence of two herbicides on the antifungal activity of some fungicides against Pythium butleri and Rhizoctonia solani causing damping-off of pea. Journal of Pesticide Sciences, 13: 585–588. Heydari, A. 2007. Biological control of turfgrass fungal diseases. In Turfgrass Management and Physiology (Ed. M. Pessarakli). CRC Press, Boca Raton, FL, 690pp. Heydari, A. and I.J. Misaghi. 1998a. Biocontrol activity of Burkholderia cepacia against Rhizoctonia solani in herbicide-treated soils. Plant and Soil, 202: 109–116. Heydari, A. and I.J. Misaghi. 1998a. Interaction between herbicides and cotton seedling damping-off in the field. Cotton Report, 112: 564–567. Heydari, A. and I.J. Misaghi. 1998b. The impact of herbicides on the incidence and development of Rhizoctonia solani-induced cotton seedling damping-off. Plant Disease, 82: 110–113. Heydari, A. and I.J. Misaghi. 2003. The role of rhizosphere bacteria in herbicide-mediated increase in Rhizoctonia solani-induced cotton seedling damping-off. Plant and Soil, 257: 391–396. Heydari, A. and A. Ghredaghli. 2007. Integrated Pest Management on Cotton in Asia and North Africa. INCANA Press, Tehran, Iran, 103pp. Heydari, A., I.J. Misaghi, and W.B. McCloskey. 1997. Effects of three soil applied herbicides on populations of plant disease suppressing bacteria in the cotton rhizosphere. Plant and Soil, 195: 75–81. Heydari, A., H. Fattahi, H.R. Zamanizadeh, N. Hassanzadeh, and L. Naraghi. 2005. Investigation on the possibility of using bacterial antagonists for biological control of cotton seedling damping-off in green house. Applied Entomology and Phytopathology, 72(1): 51–69. Jahanifar, H., A. Heydari, N. Hassanzadeh, H.R. Zamanizadeh, S. Rezaee, and L. Naraghi. 2008. A comparison between antibiotic-resistant mutants of antagonistic bacteria and their wild types in controlling cotton seedling damping-off disease. Journal of Biological Sciences, 8(5): 914–919. Janlou, H.M., S. Nasrollahnejad, and A. Heydari. 2008. Investigation of control ability of some isolates of Pseudomonas fluorescens and Bacillus subtilis on cotton seedling damping-off in the field condition. Iranian Journal of Agricultural Science and Technology, 22: 89–100. Katan, J. and Y. Eshel. 1973. Interaction between herbicides and plant pathogens. Journal of Residue Review, 45: 147–177. Khodakaramian, G., A. Heydari, and G.M. Balestra. 2008. Evaluation of pseudomonads bacterial isolates in biological control of citrus bacterial canker disease. International Journal of Agricultural Research, 3(4): 268–272. Kim, D.H. and I.J. Misaghi. 1996. Biocontrol performance of two isolates of Pseudomonas fluorescens in modified soil atmospheres. Phytopathology, 86: 1238–1241. Loper, J.E., C. Haack, and M.N. Schroth. 1984. Population dynamics of soil Pseudomonads in the rhizosphere of potato (Solanum tuberosum L.). Journal of Applied and Environmental Microbiology, 49: 416–422. Macauley, B.J. and D.M. Griffin. 1969. Effects of carbon dioxide and oxygen on the activity of some soil fungi. Transactions of British Mycology Society, 53: 53–62. Matteson, P.C. 1996. Implementing IPM-policy and institution revolution. Journal of Agricultural Entomology, 13: 173–183. McKenny, D., K.E. Brown, and D.G. Allison. 1995. Influence of Pseudomonas aeruginosa exproducts on virulence factor production in Burkholderia cepacia: Evidence of interspecies communication. Journal of Bacteriology, 177: 6989–6992. Mekwatanakarn, P. and K. Sivasithamparam. 1987. Effect of certain herbicides on soil microbial populations and their saprophytic growth in soil and pathogenicity of take-all fungus. Journal of Biology and Fertility of Soils, 5: 175–180. Miller, J.H., C.H. Carter, R.H. Garber, and J.E. DeVay. 1979. Weed and disease responses to herbicides in single- and double-row cotton (Gossypium hirsutum). Journal of Weed Science, 27: 444–449. Misaghi, I.J. 1982. Physiology and Biochemistry of Plant–Pathogen Interactions. Plenum Press, New York, 272pp.

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Misaghi, I.J., M.W. Olsen, J.M. Billotte, and R.M. Sonoda. 1992. The importance of rhizobacterial mobility in biocontrol of bacterial wilt of tomato. Journal of Soil Biology and Biochemistry, 24: 287–293. Misaghi, I.J., K. Zaki, A. Heydari, and M.N. Shatla. 1998. Non-chemical control of cotton seedling dampingoff in the field, Cotton Report, 112: 570–575. Molin, S. 1980. Effects of high concentration of carbon dioxide on growth rate of Pseudomonas fragi, Bacillus cereus and Streptococcus cremoris. Journal of Applied Bacteriology, 49: 409–416. Moorman, T.B. and C.C. Dowler. 1991. Herbicide and rotation effect on soil and rhizosphere microorganisms and crop yield. Journal of Agricultural Ecosystem and Environment, 35: 311–325. Moustafa-Mahmoud, S.M., D.R. Sumner, and M.M. Ragab. 1993. Interactions of fungicides, herbicides and planting date with seedling diseases of cotton caused by Rhizoctonia solani AG-4. Plant Disease, 77: 79–86. Mudd, P.J., M.P. Greaves, and S.J.L. Wright. 1985. Effect of isoproturon in the rhizosphere of wheat. Journal of Weed Research, 25: 423–424. Naraghi, L., A. Heydari, A. Karimi-Roozbehani, and D. Ershad. 2004. Isolation of Talaromyces flavus from Golestan cotton fields and its agonistic effects on Verticillium dahliae the causal agent of cotton verticillium wilt. Iranian Journal of Plant Pathology, 39(3–4): 109–122. Naraghi, L., H. Zareh-Maivan, A. Heydari, and H. Afshari-Azad. 2007. Investigation of the effect of heating, vesicular arbuscular mycorrhiza and thermophillic fungus on cotton wilt disease. Pakistan Journal of Biological Sciences, 10: 1596–1603. Naraghi, L., A. Heydari, and F. Azaddisfani. 2008. Study on antagonistic effects of non-volatile extracts of Talaromyces flavus on cotton verticillium wilt disease. Asian Journal of Plant Sciences, 7(4): 389–393. Neubauer, R. and Z. Avizohar-Hershenson. 1973. Effect of the herbicide, trifluralin on Rhizoctonia disease in cotton. Phytopathology, 63: 651–652. Pierson III, L.S. and E.A. Pierson. 1996. Phenazine antibiotic production in Pseudomonas aureofaciens: Role in rhizosphere ecology and pathogen suppression. FEMS Microbiology Letter, 136: 101–108. Pinckard, J.A. and L.C. Standifer. 1966. An apparent interaction between cotton herbicidal injury and seedling blight. Plant Disease Report, 50: 172–177. Rodriguez-Kabana, R. and E.A. Curl. 1980. Non-target effects of pesticides on soilborne pathogens and diseases. Annual Review of Phytopathology, 18: 311–332. Rovira, A.D. and H.J. McDonald. 1986. Effect of the herbicide chlorsulfuron on Rhizoctonia bare patch and take-all of barley and wheat. Plant Disease, 70: 879–882. Schroth, M.N. and J.G. Hancock. 1981. Selected topics in biocontrol. Annual Review of Microbiology, 35: 453–476. Shahriari, F., G. Khodakaramian, and A. Heydari. 2005. Assessment of antagonistic activity of Pseudomonas fluorescens biovars toward Pectobacterium carotovorum subsp. atrosepticum. Journal of Science and Technology of Agriculture and Natural Resources, 8(4): 201–211. Shahriari, F., G. Khodakaramian, and A. Heydari. 2006. Characterization of Pseudomonas fluorescens biovars isolated from main potato growing area of Iran and evaluation of their antibiotic and siderophore production. Iranian Journal of Agricultural Sciences, 36(4): 849–858. Shahraki, M., A. Heydari, N. Hassanzadeh, and L. Naraghi. 2008. Investigation of the possibility of biological control of sugar beet damping-off disease. Iranian Journal of Agricultural Sciences, 13(1): 23–38. Shahraki, M., A. Heydari, and N. Hassanzadeh. 2009. Study on metabolites produced by some antagonistic bacteria and their effects on two isolates of pathogenic fungus, Rhizoctonia solani. Iranian Journal of Biology, 22(1): 247–255. Starratt, A.N. and G. Lazarovits. 1996. Increase in free amino acid levels in tomato plants accompanying herbicide-induced disease resistance. Journal of Pesticide Biochemistry & Physiology, 54: 230–240. Utkhede, R.S. 1982. Effects of six herbicides on the growth of Phytophthora cactorum and a bacterial antagonist. Journal of Pesticide Sciences, 13: 693–695. Weller, D.M. 1988. Biocontrol of soil-born plant pathogens in the rhizosphere with bacteria. Annual Review of Phytopathology, 26: 379–407. Wilcox, W.F. 1996. Influence of dinitroanilin herbicides on growth, sporulation and infectivity of four Phytophthora spp. pathogenic to deciduous fruit trees. Phytopathology, 86: 906–913. Wyse, D.L., W. F. Meggitt, and D. Penner. 1976. Effect of herbicides on the development of root rot on navy bean. Journal of Weed Science, 24: 11–15. Youssef, B.A., M.N. Ragab, and S.M. Moustafa. 1987. Effect of herbicides on the incidence of Rhizoctonia solani infection and certain components of cotton plants. Agricultural Research Review, 65: 153–160. Zaki, K., I.J. Misaghi, A. Heydari, and M.N. Shatla. 1998. Control of cotton seedling damping-off in the field by Burkholderia cepacia. Plant Disease, 82: 291–293.

in Plants and Crops 31 Stress Induced by Fungal Pathogens Asghar Heydari and Giorgio M. Balestra Contents 31.1 31.2 31.3 31.4 31.5

Introduction........................................................................................................................... 787 Attachment of Fungal Pathogens to Host Plants................................................................... 788 Host Plant Penetration by Fungal Pathogens Using Mechanical Forces............................... 789 Host Plant Penetration by Fungal Pathogens by the Aid of Enzymes................................... 790 Pathogenesis of Fungal Pathogens on Host Plants by Toxin Production............................... 791 31.5.1 Non-Host-Specific Toxins.......................................................................................... 791 31.5.2 Host-Specific (Selective) Toxins................................................................................ 791 31.6 Pathogenesis of Fungal Pathogens on Host Plants by Growth Regulators............................ 792 31.7 Stressful Effects of Fungal Pathogens on Host Plants........................................................... 793 31.7.1 Effects on Photosynthesis.......................................................................................... 793 31.7.2 Effects on Respiration............................................................................................... 794 31.7.3 Effects on Water and Nutrient Translocation............................................................ 794 31.7.4 Effects on Cell Membrane Permeability................................................................... 795 31.7.5 Effects on Transcription and Translation.................................................................. 795 31.8 Summary............................................................................................................................... 796 References....................................................................................................................................... 797

31.1  Introduction Pathogenic fungi can attack a very wide range of plants, and by doing so can cause very serious losses to agricultural and horticultural crops (Agrios, 1988; Alexopolous and Mimms, 1996; Chen and Dickman, 2005; Doehlmann et al., 2006; Heydari, 2007; Heydari et al., 2005; Heydari and Misaghi, 2003; Mayer et al., 2001; Ortoneda et al., 2004; Park et al., 2005; Scully and Bidochka, 2006; St. Leger et al., 2000; Zaki et al., 1998). A great deal of research has been carried out to study the pathogens, but very little consideration has been given to the mechanisms involved in the pathogenic process of these types of organisms. As a result of fungal pathogen attack, different stressful conditions are induced and created in plants and crops. In this review, an attempt will be made to describe and discuss these stresses. In the case of fungal pathogen attack, severe damage is caused to the host, but this damage does not necessarily lead to death of the host plant (Alexopolous and Mimms, 1996; Chen and Dickman, 2005; Doehlmann et al., 2006; Heydari, 2007; Heydari et al., 2005; Heydari and Misaghi, 2003). The attack by necrotrophic fungi, in which host cells are killed, presents a slightly different situation (Alexopolous and Mimms, 1996; Chen and Dickman, 2005; Doehlmann et al., 2006; Heydari, 2007). Following location of the host, fungi in some way attach to the plant (Alexopolous and Mimms, 1996). The pathogen, after attachment, must penetrate the outer layers of the host, by mechanical breaching of the defense barriers or by using chemical mechanisms to disrupt physical barriers, for example, by the use of lytic enzymes or through existing openings such as stomata (Alexopolous and Mimms, 1996; Doehlmann et al., 2006; Heydari, 2007; Heydari et al., 2005). 787

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Following penetration of the host’s outer layers, the pathogen either enters into or fuses with the host tissue, permitting the withdrawal of nutrients from the host plant (Alexopolous and Mimms, 1996; Chen and Dickman, 2005; Doehlmann et al., 2006). In optimal parasitism, the attacking organism neutralizes the host defense responses so that a continued coexistence is possible, without death of the host cells. This process of neutralization involves a series of complex reactions, in which levels of host hormones (growth substances) are modified or hormones are secreted by the attacking organisms (Chen and Dickman, 2005; Doehlmann et al., 2006; Heydari, 2007). The pathogen creates a local environment, in which the parasite becomes a sink for the nutrients present in the host, without, however, completely depleting host resources (Alexopolous and Mimms, 1996). Fungal pathogens frequently produce toxins that have an adverse effect on the host, without necessarily killing it (Brodhagen and Keller, 2006; Daly and Knoche, 1982; Dubrin, 1981; Mitchel, 1984). In extreme cases, parasite toxins kill host cells or tissues. An important part of the pathogen–host interaction is the genetic mechanisms of compatibility or incompatibility (Belkhadir et al., 2004; Doehlemann et al., 2006; Dunkle, 1984; Feldbrugge et al., 2004). This involves the ability of the pathogen to recognize the host and the host to sense the presence of the pathogen. Incompatibility, in which the host recognizes and rejects the pathogen, is part of the resistance mechanism. The ability of the pathogen to recognize its host is also a very significant part of the attack process (Belkhadir et al., 2004; Doehlemann et al., 2006; Dunkle, 1984; Feldbrugge et al., 2004). The number of fungi is extremely large and the number of pathogens among them comprises probably thousands of species (Alexopolous and Mimms, 1996; Chen and Dickman, 2005; Doehlmann et al., 2006; Heydari, 2007; Heydari et al., 2005; Heydari and Misaghi, 2003; Mayer et al., 2001; Ortoneda et al., 2004; Park et al., 2005). It is characteristic of most pathogenic fungi to show a great deal of species specificity. Most of the biotrophic fungi are able to infect only a very limited number of plant species, whereas the necrotrophic fungi are far more versatile and are often able to infect hundreds of plant species (Alexopolous and Mimms, 1996; Chen and Dickman, 2005; Doehlmann et al., 2006; Heydari and Misaghi, 2003; Mayer et al., 2001; Ortoneda et al., 2004; Park et al., 2005). Fungal plant pathogens and the infection and diseases they cause in host plants can result in various stressful conditions. They may affect the physiological functions of plants and cause serious damage and disruptions to these functions. Almost all essential physiological functions of plants, including photosynthesis, translocation of water and nutrients, transpiration, respiration, permeability of cell membrane, and transcription and translation, can be affected by fungal pathogens (Allakhverdieva et al., 2001; Antunes and Sfakiotakis, 2000; Camejo et al., 2005; Crafts-Brander and Salvucci, 2002; Hancock and Huisman, 1981; Manners and Scott, 1983). As a result, serious stressful conditions may be induced and created in plants. In this chapter, the pathogenesis and the ways that fungal pathogens attack and cause diseases in their hosts will be first discussed and then their stressful impacts on the structures and functions of plants will be reviewed.

31.2  Attachment of Fungal Pathogens to Host Plants The manner in which fungal pathogens reach their host plants depends on the part of the host that is attacked (Alexopolous and Mimms, 1996). If above-ground parts of the plants are attacked by pathogens, frequently the fungal pathogen reaches them in a fortuitous manner, due to the accidental spread of the fungal structural elements such as mycelium or spores, by wind, rain, or other environmental factors (Agrios, 1988; Podila et al., 1993). In some cases, the infecting fungal pathogen may be carried by insects or other organisms, but again, contact occurs in an accidental fashion. When plant roots are attacked, there is often an actual active mechanism by which the fungus reaches the host (Agrios, 1988; Alexopolous and Mimms, 1996). In some cases, fungal mobile structures such as zoospores are attracted to the roots by chemotactic mechanisms. In other instances, the infecting pathogen may reach the root accidentally or it may be carried there by organisms present in the soil (Agrios, 1988).

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Usually, fungal infection of a host plant is preceded by germination of a fungal spore (Alexopolous and Mimms, 1996). The spore lands on the surface of its host, germinates, and then one of a number of alternative pathways is followed. In the case of zoospores, after encystment and germination, growth is towards the host, probably by a chemotactic mechanism, although much remains to be elucidated in this respect (Alexopolous and Mimms, 1996). Hyphae may enter the host through natural openings, such as stomata, lenticels, or hydathodes, or through existing lesions or wounds in the surface. Sometimes the hyphae form an appressorium, a rigid structure, often containing melanin, which sticks to the surface of the host (Agrios, 1988; Alexopolous and Mimms, 1996; Dunkle, 1984; Francis et al., 1996; Hoch and Staples, 1987; Podila et al., 1993; Thines et al., 2000; Veneault-Fourrey et al., 2006). The appressorium sticks to the surface of the host with the aid of adhesive compounds, which include glycoproteins, polysaccharides, polymers of hexosamines and xylans, and perhaps also lipids (Agrios, 1988). The nature of the adhesive can be quite variable, depending on the host and the infecting fungus (Thines et al., 2000; Veneault-Fourrey et al., 2006). Eventually, the appressorium develops sufficient turgor pressure so that a mechanical breach of the cuticle is possible (Hoch and Staples, 1987; Thines et al., 2000; Veneault-Fourrey et al., 2006). The cuticle on the surface of the host may be softened by the action of enzymes, such as cutinase, enabling penetration.

31.3  Host Plant Penetration by Fungal Pathogens Using Mechanical Forces Fungal Plant pathogens are very small and microscopic organisms that cannot generally apply a voluntary force to plant surface (Alexopolous and Mimms, 1996). In order to enter plant tissues and cells, they need to be equipped with some mechanical structures by which they reach and enter their host plants (Goodman et al., 1986; Isaac, 1992; Misaghi, 1982; Strange, 2003). The penetration of fungal pathogens to the tissue of their host has been well studied (Alexopolous and Mimms, 1996; Goodman et al., 1986; Isaac, 1992; Strange, 2003). Pathogenic fungi are facilitated with some mechanical structures such as haustorium and appressorium, which can be used when these pathogens penetrate host plants (Agrios, 1988; Alexopolous and Mimms, 1996; Thines et al., 2000; Veneault-Fourrey et al., 2006). The fungal haustorium is a well-recognized structure, which is basically an extension of part of a fungal hypha that penetrates into the cell or cells of the host, without lesion or lysis of the host cell wall (Alexopolous and Mimms, 1996). It can be likened to the finger of a glove, which deforms the cell wall of the host cell, but does not damage it. The fungal haustorium is well described and it is sufficient here to cite the general literature (Agrios, 1988; Alexopolous and Mimms, 1996; Thines et al., 2000; Veneault-Fourrey et al., 2006). The fungal haustorium is able to obtain nutrients from the host cells (Alexopolous and Mimms, 1996). Mechanical forces and stress induced by them in plants attacked by fungal pathogens are also mediated by appressorium, which is a swollen tip of a hyphae or germ tube that facilitates attachment and penetration of the host by the fungus (Agrios, 1988; Alexopolous and Mimms, 1996). Appressoria are either melanized, such as those of Colletotrichum and Magnaporthe, or non-melanized such as that belongs to Erysiphe, which is an obligate parasite and the causal agent of powdery mildew disease on many plants (Agrios, 1988; Alexopolous and Mimms, 1996). After penetration of the host plants, a considerable amount of mechanical force is exerted on host tissues by fungal pathogens. Through increased pressure created by this force, plant tissues, cell walls, and cuticle are expanded and pushed out, and finally break down (Alexopolous and Mimms, 1996; Goodman et al., 1986; Isaac, 1992; Strange, 2003). Once a pathogenic fungus has entered a plant cell, it usually secretes increased amounts of enzymes that complete the process of penetration and assist the pathogen to establish and settle down in the host plant (Alexopolous and Mimms, 1996; Goodman et al., 1986; Isaac, 1992; Misaghi, 1982; Strange, 2003).

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31.4  Host Plant Penetration by Fungal Pathogens by the Aid of Enzymes In addition to mechanical penetration, fungal pathogens also use biochemical weapons including different enzymes to enter plant tissues (Francis et al., 1996; Idnurm and Howlett, 2002; Jennings et al., 1998; Kolattukudy, 1981, 1985; Koller et al., 1982, 1995; Kosuge and Nester, 1984; Mohavedi and Heale, 1990; Thines et al., 2000; Tian et al., 2004; Van Kan, 2006; Ten Have et al., 2004). Initial penetration may be through natural openings, such as stomata, lenticels, or cracks in the surface of the host. However, even when this occurs, enzymes are subsequently used to continue penetration. Such penetration is achieved by the formation of several groups of enzymes (Francis et al., 1996; Jennings et al., 1998; Kolattukudy, 1981, 1985; Koller et al., 1995). The first group comprises those that degrade the surface layers of the host, such as layers of waxes, cutins, and suberins (Francis et al., 1996; Koller et al., 1982, 1995). Degradation of cell wall substrates is the most important mode of action and mechanism of enzymes that enable fungal pathogens to penetrate plant tissues (Agrios, 1988; Idnurm and Howlett, 2002). The most thoroughly researched of these enzymes are the cutinases. Although the evidence of the function of cutinases in fungal penetration is not unambiguous, there is a great deal of evidence to show that they do play a role in penetration into the host (Francis et al., 1996; Koller et al., 1995). The next group of enzymes secreted by fungi that are well researched and extensively described are those involved in the degradation of pectins (Agrios, 1988; Kolattukudy, 1981, 1985). A wide range of enzymes varying in activity, specificity, distribution, and function are known. One way of discriminating among them is by a definition of their activity. On the one hand, there are the polygalacturonases, which cleave the galacturonide bonds in the pectin skeleton (Alexopolous and Mimms, 1996). These enzymes may be endo- or exo-polygalacturonases or in some cases endoor exo-methyl polygalacturonases (Alexopolous and Mimms, 1996; Mohavedi and Heale, 1990). Although having an entirely different mode of action, the pectin lyases or pectin trans-eliminases also break down the galacturonide bond of the pectin (Agrios, 1988). The number of these enzymes is very large, and they usually exist as a number of isozymes that are coded by a series of genes, the expression of which depends on the pathogen, its stage of development, and the host (Agrios, 1988). However, their importance in pathogen penetration into the host is not in doubt, although often, the specific isozyme that is important has not been determined. Frequently, the action of the polygalacturonases is preceded by the action of the pectin methyl esterases, which cleave the methyl group attached to the carboxylic acid present in pectins (Mohavedi and Heale, 1990). Again, endo- and exo-pectin methylesterases are known that are coded by a large number of genes. Many isozymes are known and in general it can be stated that the pectin methylesterases are an essential part of penetration of the fungal pathogen into its host. Also in this case, the literature is extensive and will not be reviewed here. Other enzymes present in many pathogens are the cellulases and xylanases, which act directly on the glycosidic bonds of cellulose or on those of arabinoxylans present in the plant cell wall (Agrios, 1988; Alexopolous and Mimms, 1996). These enzymes have been less well described and their role in fungal penetration is less clear. Although the cell wall of many plant cells is lignified, the degradation of lignin is probably not a crucial step in fungal penetration (Agrios, 1988). Lastly, proteases produced by fungal pathogens must be mentioned. These are apparently involved in pathogenicity and may be responsible for the initial damage to host cells (Alexopolous and Mimms, 1996; Doehlemann et al., 2006). Overall, the concept emerging from the studies of these enzymes reveal that fungi use an array of enzymes to break down host cell walls to separate host tissue and to permit penetration into host tissue, and, in addition, are often able to inactivate or kill the host cells, thereby liberating nutrients that are then available for growth and development of the fungal pathogen. In addition to fungal enzymes, hormones formed by the pathogen also often play a role in redirecting the flow of nutrients toward the site of infection, thereby improving the sink from which the pathogen obtains its nutrients (Agrios, 1988).

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31.5  Pathogenesis of Fungal Pathogens on Host Plants by Toxin Production In phytopathology, the term “toxin” is reserved for toxic chemicals (chemopathogens) that are able to cause effects similar to those of disease symptoms induced by microorganisms (Gardiner et al., 2005; Scheffer, 1983). This may be confused with two other names: phytotoxins (for any product of a living organism toxic to plants) and mycotoxins (produced by several fungi in infected seeds, feeds, or foods and capable of causing illnesses of varying severity and death to animals and humans that consume such substances). Most microbe toxins are low molecular weight compounds with diverse structures that act as positive agents of virulence or pathogenicity (Brodhagen and Keller, 2006; Daly and Deverall, 1983; Gardiner et al., 2005; Nishimura and Komoto, 1983; Palmer et al., 2004; Scheffer, 1983). Pathogenic fungi can produce toxins in infected plants as well as in culture medium. The toxin is produced only in the plant or under specific inductive conditions are much less likely to have been discovered. Toxins are usually effective in very low concentrations, target the cellular membranes by affecting the permeability, inactivate or inhibit enzymes, and inhibit or disturb the signal transduction (Brodhagen and Keller, 2006; Dally and Deverall, 1983; Mitchel, 1984; Nishimura and Komoto, 1983; Scheffer, 1983).

31.5.1  Non-Host-Specific Toxins Toxins produced by phytopathogenic microbes have been shown to produce all or part of the disease syndrome, not only in the host plant but also in other species of plants that are not normally attacked by the pathogen in nature (Scheffer, 1983). These toxins increase the extent of disease caused by a pathogen but are not essential for the pathogen to cause disease (Scheffer, 1983). By definition, non-host-specific toxins are not primary determinants of host range. One of the typical fungal non-host-specific toxins is fusicoccin, which is a diterpenoid glycoside synthesized via the mevalonic acid pathway and is produced by Fusicoccum amygdali, causing the wilting syndrome in peach and almond trees (Agrios, 1988; Brodhagen and Keller, 2006; Scheffer, 1983). The fusicoccin toxin causes the opening of the stomata and increased transpiration and subsequent wilting in plants. The site and mode of action of this toxin is the direct activation of plant H+-ATPase and reversible interaction between its C-terminal region and regulatory 14-3-3. The 14-3-3 proteins are present in all eukaryotes and act as regulators in various signal transduction pathways (Brodhagen and Keller, 2006). Different Fusarium species produce a complex of several toxins including fusaric acid (5-butylpicolinic acid), fumonisin B1 (produced by F. moniliform and by Alternaria alternata f. sp. lycopersici as AAL-toxin), trichothecene vomitoxin-deoxynivalenol (DON), zearalenone (ZEN), T-2 toxin, etc. (produced by F. graminearum and other Fusarium species). These toxins are phytotoxic to a broader range of plants (Agrios, 1988). A number of other NHS-toxins such as oxalic acid have been isolated from phytopathogenic fungi (sclerotium and sclerotinia) and many others.

31.5.2  Host-Specific (Selective) Toxins A host-specific toxin is a substance produced by a pathogenic microbe that, at physiological concentrations, is toxic to the hosts of that pathogen and shows little or no toxicity against non-susceptible plants (Brodhagen and Keller, 2006; Mitchel, 1984; Nishimura and Komoto, 1983; Scheffer, 1983). In general, host-specific (selective) toxins (HSTs) are determinants of host range or specificity in that plant species, varieties, or genotypes. HSTs have been critical factors in two major epidemics of crops in the United States in the twentieth century, including the southern corn leaf blight epidemics of 1970 that destroyed about 15% of that year’s crop (Mitchel, 1984). They are also important factors in several other economically significant diseases. In general, all known HSTs are

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produced by certain fungi. Even among the fungi, most known HSTs are produced by species or pathotypes in just two genera: Cochliobolus (also known by the old and new names for its imperfect stage, Helminthosporium or Bipolaris, respectively) and Alternaria (Brodhagen and Keller, 2006; Mitchel, 1984; Nishimura and Komoto, 1983; Scheffer, 1983). Victorin is one of the first host-specific toxins discovered, and its remarkable toxic effects have been recorded (Agrios, 1988). Its structure is a polypeptide linked to a nitrogen-containing sesquiterpene and is produced by Cochliobolus victoriae, which causes foot and root rot and leaf blight in certain oat varieties (Agrios, 1988; Brodhagen and Keller, 2006). Plant phenotypical reactions and stresses caused by this toxin include some general changes in the physiology of host that are common to infectious plant diseases. The site and mode of action of this toxin is the major 100-kD victorin-binding protein that has been purified, and its gene encodes the pyridoxal-phosphate-containing P subunit of glycine decarboxylase (GD) (Brodhagen and Keller, 2006). Another important toxin in this group is T-toxin (HMT-toxin). It is produced by race T isolate of C. heterostrophus, the causal agent of the southern corn leaf blight that is believed to be one of the most serious diseases in the recent history of plant pathology (Agrios, 1988; Brodhagen and Keller, 2006). Race O of C. heterostrophus, however, which does not produce T-toxin, is a minor pathogen of corn regardless of cytoplasm. Site and mode of action of T-toxin is active at about 10 nM against Tcms maize and at about 10 μM against maize with normal cytoplasm. Mitochondria in vitro and in situ are quickly, specifically, morphologically, and biochemically affected by T-toxin (Agrios, 1988; Brodhagen and Keller, 2006). HC-toxin is another host-specific toxin, which is a cyclic tetrapeptide produced by C. carbonum race 1, a causative agent of the Northern corn leaf spot (Agrios, 1988; Brodhagen and Keller, 2006). This toxin inhibits root growth of compatible maize, but is not toxic to cells. Current evidence indicates that the site of action of HC-toxin is histone deacetylase (HD), an enzyme that reversibly deacetylates the core histone (H3 and H4) while they are assembled in chromatin (Brodhagen and Keller, 2006). Acetylation and deacetylation of the core histones alter the inducibility and suppressibility of certain classes of genes. Current research is aimed at understanding how the inhibition of HD activity promotes the infection of maize by C. carbonum race 1. Finally, AAL-toxin is the last host-specific toxin discussed in this section. The structure of this toxin is related to sphinganine and is produced by Alternaria alternata f. sp. lycopersici as AAL-toxin and by Fusarium moniliform as fumonisin B1 toxin (Agrios, 1988; Brodhagen and Keller, 2006). The AAL-toxin is essential for the pathogenesis of AAL in tomato, because the toxin-deficient mutants cannot infest healthy compatible tomato leaves. However, all strains of F. moniforme are not pathogenic on the AAL compatible tomato isoline, indicating that the toxin produced by F. moniforme is not sufficient for virulence on tomato (Brodhagen and Keller, 2006). Plant phenotypical reactions, toxicity, and resistance of this toxin indicates that it acts as a phytotoxin against plants. The AAL-toxin is toxic to all tissues of sensitive tomato cultivars at low concentrations and induces apoptosis in sensitive tomato lines (Brodhagen and Keller, 2006).

31.6  Pathogenesis of Fungal Pathogens on Host Plants by Growth Regulators Growth regulators are naturally occurring compounds in plants and act in very low concentrations (Agrios, 1988; Ashraf and Foolad, 2007; Cessna et al., 2000; Hamer and Holden, 1997; Mur et al., 2006; Tsitsigiannis and Keller, 2006; Viaud et al., 2002; Walters et al., 2006; Wang et al., 2003). The production and occurrence of growth regulators are usually changed and altered after fungal pathogens attack and infect plants. The most important growth regulators are discussed below. Auxin, which is also called IAA, is one of the most important growth regulators involved in fungal pathogenicity (Agrios, 1988; Ashraf and Foolad, 2007; Cessna et al., 2000; Hamer and Holden, 1997). Studies have shown that IAA levels are changed in many diseased plant tissues. Several fungal plant diseases such as corn smut (Ustilago maydis) and clubroot of cabbage (Plasmodiophora

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brassicae) show a typical gall symptom due to the increased levels of IAA (Agrios, 1988; Mur et al., 2006; Tsitsigiannis and Keller, 2006; Viaud et al., 2002). Many other fungal pathogens are also capable of production of IAA. Gibberellin is another growth regulator that is directly related to fungal pathogen infection (Agrios, 1988; Cessna et al., 2000; Hamer and Holden, 1997; Mur et al., 2006). A century ago, rice farmers in Asia noticed some exceptionally tall seedlings growing in their paddies (Agrios, 1988). Before these rice seedlings could mature and flower, they grew so tall and spindly that they toppled over. In Japan, this aberration in growth pattern became known as bakanae (foolish seedling disease) of rice. In 1926, Kurosawa, a Japanese scientist discovered that the disease was caused by a fungal pathogen, Gibberella fujikuroi (Agrios, 1988). By the 1930s, Japanese scientists had determined that this fungus produced hyper-elongation of rice stems by secreting a chemical, which was given the name gibberellin. Gibberellins are normal constituents of green plants and also produced by several other microorganisms. The best known gibberellin is gibberellic acid. In the past years, scientists have identified more than 80 different gibberellins, many of them occurring naturally in plants. Spraying of diseased plants with gibberellin overcomes some of the symptoms (Agrios, 1988). Ethylene is another important growth regulator that is induced and produced in fungal-pathogeninfected plants (Cessna et al., 2000; Hamer and Holden, 1997; Mur et al., 2006). Ethylene production in infected tissues can be dramatically induced. This induction is largely dependent on activation of the ethylene biosynthetic pathway in plant tissues. Genes encoding several key enzymes involved in the ethylene biosynthesis are highly activated at the transcriptional level (Cessna et al., 2000; Hamer and Holden, 1997; Mur et al., 2006). Ethylene has been considered as a signal in plants for wounding and senescent responses. Recent studies show that ethylene, together with another signal component jasmonic acid, may play an essential role in plant defense responses of several pathosystems (Mur et al., 2006). In addition to the chemicals described above, fungal pathogens use other chemical weapons in their pathogenesis including polysaccharides, plant defense suppressors, transporters, etc.

31.7  Stressful Effects of Fungal Pathogens on Host Plants 31.7.1  Effects on Photosynthesis Photosynthesis is defined as a function that enables green plants to convert light energy to chemical energy. Plants then use this chemical energy in their processes. In photosynthesis, carbon dioxide taken from the atmosphere and water taken from the soil come together in the chloroplast of the plant cells and with the aid of light energy react and form glucose and release oxygen (Ellis et al., 1981; Wise et al., 2004). As a very important and basic function of green plants, any disruption or interference in photosynthesis can create and induce stressful conditions in plants. Fungal pathogens are among the most important biotic agents that can infect plants, disrupt the photosynthesis process, and induce serious stresses in plants (Agrios, 1988). A very obvious example of the interference of fungal pathogens with the photosynthesis is the chlorosis and necrosis that they cause on the green parts of plants, which then result in reduced growth of many infected plants (Agrios, 1988). In some diseases caused by fungal pathogens, such as blights or leaf spots, plant photosynthesis is significantly reduced due to the lessening of leaf surface area that is the main photosynthetic part of plants (Rollins, 2003). Photosynthesis in other fungal diseases is also reduced because of the impact of the disease on infected plant tissues and cells. It has been shown that in some fungal diseases, toxins that are produced by these fungal pathogens cause serious inhibition in the production of plant enzymes that are directly or indirectly involved in the photosynthesis (Agrios, 1988). In wilt diseases that are caused by vascular plant fungal pathogens, the stomata are partially closed, chlorophyll is reduced, and photosynthesis is stopped even before wilting symptoms are observed (Agrios, 1988; Van Kan, 2006). All the above-mentioned conditions and negative impacts of fungal plant pathogens on the photosynthesis result in serious pathological conditions in the infected and diseased plants.

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31.7.2  Effects on Respiration The most evident effect of fungal diseases on infected plant respiration is the general increase of respiration as a result of the faster use of reserved carbohydrates by plant tissues (Agrios, 1988). An increase in the respiration usually begins shortly after pathogen attack and continues during multiplication and infection stages. When resistant plants are attacked by fungal pathogens, their respiration increases rapidly compared to susceptible ones, because in resistant plants, higher levels of energy are needed to activate the mechanism of resistance to the pathogen (Agrios, 1988). It has been shown that in infected plants, the activity and concentration of some enzymes involved in the respiration are significantly increased. In infected plants, the accumulation of phenolic compounds that are involved in respiration seems to be higher than healthy plants (Agrios, 1988). The increase of respiration in plants infected by fungal pathogens can also be explained by an increase in plant metabolism. Studies have indicated that when a plant is infected by a fungal pathogen, its growth is stimulated at first, protoplasmic streaming is increased, and materials are synthesized, translocated, and accumulated in infected tissues (Van Kan, 2006). The energy required for these activities derives from ATP produced during respiration. Since part of this energy is wasted during pathogen infection, an increased respiration is therefore induced to enable the plant to carry out its normal metabolism and activity. Infection of plants by fungal pathogens also results in the activation of the pentose pathway, compared to those of healthy ones (Palmer et al., 2004). Since the pentose pathway is not directly linked to ATP production, the increased respiration through this pathway fails to produce utilizable energy and is therefore a less efficient source of energy in fungal pathogen-infected plants. As discussed above, an alteration in the normal respiration of plants is also another stressful condition created in plants by fungal pathogens.

31.7.3  Effects on Water and Nutrient Translocation Most of the plant pathogenic fungi negatively affect the translocation and the movement of water and nutrients in their host plants (Mace et al., 1981). Some fungal pathogens infect plant root and cause a reduced water uptake by the root cells, while many others may grow in the xylem vessels, which results in the blockage of water pathway (Mace et al., 1981). Increased and excessive respiration caused by some fungal pathogens may also create a stressful condition in plants and cause reduced water uptake and movement through the plant system (Mace et al., 1981). It is thought that many plant pathogenic fungi such as damping-off causal agents and the root-rotting fungi cause serious destructions in root tissues before appearance of above-ground symptoms (Mace et al., 1981). Root injuries directly affect the functioning of root cells in the absorption and uptake of water. Some vascular pathogens may affect and reduce water absorption by decreasing root hair production. The causal agents of fungal damping-off, stem rot, and canker diseases may reach the xylem vessels in the infected area and cause xylem tissue destruction in young plants (Mace et al., 1981). Affected vessels may also be filled with the pathogen structures and with substances produced by the pathogen or by the materials produced by the plant host in response to pathogen infection. The most typical and destructive damage to the xylem system in translocating water has been observed with some wilt-causing pathogens including Fusarium (Mace et al., 1981). These pathogenic fungi invade the root xylems and cause diseases primarily by interfering with upward movement of water through the xylem vessels. In addition to interference with water uptake, fungal pathogens may also affect and damage the plant nutrient translocation system seriously (Mace et al., 1981). Movement and translocation of nutrients produced in leaf cells through photosynthesis occurs through plasmodesmata into adjoining phloem elements and then they move to non-photosynthetic plant cells. Plant pathogens including fungi can potentially interfere with these processes and create stressful conditions in plants.

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It is believed that obligate fungal pathogens such as rust and powdery mildew causal agents, can cause accumulation of photosynthetic products and inorganic nutrients in the infected tissues of the host (Mace et al., 1981). Plant pathogens may infect phloem tissues and interfere with the movement of organic nutrients from the leaf cells to phloem or with translocation through phloem elements and possibly with their movement from the phloem into the cells that need to utilize them. However, the synthesis of starch and dry weight are temporarily increased due to the movement and translocation of organic nutrients from uninfected areas of the leaves or from healthy leaves to the diseased tissues and parts of the host plants.

31.7.4  Effects on Cell Membrane Permeability Plant cell membrane, consists of a double layer of lipid molecules and functions as a permeability barrier that allows passage into a cell only of substances needed by the cell and inhibits passage out of them (Agrios, 1988). Small water-soluble molecules such as ions, sugars, and amino acids flow through or are pumped through special membrane channels made of proteins. In plant cells, only small molecules reach the membrane because of their cell wall. Disruption or disturbance of cell membrane by chemicals or biological factors such as fungal pathogens usually increases permeability of cell membrane (Agrios, 1988; Van Kan, 2006). This results in the loss of useful substances by flowing out and excessive inflow of any substance. When a fungal pathogen attacks and infects its host plant, alteration in the permeability of plant cell membrane is the first detectable response of the plant to the pathogen invasion. The loss of electrolytes, which are small water-soluble molecules, is the most common result of alteration in cell membrane permeability (Agrios, 1988). Enzymes and toxins produced by fungal pathogens are the most important weapons of pathogens, which are used in this phenomenon. As a result of cell membrane permeability, essential and useful nutrients required for plant growth and reproduction are lost and this creates a stressful condition in plants, which most of the time causes serious damages to plant health and productivity.

31.7.5  Effects on Transcription and Translation Transcription and translation are two important processes in plants and play very important roles in plant biology and metabolism (Agrios, 1988, Manners and Scott, 1983; Rollins, 2003). Proteins are made and produced by translation of messenger RNA that is made by transcription of plant cellular DNA. Any disturbance or interference with these processes results in serious damages to normal plant life and induces stressful conditions in plant environment. Fungal plant pathogens, particularly those that are obligate parasites such as powdery mildew and rust causal agents, can potentially affect and interfere with the transcription process in their host plants (Agrios, 1988). In some fungal infections, the composition, structure, and function of the chromatin associated with the cell DNA are changed by pathogen-induced irritations and this can affect the transcription process significantly. It has been shown that in some plants infected by fungal pathogens, especially in resistant ones, higher levels of RNA are observed compared to the healthy plants (Agrios, 1988). This is particularly true in the early stages of infection. The higher levels of RNA present in infected plants, therefore, cause an increase in transcription rate and the substances synthesized by plant cells. In addition to transcription, fungal plant pathogens may also affect the translation of messenger RNA to proteins (Manners and Scott, 1983; Van Kan, 2006). Fungal infection usually results in an increase in the level and production of plant enzymes, particularly those involved in the respiration or oxidation and production of phenolic compounds (Agrios, 1988). In addition to this, and as was described before, the level of RNA produced by plant cells are also increased after a plant is infected by fungal pathogens. The increased levels of RNA causes an additional synthesis of proteins through the translation process. An increased production of proteins by plant cells is reflected

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by additional levels of enzymes and creates an unusual situation in plant structure and function, which may induce serious stresses in the fungal pathogen-infected plants (Agrios, 1988).

31.8  Summary Fungal plant pathogens attack plants and cause very serious losses to agricultural and horticultural crops. Plant pathogenic fungi and the infection and diseases they cause in the host plants can result in the induction of serious stresses in their hosts. They may affect physiological functions of plants and cause serious damages and disruptions in these functions. Penetration of fungi into the tissue of their host has been well researched. Pathogenic fungi are facilitated with some mechanical structures such as haustorium and appressorium that can be used in the penetration of fungus into the host plant. In addition to mechanical penetration of host plants, enzymes are also used as biochemical weapons in fungal penetration. Enzymes are subsequently used to continue penetration. Such penetration is achieved by the formation of several groups of enzymes that degrade plant cell walls and enable fungal pathogens to enter their host plant tissues. After penetration of the fungal pathogens into the host, disease-symptom induction takes place by means of toxins and growth regulators. Fungal toxins may be either non-host specific or host specific. Non-host-specific toxins are produced by phytopathogenic fungi not only in the host plant but also in other species of plants that are not normally attacked by the pathogen in nature. Pathogenic fungi produce host-specific-toxins only when they infect certain hosts. Growth regulators, which are chemical compounds that occur naturally in plants and act in very low concentrations are also very important in fungal pathogenesis. After fungal pathogens attack and infect plants, the production and occurrence of growth regulators are usually changed and altered and create stressful conditions in plants and crops. Auxin is one of the most important growth regulators. Studies have shown that auxin levels are changed in many diseased plant tissues. Several fungal plant diseases such as corn smut (Ustilago maydis) and clubroot of cabbage (Plasmodiophora brassicae) show a typical gall symptom due to the increased levels of auxin. Gibberellin is another plant growth regulator produced in some plants. In addition to auxin and gibberellin, ethylene is also produced as a growth regulator in plants. Ethylene has been considered as a signal in plants for wounding and senescent responses. Plant pathogenic fungi and their penetration, infection, and pathogenesis in plants affect basic plant functions and physiology and induce and create serious stressful conditions. Photosynthesis is the first plant function affected by pathogenic fungi. Fungal pathogens are among the most important agents that can disrupt photosynthesis and induce serious stresses in the plants. A very obvious example of the interference of fungal pathogens in the photosynthesis is the chlorosis and necrosis that they cause on the green parts of plants, which results in reduced growth of many infected plants. In addition to photosynthesis, the respiration of plants is also affected by fungal pathogens. The most evident effect of fungal diseases on infected plant respiration is the general increase of respiration as a result of faster use of reserved carbohydrates by plant tissues. Plant pathogenic fungi also negatively affect the translocation and the movement of water and nutrients in their host plants. Some fungal pathogens infect the plant root and cause a reduced water uptake by the root cells, while many others may grow in the xylem vessels, which results in the blockage of the water pathway. It is thought that many plant pathogenic fungi such as damping-off causal agents and the root-rotting fungi cause serious destructions in root tissues before appearance of above-ground symptoms. In addition to interference with water uptake, fungal pathogens may also affect and damage the plant nutrient translocation system seriously. It is believed that obligate fungal pathogens such as rust and powdery mildew causal agents, can cause accumulation of photosynthetic products and inorganic nutrients in the infected tissues of the host plant. Plant pathogens may infect phloem tissues and interfere with the movement of organic nutrients from the leaf cells to phloem or with translocation through phloem elements and possibly with their movement from the phloem into the cells that need to utilize them.

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Plant cell membrane consists of a double layer of lipid molecules and functions as a permeability barrier that allows passage into a cell only of substances that are needed by cell and inhibit passage out of them. Disruption or disturbance of cell membrane by chemicals or biological factors such as fungal pathogens usually increases permeability of cell membrane. This results in the loss of useful substances by flowing out and excessive inflow of any substances. Transcription and translation are two major processes in plants and play very important roles in plant biology and metabolism. Proteins are made and produced by translation of messenger RNA that is made by transcription of plant cellular DNA. Any disturbance or interference with these processes results in serious damages to plant normal life and induces stressful conditions in plant environment. In addition to transcription, fungal plant pathogens may also affect the translation of messenger RNA to proteins. Fungal infection usually results in an increase in the level and production of plant enzymes, particularly those involved in the respiration or oxidation and production of phenolic compounds. Additional levels of enzymes create an unusual situation in plant structure and function that may induce serious stresses in the fungal pathogen-infected plants. As was discussed in this review, plant pathogenic fungi are among the most important biotic agents that can seriously affect plant and crop health and cause serious damages and injuries to different parts of plants during various stages of their life cycle. These injuries and damages can result in the induction of stressful conditions in plants that make plants to suffer and even die due to the diseases and disorders caused by fungal pathogens. In order to have healthy and productive plants and crops, it is extremely important to protect them against fungal pathogens as was reported by several workers cited in this chapter. This can be done by using a combination of different strategies including chemical, cultural, biological, and genetic control methods to combat and overcome fungal plant diseases as was reported by several workers cited in this chapter. The successful application and implementation of control strategies will hopefully reduce damages and stresses caused and induced by fungal plant pathogens and will be a promising approach to a sustainable agriculture.

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Daly, J.M. and B.J. Deverall. 1983. Toxins in Plant Pathogenesis. Academic Press, New York. Doehlemann, G., P. Berndt, and M. Hahn. 2006. Different signaling pathways involving a G alpha protein, cAMP and a MAP kinase control germination of Botrytis cinerea conidia. Molecular Microbiology, 59: 821–835. Dunkle, L.D. 1984. Factors in pathogenesis. In Plant–Microbe Interactions: Molecular and Genetic Prospectives, T. Kosuge and E.W. Nester (eds.), vol. 1, pp. 19–41. McMillan, New York. Durbin, R.D. (ed.). 1981. Toxin in Plant Disease. Academic Press, New York. Ellis, M.A., D.C. Ferree, and D.E. Spring. 1981. Photosynthesis, transpiration and carbohydrate content of apple leaves infected by Podosphaera leucotricha. Phytopathology, 71: 392–395. Feldbrugge, M., J. Kamper, G. Steinberg, and R. Kahmann. 2004. Regulation of mating and pathogenic development in Ustilago maydis. Current Opinion in Microbiology, 7: 666–672. Francis, S.A., F.M. Dewey, and S.J. Gurr. 1996. The role of cutinase in germ tube development and infection by Erysiphe graminis f. sp. hordei. Physiological and Molecular Plant Pathology, 49: 201–211. Gardiner, D.M., P. Waring, and B.J. Howlett. 2005. The epipolythiodioxopiperazine (ETP) class of fungal toxins: Distribution, mode of action, functions and biosynthesis. Microbiology, 151: 1021–1032. Goodman, R.N., Z. Kiraly, and K.R. Wood. 1986. The Biochemistry and Physiology of Plant Disease. University of Missouri Press, Columbia, MO. Hall, A.E. 2001. Crop Responses to Environment. CRC Press LLC, Boca Raton, FL. Hamer, J.E. and D.W. Holden. 1997. Linking approaches in the study of fungal pathogenesis: A commentary. Fungal Genetics and Biology, 21: 11–16. Hancock, J.G. and O.C. Huisman. 1981. Nutrient movement in host-pathogen system. Annual Review of Phytopathology, 19: 309–331. Heydari, A. 2007. Biological control of turfgrass fungal diseases. In Handbook of Turfgrass Management and Physiology, M. Pessarakli (ed.). CRC Press, Boca Raton, FL. Heydari, A. and I.J. Misaghi. 2003. The role of rhizosphere bacteria in herbicide-mediated increase in Rhizoctonia solani-induced cotton seedling damping-off. Plant and Soil, 257: 391–396. Heydari, A., H. Fattahi, H.R. Zamanizadeh, N. Hassanzadeh, and L. Naraghi. 2005. Investigation of the possibility of using bacterial antagonists for biological control of cotton seedling damping-off in greenhouse. Applied Entomology and Phytopathology, 72: 51–69. Hoch, H.C. and R.C. Staples. 1987. Structural and chemical changes among the rust fungi during appressorium development. Annual Review of Phytopathology, 25: 231–247. Idnurm, A. and B.J. Howlett. 2002. Isocitrate lyase is essential for pathogenicity of the fungus Leptosphaeria maculans to canola (Brassica napus). Eukaryotic Cell, 1: 719–724. Isaac, S. (ed.). 1992. Fungal–Plant Interactions. Chapman and Hall, London, U.K. Jennings, D.B., M. Ehrenshaft, D.M. Pharr, and J.D. Williamson. 1998. Roles for mannitol and mannitol dehydrogenase in active oxygen-mediated plant defense. Proceedings of National Academy of Science, 95: 15129–15133. Kolattukudy, P.K. 1981. Structure, biosynthesis and degradation of cutin and suberin. Annual Review of Plant Physiology, 32: 539–567. Kolattukudy, P.E. 1985. Enzymatic penetration of plant cuticle by fungal pathogens. Annual Review of Phytopathology, 23: 223–250. Koller, W., C.R. Allan, and P.E. Kolattukudy. 1982. Role of cutinase and cell wall degrading enzymes in infection of Pisum sativum by Fusarium solani f. sp. pisi. Physiological Plant Pathology, 20: 47–60. Koller, W., C. Yao, F. Trial, and D.M. Parker. 1995. Role of cutinases in the invasion of plants. Canadian Journal of Botany, 73: S1109–S1118. Koskela, T., S. Puustinen, V. Salonen, and P. Mutikainen. 2002. Resistance and tolerance in a host plant: Genetic variations and costs. Evolution, 56: 899–908. Kosuge, T. and E.W. Nester (eds.). 1984. Plant–Microbe Interactions: Molecular and Genetic Perspectives, vol. 1. Macmillan, New York. Lengeler, K.B., R.C. Davidson, C. D’Souza, T. Harashima, W.C. Shen, P. Wang, X. Pan, M. Waugh, and J.  Heitman. 2000. Signal transduction cascades regulating fungal development and virulence. Microbiology and Molecular Biology Review, 64: 746–785. Mace, M.E., A.A. Bell, and C.H. Beckman (eds.). 1981. Fungal Wilt Diseases of Plants. Academic Press, New York. Manners, J.M. and K.H. Scott. 1983. Translational activity of polysomes of barley leaves during infection by Erysiphe graminis f. sp. hordei. Phytopathology, 73: 1386–1392.

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Mayer, A.M., R.C. Staples, and N.L. Gilad. 2001. Mechanism of survival of necrotrophic fungal plant pathogens in hosts expressing the hypersensitive response. Phytochemistry, 58: 33–41. Misaghi, I.J. 1982. Physiology and Biochemistry of Plant–Pathogen Interactions. Plenum, New York. Mitchel, R.E. 1984. The relevance of host-specific toxins in the expression of virulence by pathogens. Annual Review of Phytopathology, 22: 215–245. Mohavedi, S. and J.B. Heale. 1990. The roles of aspartic proteases and endopectin lyase enzymes in the primary stages of infection and pathogenesis of various tissues by different isolates of B. cinerea. Physiological and Molecular Plant Pathology, 36: 303–324. Mur, L.A., T. Carver, and E. Prats. 2006. No way to live; the various roles of nitric oxide in plant-pathogen interactions. Journal of Experimental Botany, 57: 489–505. Nishimura, S. and K. Komoto. 1983. Host-specific toxins and chemical structures from Alternaria species. Annual Review of Phytopathology, 21: 87–116. Ortoneda, M., J. Guarro, M.P. Madrid, Z. Caracuel, M.I.G. Roncero, E. Mayayo, and A. Di Pietro. 2004. Fusarium oxysporum as a multihost model for the genetic dissection of fungal virulence in plants and mammals. Infectious Immunology, 72: 1760–1766. Palmer, A.G., R. Gao, J. Maresh, W.K. Erbil, and D.G. Lynn. 2004. Chemical biology of multihost/pathogen interactions: Chemical perception and metabolic complementation. Annual Review of Plant Pathology, 42: 439–464. Park, J.-H., G.J. Choi, K.S. Jang, H.K. Lim, H.T. Kim, K.Y. Cho, and J.C. Kim. 2005. Antifungal activity against plant pathogenic fungi of chaetoviridins isolated from Chaetomium globosum. Microbiology Letter, 252: 309–313. Podila, G.K., L.M. Rogers, and P.E. Kolattukudy. 1993. Chemical signals from avocado surface wax trigger germination and appressorium formation in Colletotrichum gloeosporioides. Plant Physiology, 103: 267–272. Rollins, J.A. 2003. The Sclerotinia sclerotiorum pac1 gene is required for sclerotial development and virulence. Molecular Plant–Microbe Interactions, 16: 785–795. Scheffer, R.P. 1983. Toxins as chemical determinants of plant disease. In Toxins and Plant Pathogenesis, J.M. Daly and B.J. Deverall (eds.). Academic Press, New York. Scully, L. and M. Bidochka. 2006. A cysteine/methionine auxotroph of the opportunistic fungus Aspergillus flavus is associated with host-range restriction: A model for emerging diseases. Microbiology, 152: 223–232. Staples, R.C. and A.M. Mayer. 2003. Suppression of host resistance by fungal plant pathogens. Israel Journal of Plant Sciences, 51: 173–184. St. Leger, R.J., S.E. Screen, and B. Shams-Pirzadeh. 2000. Lack of host specialization in Aspergillus flavus. Applied and Environmental Microbiology, 66: 320–324. Strange, R.N. 2003. Introduction to Plant Pathology. John Wiley & Sons, Chichester, U.K. Ten Have, A., E. Dekkers, J. Kay, L.H. Phylip, and J.A. van Kan. 2004. An aspartic proteinase gene family in the filamentous fungus Botrytis cinerea contains members with novel features. Microbiology, 150: 2475–2489. Thines, E., R. Weber, and N.J. Talbot. 2000. MAP kinase and protein kinase A-dependent mobilization of triacylglycerol and glycogen during appressorium turgor generation by Magnaporthe grisea. Plant Cell, 12: 1703–1718. Tian, M., E. Huitema, L. da Cunha, T. Torto-Alalibo, and S. Kamoun. 2004. A Kazal-like extracellular serine protease inhibitor from Phytophthora infestans targets the tomato-related protease P69B. Journal of Biological Chemistry, 279: 26370–26377. Tsitsigiannis, D.I. and N.P. Keller. 2006. Oxylipins act as determinants of natural product biosynthesis and seed colonization in Aspergillus nidulans. Molecular Microbiology, 59: 882–892. Van Kan, J.A. 2006. Licensed to kill: The lifestyle of a necrotrophic plant pathogen. Trends in Plant Sciences, 11: 247–253. Veneault-Fourrey, C., K. Lambou, and M.-H. Lebrun. 2006. Fungal Pls1 tetraspanins as key factors of penetration into host plants: A role in re-establishing polarized growth in the appressorium? Microbiology Letter, 256: 179–184. Viaud, M.C., P.V. Balhadere, and N.J. Talbot. 2002. A Magnaporthe grisea cyclophilin acts as a virulence determinant during plant infection. Plant Cell, 14: 917–930. Walters, D.R., T. Cowley, and H. Weber. 2006. Rapid accumulation of trihydroxy oxylipins and resistance to the bean rust pathogen Uromyces fabae following wounding in Vicia faba. Annual Botany, 97: 779–784.

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Wang, Z.Y., C.R. Thornton, M.J. Kershaw, L. Debao, and N.J. Talbot. 2003. The glyoxylate cycle is required for temporal regulation of virulence by the plant pathogenic fungus Magnaporthe grisea. Molecular Microbiology, 47: 1601–1612. Wise, R.R., A.J. Olson, S.M. Schrader, and T.D. Sharkey. 2004. Electron transport is the functional limitation of photosynthesis in field-grown Pima cotton plants at high temperature. Plant Cell Environment, 27: 717–724. Young, N. 2000. The genetic architecture of resistance. Current Opinion in Plant Biology, 3: 285–290. Zaki, K., I.J. Misaghi, A. Heydari, and M.N. Shatla. 1998. Control of cotton seedling damping-off in the field by Burkholderia cepacia. Plant Disease, 82: 291–293.

Part VI Genetic Factors and Plant/ Crop Genomics under Stress

Factors Affecting 32 Genetic Abiotic Stress Tolerance in Crops Arun Kumar Joshi Contents 32.1 Introduction...........................................................................................................................804 32.2 Drought Tolerance................................................................................................................. 805 32.2.1 Morphophysiological Traits: Genetic Basis...............................................................805 32.2.1.1 Earliness......................................................................................................806 32.2.1.2 Roots...........................................................................................................806 32.2.1.3 Stomatal Conductance................................................................................806 32.2.1.4 Epicuticular Wax.........................................................................................806 32.2.1.5 Osmotic Adjustment...................................................................................807 32.2.1.6 Transpiration Efficiency..............................................................................807 32.2.2 Genetics of Drought Tolerance..................................................................................808 32.2.3 Gene Expression........................................................................................................809 32.2.4 Breeding..................................................................................................................... 811 32.3 Submergence Tolerance......................................................................................................... 812 32.3.1 Morphophysiological Traits: Genetic Basis............................................................... 813 32.3.2 Genetics..................................................................................................................... 813 32.3.3 Gene Expression........................................................................................................ 814 32.3.4 Breeding..................................................................................................................... 815 32.4 Heat Tolerance....................................................................................................................... 816 32.4.1 Genetic Basis............................................................................................................. 816 32.4.2 Gene Expression........................................................................................................ 817 32.4.3 Breeding..................................................................................................................... 818 32.5 Cold Tolerance....................................................................................................................... 819 32.5.1 Morphophysiological Traits: Genetic Basis............................................................... 819 32.5.2 Genetics..................................................................................................................... 820 32.5.3 Gene Expression........................................................................................................ 821 32.5.4 Breeding..................................................................................................................... 822 32.6 Salinity Resistance................................................................................................................ 823 32.6.1 Morphophysiological Traits: Genetic Basis...............................................................824 32.6.2 Genetics..................................................................................................................... 825 32.6.3 Gene Expression........................................................................................................ 826 32.6.4 Breeding..................................................................................................................... 826 32.7 Acid Soil Tolerance................................................................................................................ 827 32.7.1 Morphophysiological Traits: Genetic Basis............................................................... 828 32.7.2 Genetics..................................................................................................................... 828

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32.7.3 Gene Expression........................................................................................................ 829 32.7.4 Breeding..................................................................................................................... 829 32.8 Conclusions............................................................................................................................ 830 Acknowledgment............................................................................................................................ 831 References....................................................................................................................................... 831

32.1  Introduction Plant growth and development is a result of the interplay between the genetically governed potential of the plant and the plant environment in which it grows (Lewis 1976). Therefore, plant performance is often affected by a number of unfavorable environmental factors, among which abiotic factors are of crucial importance. Since plants are immobile, they suffer more compared to other organisms. The significance of abiotic stresses can be understood from the fact that the earth’s surface, which is 70% salt water and 30% land, possesses only half of its land area free from extremes of water and temperature, and to soil erosions or difficult geography (Lewis and Christiansen 1981). The other half that is used for agricultural production has to face a number of abiotic stresses that are considered to be the main source of yield reductions all over the world (Boyer 1982, Cushman and Bohnert 2000). The recent reports of climate change (IPCC 2007, Battisti and Naylor 2009) are expected to further aggravate the threat of abiotic stresses. Abiotic stresses are generally of two types: stable and fluctuating. Stable stresses are caused by abnormal pH or metal toxicity, whereas unstable or fluctuating stresses include abnormal levels of water (drought/flooding), temperature (cold/hot), and other factors such as pollutants. Abiotic stresses are characterized by the occurrence of more than one stress at a particular growth stage or throughout the growing cycle, even though one stress may dominate. Since the whole biotic world, directly or indirectly, is dependent on plants for survival, any stress to plants gets reflected in their own stress. This is of utmost importance for human beings and the present-day agriculture when our major natural resources are shrinking except the number of mouths to feed. Therefore, providing relief to crop plants from abiotic stresses is providing relief to man himself. Relief from abiotic stresses is possible either by changing/avoiding the environment or changing the genotype of the plant itself. Man has tried to improve stress tolerance of plants from the day he started crop domestication and cultivation. However, probably abiotic stress tolerance was not as important for food security as it is today. The improvement in the abiotic stresses tolerance of crops was rather slow for a long time due to lack of knowledge about their genetic control. With the advancement of knowledge about the genetic factors affecting abiotic stress tolerance, there is a new hope for developing significantly better stress tolerance crops. Breeding for abiotic tolerance may be direct (selection pressure under stress) or indirect (selection pressure under stress-free environment) (Lewis and Christiansen 1981). The simplest approach to breeding for stress tolerance is to select for yield, which is the integrating trait, and to carry out the selection in a representative stress environment. It can be enhanced by carefully managed stresses (Bänziger et al. 2006, Lafitte et al. 2006) and by intelligent choice of parents to pyramid desirable traits (Yeo and Flowers 1986). The approach of using fewer crosses with larger population (Witcombe and Virk 2001) can be very effective, which has been proved in breeding of stresstolerant, widely adapted rice (Joshi et al. 2007a–e). Recently, molecular tools, especially molecular markers, have been added in this direction. The advent of molecular markers has revolutionized the genetic analysis of crop plants and provided not only geneticists, but also physiologists, agronomists, and breeders with valuable new tools to identify traits of importance in improving resistance to abiotic stresses (Rudd et al. 2005), and is seen as an approach for precision plant breeding in the twenty-first century (Collard and Mackill 2008). Marker-assisted selection can be effective in enhancing efficiency, but, at least so far, the selection for markers linked to component traits of low heritability has not produced predicted outcomes (Witcombe et al. 2008). Fine-mapping and map-based cloning of quantitative trait loci

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(QTLs) (Price 2006) will allow them to be used more effectively in breeding by eliminating the effects of unwanted linked alleles. Marker Assisted Selection (MAS) need to be fine-tuned, so that precise combinations of alleles can be combined for maximum effect. Marker-assisted breeding is now increasingly targeted toward tracking the candidate genes responsible for stress tolerance through gene identification and functional studies (Tuberosa and Salvi 2004, Mantri et al. 2007). Till date, the expression profiles of all the genes in genome have been investigated in some of the model crops, such as Arabidopsis thaliana (thale cress), Oryza sativa (rice), Medicago truncatula (barrel medic), and Populus trichocarpa (black cottonwood). Other crops where this work has been possible are Brachypodium distachyon, Lotus japonicus (lotus), Manihot esculenta (cassava), Solanum lycopersicum (tomato), Solanum tuberosum (potato), Sorghum bicolor, Zea mays (corn), and soybean (http://www.ncbi.nlm.nih.gov/genomes/PLANTS/PlantList.html). Transcription factors (TFs) that bind to DNA through specific cis-regulatory sequences and either activate or repress gene transcription have been reported to act as control switches in stress signaling (Tran and Mochida 2010). The recent completion of the soybean genomic sequence has open wide opportunities for large-scale identification and annotations of regulatory TFs in soybean for functional studies. Transgenic and functional genomics approaches hold tremendous promise for the future and are being pursued vigorously to improve qualitative and quantitative traits, including tolerance to biotic and abiotic stresses in different crops (Ashraf 2010).

32.2  Drought Tolerance The ability of a crop to grow satisfactorily in areas subjected to water deficits has been termed drought resistance (Turner 1986). Crops all over the world are exposed to chronic or sporadic periods of drought (Boyer 1982), a multidimensional stress affecting plants at various levels of organization (Blum 1996). Drought stress affects the yield by reducing both sink and source. It can be a result of stress affecting either one of these directly or their interaction with one another (Blum 1996). Before seeing the external world, the living plant (the dormant embryo) within the seed is highly tolerant to desiccation, but loses its tolerance upon germination and emergence. It has been suggested that a unified abiotic stress resistance mechanism for drought at the level of the whole plant or the single gene is probably not present (Blum 2004). Plants survive under drought through the avoidance or postponement of dehydration or tolerance (Turner 1986). The traits associated with avoidance and tolerance can be constitutive (differing between genotypes) or adaptive (varying with the stage of the life cycle) (Witcombe et al. 2008). Drought avoidance and drought tolerance involve different mechanisms and processes, and phenology is the singlemost important factor influencing whether a plant avoids drought. In other words, plants tolerate drought stress through various morphological, biochemical, and molecular adjustments at the whole plant level.

32.2.1  Morphophysiological Traits: Genetic Basis Though there are no traits that confer global drought tolerance (Passioura 1996), numerous constitutive traits carry a large impact on crop performance under drought stress (Blum 1996). According to Wilson (1981), traits associated with water use efficiency act through their effect on (1) the timing of crop development, (2) the efficiency of root to harvest water, (3) the effective transpiration control by shoots and the relationship of transpiration and photosynthesis, and (4) the ability of plants to endure stress. Any trait that reduces transpiration or increases photosynthesis will increase the water use efficiency. Ludlow and Muchow (1990) have reviewed important traits in crops for success in water-limited environments. Any trait that reduces transpiration or increases photosynthesis will increase the water use efficiency. Though several traits join together to fight drought, the genetics of some of the traits useful under drought conditions is given below.

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32.2.1.1  Earliness Rapid plant development and early maturation require less water, and thus work through drought avoidance (Turner 1979, 1986, Ludlow and Muchow 1990, Boyer 1996). There is genetic variation for earliness, both across and within species (Hall and Grantz 1981, Richards and Passioura 1989, Richards 2004). For example, in wheat, there is much genetic variation for flowering time and maturity (Innes and Quarrie 1987). The Rht genes in wheat possibly possess a pleiotropic effect on earliness (Innes and Quarrie 1987). In maize, evidence for numerous small-effect QTLs was found to control the flowering time (Buckler et al. 2009); however, no individual QTLs were identified at which allelic effects are determined by the geographic origin or large effects for epistasis or environmental interactions. Thus, in contrast to rice and Arabidopsis, a simple additive model was suggested to accurately predict the flowering time for maize (Buckler et al. 2009). 32.2.1.2  Roots An extensive root system is desirable for efficient water extraction in different crops (Hurd 1974, Lorens et al. 1987, Boyer 1996). Genetic variation in root characteristics does exist in crop plants (Hurd 1974, Taylor et al. 1974, Boyer et al. 1980, Lehman and Engelke 1993, Sarker et al. 2005). Many root characteristics have been shown to be under genetic control and are quantitatively inherited. But in rice, the difference in the depth of rooting was controlled by only a few genes (ArmentaSoto et al. 1983, Ekanayke et al. 1985). In wheat, genetic variation for root hydraulic conductance is present and is heritable (Blum and Johnson 1993). Genetic variability for root size was found in sorghum (Blum et al. 1997), wheat (O’Brien 1979), rice (Larsson and Svenningson 1986), soybean (Boyer et al. 1980), and oat (Murphy and Nelson 1982). The density of root hairs also shows considerable genetic variability (200 cm2 in trees to 2500 cm2 in cereals), but little is known about intraspecific variability and the genetics of this trait (Monneveux and Belhassen 1996). Root traits (root length, root number, root-tip thickness, and root/shoot weight ratio) in rice have been found to possess moderate heritability and are under the control of both additive and dominant gene effects (Armenta-Soto 1983, Ekanayke 1985). Some of these traits also showed heterosis in several crop plants (Sinha and Khanna 1975, Blum 1996). The growth angle and the number of seminal roots in wheat are also reported to show a significant genotypic variation (Manschadi et al. 2008). 32.2.1.3  Stomatal Conductance The control of leaf stomatal conductance (gs) is a crucial mechanism for plants, since it is essential for both CO2 acquisition and desiccation prevention (Dodd 2003, Medici et al. 2007). A reduced stomatal conductance through various characteristics, such as stomatal frequency, length, and behavior (Ludlow and Muchow 1990), increases the water use efficiency. Genetic variation is reported for various stomatal characters (Clarke and Townlet-Smith 1984, Jones 1987), and they seem to be highly heritable (Roark and Quisenberry 1977). In addition, dimensions, and especially frequency, can change more than twofold in response to radiation or to water status, or according to developmental stages (Jones 1987, Roark and Quisenberry 1977). Monneveux et al. (2006) reported that durum wheat breeding at maturity would lead, under Mediterranean drought conditions, to higher stomatal conductance, lower transpiration efficiency, and higher grain yield. Medici et al. (2007) reported that in maize, the stomatal conductance of drought-tolerant varieties was lower compared to other cultivars. 32.2.1.4  Epicuticular Wax Many characteristics of the leaf affect drought tolerance, which include cuticular wax characteristics (Cameron et al. 2006). Likewise, a waxy layer covering the plant parts, for example, the glaucous character in wheat and the bloomed trait in sorghum, improves the water use deficiency. Genetic variation is reported for the bloomed trait in sorghum (Ebercon et al. 1977, Jordan et al. 1983) and the glaucousness in wheat (Nizam Uddin and Marshall 1988). Variation for cuticular wax is also reported in other crops, for example, oat (Bengtson et al. 1978) and rice (O’Toole et al. 1979).

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The genetics of epicuticular wax has been investigated in several crop species. The presence of waxy bloom was found to be controlled by a single dominant gene in sorghum (Monneveux and Belhassen 1996). However, heritability for the bloomed trait of sorghum was low (Jordan et al. 1983). In tobacco, a sixfold increase of tree tobacco lipid transfer proteins (LTPs) gene transcripts was observed after three drying events, providing further evidence that LTP is involved in cuticle deposition (Cameron et al. 2006). In wheat also, a dominant non-glaucousness gene was reported from synthetic hexaploid wheat (Triticum aestivum L.), which has been mapped in chromosome 2DS (Liu et al. 2007). 32.2.1.5  Osmotic Adjustment Osmotic adjustment (OA) reduces the rate of leaf senescence (stay-green trait) because it increases both avoidance and tolerance to dehydration. Genetic variation for this trait has been found in wheat (ms gene) (Morgan 1977, 1984, Blum et al. 1983, Morgan and Condon 1986), sorghum (Akerson et al. 1980, Wright and Smith. 1983, Blum and Sullivan 1986), millet (Hensen 1982), cotton (Karami et al. 1980), rice (Lilley et al. 1996), and pigeon pea (Flower and Ludlow 1987). OA is simply inherited and only one or few genes are involved in wheat (Morgan 1977, 1991) and soybean (Basnayake et al. 1995). In rice, the indica cultivars tend to be more dehydration tolerant than japonica cultivars (Lilley et al. 1992). A gene for OA was located in chromosome 7A of wheat (Morgan 1991). On the basis of homology between a small segment of chromosome 7 of wheat and chromosome 8 of rice (Ahn et al. 1993, Van Deynze et al. 1993), it has been suggested that there might be an association between the OA gene of wheat and rice. Jiang and Huang (2001) demonstrated that drought preconditioning enhanced heat tolerance in Kentucky bluegrass (Poa pratensis L.), which could be related to the maintenance of higher OA associated with the accumulation of ion solutes and water-soluble carbohydrates and the development of extensive roots deeper in the soil profile. In maize, the relationship of OA and yield maintenance under drought was not established for a long time. However, a study (Chimenti et al. 2006) compared two S4 populations derived from a cross between inbred lines exhibiting the highest and lowest capacities for OA in a screening applied to 20 inbred lines. Crops of these populations were grown under a rainout shelter and subjected to 30-day droughts either before or during flowering. They concluded that OA can contribute to drought tolerance in maize crops exposed to water deficit both before and during flowering, and that the trait carries no yield penalty under irrigation. 32.2.1.6  Transpiration Efficiency The transpiration efficiency of C4 plants is greater than C3 plants (El-Sharkawy 2009). Genetic variation for transpiration efficiency has been reported in wheat, barley, cotton, peanut, and sunflower (Blum and Johnson 1993). Its inheritance is complex (Ludlow and Muchow 1990) but heritability is high (Martin and Thorstenson 1988). High heritability has been noted in crested wheat grass (Johnson et al. 1990), peanut (Hubic et al. 1988), and wheat (Ehdaie et al. 1991), and moderate heritability in cowpea (Hall et al. 1993). Several other traits, such as mobilization of preanthesis assimilates, leaf movements, epidermal conductance, developmental plasticity, and leaf area maintenance, are also important for drought tolerance (Ludlow and Muchow 1990). Genetic variation for some of these traits is also reported, for example, for the mobilization of preanthesis assimilates (Blum et al. 1983, Constable and Hearn 1978) and leaf area maintenance (Duncan et al. 1981, Rosenow et al. 1983). Richards and Passioura (1981) found intraspecific variability in bread wheat for the xylem vessel diameter, which possesses high heritability. Leaf rolling showed genetic variation in sorghum (Chang et al. 1974, Beg 1980) and rice (Chang et al. 1974, Turner 1986). Leaf rolling was also found to have an association with drought tolerance in rice (Efisue et al. 2009). Genetic variation for epidermal conductance has been observed in rice (Yoshida and Reyes 1976), sorghum (Jordan et al. 1984, Blum 1979) and soybean (Manavalan et al. 2009).

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32.2.2  Genetics of Drought Tolerance Genetics of drought resistance can be partly understood through the inheritance of traits responsible for drought avoidance or postponement or tolerance, as described above. However, the situation is complex as single genes that substantially change water use efficiency are difficult to find and it generally involves many genes and many interactions (Boyer 1996). Tolerance to drought is rare in vegetative parts of plants, while angiospermous seeds and pollen are able to survive extreme dehydration (Leopold et al. 1992). Genes responsive to drought, desiccation, high osmoticum, or wilting have been identified in tomato (Cohen and Bray 1990), Craterostigma plantagineum (Bartels et al. 1990, Bartels and Salamini 2001, Bartels 2005), maize (Close et al. 1989, Tuberosa et al. 2002), barley (Close et al. 1989), rice (Mundy and Chua 1988), Arabidopsis (Gilmour et al. 1992), tobacco (La Rosa et al. 1989), soybean (Surowy and Boyer 1991), cotton (Baker et al. 1988), and wheat (Kirigwi et al. 2007, Gupta et al. 2009). The location of genes having a major effect on drought-induced abscisic acid (ABA) accumulation in wheat was determined by using molecular markers, a set of single chromosome substitution lines and populations derived from a cross between high and low ABA-producing genotypes (Quarrie et al. 1994). A similar drought test with detached and partially dehydrated leaves confirmed the location of gene(s) regulating ABA accumulation in the long arm of chromosome 5A (Quarrie et al. 1994). The MAPMAKER QTL showed the most likely position for the ABA QTL to be between the loci Xpsr575 and Xpsr426, about 8 cm from Xpsr 426; another QTL for ABA accumulation may be present on chromosomes 3BS and 6AL (Quarrie et al. 1994). QTL for response to drought has been reported in different crops, such as maize (Paterson et al. 1991, Lebreton et al. 1995, Chenu et al. 2009), sorghum (Tuinstra et al. 1998, Crasta et al. 1999, Sanchez et al. 2002), rice (Kato et al. 2008, Bernier et al. 2009), wheat (Quarrie et al. 1994, Kirigwi et al. 2007, Matthews et al. 2008, Gupta et al. 2009), and barley (Sanguineti et al. 1994). There is clear evidence now that all the major cereal species have extensive linkage blocks where the gene order is conserved (Moore et al. 1995). QTLs have been identified for several drought resistance component traits in rice (McCouch 1995, Lilley et al. 1996, Price and Tomos 1997, Yadav et al. 1997, Ali et al. 2000, Price et al. 2000, Tripathy et al. 2000, Zheng et al. 2000, Zhang et al. 2001, Li et al. 2005, Gomez et al. 2006, Kato et al. 2008). In one of such studies (Lilley et al. 1996) to understand the genetic mechanism of OA and dehydration tolerance in rice, a recombinant inbred (F7) population was mapped with 127 restriction fragment length polymorphism (RFLP) markers; a major locus was found to be associated with OA in rice (Lilley et al. 1996). This locus may be homologous with a single recessive gene previously identified for the same trait in wheat; the putative OA locus and two of five QTLs associated with the dehydration tolerance were close to the chromosome regions associated with root morphology (Lilley et al. 1996). Specht et al. (2001), using molecular marker analyses of the 236 RILs (recombinant inbredline), reported the detection of three QTLs in soybean with very strong effects on the transpiration efficiency associated with drought. In sorghum, Sanchez et al. (2002) identified four genomic regions associated with the stay-green trait using an RIL population developed from B35 × T × 7000. These four major stay-green QTLs were consistently identified in all field trials and accounted for 53.5% of the phenotypic variance. Likewise, QTLs linked to morphophysiological and plant production traits in rice under drought stress in the field were mapped by evaluating 177 F6 recombinant inbred lines of Bala × Azucena under rainfed (Gomez et al. 2006). A total of 24 QTLs were identified for various traits under stress, which individually explained 4.6%–22.3% of phenotypic variation. Composite interval mapping detected three markers, namely, RM3894, RG409, and G1073, on chromosomes 3 and 8 linked to grain yield under drought stress in temperature extreme (TE), respectively, explaining 22.3%, 17.1%, and 10.9% of phenotypic variation (Gomez et al. 2006). QTLs for leaf drying, days to 50% flowering, and number of productive tillers under drought stress colocated at certain of these regions. Further, QTLs for several root traits overlapped with QTLs for grain yield under stress in these recombinant inbred (RI) lines, indicating the pleiotropic effects of root trait QTLs on rice performance under stress (Gomez et al. 2006).

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Kato et al. (2008) reported three QTLs on chromosomes 2, 4, and 7 for the relative growth rate of rice. The QTL on chromosome 7 had a constant effect across environments, while the QTL on chromosome 4 had an effect only under nonstressed conditions, and that on chromosome 2 only under stressed conditions. The stress-specific QTL on chromosome 2 was not colocated with any QTLs for the root system depth previously reported from the same mapping population. However, this QTL was colocated with a stress-specific QTL for specific water use (SWU), suggesting that the control of transpiration was relevant to dry matter production under drought. Recently, Bernier et al. (2009) reported a large-effect QTL (qtl12.1) for grain yield under drought conditions tested across 21 field trials: 10 at IRRI (International Rice Research Institute) in the Philippines and 11 in eastern India. The relative effect of the QTL on grain yield increased with the increasing intensity of drought stress, from having no effect under well-watered conditions to having an additive effect of more than 40% of the trial mean in the most severe stress treatments. The QTL improved grain yield in 9 out of 10 direct-seeded upland trials where drought stress was severe or moderate, but no effect was measured under well-watered aerobic conditions or under transplanted lowland conditions. This confirmed that qtl12.1 has a large and consistent effect on the grain yield under upland drought stress conditions, in a wide range of environments (Bernier et al. 2009). Chenu et al. (2009) proposed a modeling approach to bridge the “gene-to-phenotype” gap caused by the genotype × environment interaction in maize under drought. They simulated the impact of QTLs controlling two key processes (leaf and silk elongation) that influence crop growth, water use, and grain yield. The simulations obtained illustrated the difficulty of interpreting the genetic control of yield for genotypes influenced only by the additive effects of QTLs associated with leaf and silk growth (Chenu et al. 2009).

32.2.3  Gene Expression Water deficit is one of the most common abiotic stresses that affects the growth and development of plants through alterations in metabolism and gene expression (Leopold 1990). The molecular basis of dehydration stress responses in cells and organisms has been intensively researched over the past years (Somvanshi 2009). Molecular and genomic analyses have facilitated new gene discovery and enabled genetic engineering to deploy several functional or regulatory genes to activate specific or broad pathways related to drought tolerance in plants (Umezawa 2006). Genetic information to withstand drought is present in plants in stress genes, but these genes are expressed only in particular developmental stages (Mckersie and Leshem 1994). The molecular studies of dehydration stress are mainly based on (1) the desiccation tolerance of the maturing embryo (Quatrano 1989) and resurrection plants (angiosperms that are able to survive dehydration and revive upon hydration) (Bartels 1990, Gaff 1971), (2) A. thaliana (Yamaguchi-Shinozaki 1993, Kasuga et al. 1993), and (3) other crops such as tomato, pea, wheat, and barley (Bartels et al. 1996). Genes regulated by drought stress can be divided into three groups (Bartels et al. 1996): genes encoding polypeptides of unknown functions, genes encoding Lea proteins and related polypeptides, and genes encoding polypeptides of known functions. The DNA sequence analysis of osmotic stress-inducible cDNAs indicates that genes responsible to drought encode a variety of proteins (Wood and Goldsbrough 1997). Many of the proteins encoded by these cDNAs have been classified into various groups, namely, LEA (late embryogenesis abundant) (Baker et al. 1988), RAB (responsive to ABA) (Skriver and Mundy 1990), and dehydrin (Close et al. 1989) proteins. LEA proteins and dehydrins are classified on the basis of their characteristic amino acid motifs, while RAB proteins are classified based upon the expression in response to ABA (Wood and Goldsbrough 1997). LEA proteins, first identified during seed maturation and desiccation, express in water-stressed vegetative tissues in almost all plants (Wood and Goldsbrough 1997). They are supposed to protect the dehydrating cells by a variety of mechanisms, including renaturation of unfolded proteins, sequestration of ions, and stabilization of the native protein structure (Wood and Goldsbrough 1997). Recently, it has been demonstrated that the

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accumulation of the barley HVA1 protein, a group of three LEA proteins, in transgenic rice confers an increased tolerance to water deficit as well as to salt stress (Xu et al. 1996). Genes have also been isolated from the resurrection plant C. plantagineum, which can recover completely from complete dryness within 24 h of contact with water (Schneider et al. 2004). Many of the desiccation-induced genes share sequence homology with lea genes (Galau et al. 1986, Piatkowski et al. 1990). ERD genes (early responsive to dehydration stress genes) in comparison to aba-responsive genes are preferentially responsive to the dehydration stress (Jensen et al. 1996). The dynamics of water transport in plants is influenced by water channel proteins in plants (Verkman 1992, Chrispeels and Maurel 1994), which are related to the super family of membraneintrinsic proteins (MIP) first characterized in Escherichia coli. The functions of different MIP channels vary (Reizer et al. 1993). Several MIP-related proteins have been identified in plants. A member of this family, Trg 31 (in pea), was initially identified when the gene expression was induced in partially dehydrated leaves (Guerrero et al. 1990). Another dehydration-inducible gene was isolated from A. thaliana (Yamaguchi-Shinozaki 1992). NOD 26, the first plant MIP protein to be characterized (Miao et al. 1992), is abundant in the prebacteroid membrane root nodules. MIP-like tonoplast intrinsic proteins (TIPS) and their corresponding genes have been identified (Chrispeels and Maurel 1994). Water deficit results in diminished growth of young leaves. In soybean, water-stressed plants show an increased expression of genes encoding the vegetative storage proteins (Vsp) (Mason and Mullet 1990). But the same does not occur in mature leaves even though they possess the potential to express Vsps when leaves are wounded (Mullet and Whitsitt 1996). In a study (Rizhsky et al. 2002), the combination of drought and heat shock resulted in the closure of stomata, suppression of photosynthesis, enhancement of respiration, and increased leaf temperature. Some transcripts induced during drought, for example, those encoding dehydrin, catalase, and glycolate oxidase, and some transcripts induced during heat shock, for example, thioredoxin peroxidase and ascorbate peroxidase, were suppressed during a combination of drought and heat shock. In contrast, the expression of other transcripts, including alternative oxidase, glutathione peroxidase, phenylalanine ammonia lyase, pathogenesis-related proteins, a WRKY TF, and an ethylene response transcriptional coactivator, was specifically induced during a combination of drought and heat shock (Rizhsky et al. 2002). Photosynthetic genes were suppressed, whereas transcripts encoding some glycolysis and pentose phosphate pathway enzymes were induced, suggesting the utilization of sugars through these pathways during stress (Rizhsky et al. 2002). In chickpea, transcriptional profiling indicated that genes are differentially regulated in response to drought tolerance (Mantri et al. 2007), although the same behavior was displayed under high-salinity and cold stresses (Mantri et al. 2007). Transgenic breeding is also being viewed as a science of the future for drought tolerance. In a recent study (Wang 2009), using Agrobacterium-mediated transformation, a wheat LEA gene, TaLEA3, was integrated into Leymus chinensis (an important grassland perennial grass). The transgenic lines showed an enhanced growth ability under drought stress, during which transgenic lines had increased the relative water content, the leaf water potential, and the relative average growth rate, but decreased the malondialdehyde content compared with non-transgenic plants (Wang 2009). A large number of drought-tolerant genes are being patented around the world. It has been reported (Somvanshi 2009) that almost all of the patented dehydration stress tolerance genes from different organisms were used in engineering drought tolerance in crop plants. Several lines of evidence have indicated that molecular tailoring of genes has the potential to overcome a number of limitations in creating drought-tolerant transgenic plants (Umezawa 2006). This suggests that in future, the transgenic approach would be playing a much greater role in drought tolerance than is visible today. However, the evaluation of the transgenic plants under stress conditions, and understanding the physiological effect of the inserted genes at the whole plant level remain the major challenges to overcome (Bhatnagar-Mathur et al. 2008).

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32.2.4  Breeding The understanding of the genetic basis of drought tolerance is poor (Ortiz et al. 2008a). Despite many decades of research, drought continues to be a major challenge to agricultural scientists, probably due to the difficult nature of the target environment and the interaction of drought with other abiotic as well as biotic stresses (Blum 1988). This is supported by the observation that in water-limited environments, the yield of the biomass of current cultivars is about the same as that of cultivars from over a century ago (Siddique et al. 1989). However, the wondrous display of plant adaptations to dry habitats points to the fact that a substantial genetic variation for drought tolerance exists, and this may be used for plant breeding (Richards 1996). The problem of breeding crops for drought environments is not due to the want of enough genetic variation, but probably lies in the elusive design of the ideal plant/the ideotype (Donald 1968), which has been in attention (Sedgley 1991) for both normal and stress conditions (Blum 1996). The demand of genes depends on the type of ideotype required for the water stress area. Since a large number of traits take part in plants’ stress response, the task is not easy in practical terms. The water status of a plant is a function of uptake (by roots) and loss (via stomata and cuticle) of water; therefore, breeding strategies may broadly focus on either of these two parameters (Kuckuck et al. 1991). So far, the breeding strategies for drought areas have been suggested to depend on (1) selecting genotypes with an improved yield in water stress environments (Mullet and Whitsitt 1996), (2) identifying and selecting traits that contribute to drought avoidance, drought tolerance, or water use efficiency (Ludlow and Muchow 1990), and (3) even selecting genotypes under nonstress environments and then trying in stress areas (Sayre et al. 1995, Richards 1996). Drought stress is highly variable in its timing, duration, and severity, and therefore displays high environmental variation and G × E variation (Witcombe et al. 2008). The use of managed stress environments can be very effective in breeding for drought tolerance; however, it is important to apply a sufficient drought stress intensity to maximize G × E (Bänziger et al. 2006). In other words, without plant selection under water deficient conditions, traits beneficial under water stress may be missed (Boyer 1996). However, in favorable environments, there is less error, and thus, a high yield potential expressed in favorable environments can also have a spin-off in less favorable environments (Richards 1996). In Australia, where wheat is grown in a water-limited environment, 95% of current cultivars can be traced to the CIMMYT germplasm where breeding is done under highly favorable environments (Richards 1996). In wheat, considerable progress has been made in yield improvement under drought in recent decades using the available gene pool and selecting under drought stress (Trethowan et al. 2002). Opportunities exist to improve the tolerance further, if new genetic variability can be combined with existing variability and if the underlying genetic control of tolerance can be better understood (Ortiz et al. 2008b). The resynthesized wheat lines developed by crossing the modern durum wheat with Aegilops tauschii, the probable donor of the D genome in hexaploid wheat, have introduced new genetic variation into the wheat gene pool for many characters. Not surprisingly, resynthesized wheat lines have also been a source of variation for drought and heat tolerance (Trethowan et al. 2002). Some advanced materials derived from resynthesized wheat lines have improved adaptation worldwide, especially in drought-stressed environments. Recognizing water productivity and water use efficiency as priorities for wheat, CIMMYT researchers disaggregated grain yield under water stress into distinct components to apply these findings to the genetic enhancement of this crop (Reynolds and Borlaug 2006). Ongoing research is providing a better understanding of traits with major effects on the water productivity in dryland wheat areas (Reynolds et al. 2007). These include root architecture and physiological traits, resistance to soil-borne pests and diseases, tolerance to heat and salinity, and zinc-deficient and borontoxic soils. The combination of improved germplasm; the Center and partners’ expertise in drought physiology, soil-borne diseases, and agronomy; and the availability of DNA markers for various traits place CIMMYT in a unique position to develop water-productive wheat with resistance to the important stresses for use by partners throughout the developing world (Ortiz et al. 2008b).

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Some important attributes for drought-prone environments are available in the wild relatives of wheat (Reynolds et al. 2007). Resynthesizing hexaploid wheat with wild ancestors has been used at CIMMYT for tapping this useful variation and incorporating such genetic resources into wheatbred germplasm (Dreccer et al. 2007). Recently, Ogbonnaya et al. (2007) found that such lines derived from resynthesizing wheat yielded 8%–30% higher than the best local check in multisite trials across diverse regions of Australia. Their results reinforce previous research conducted at CIMMYT that lines derived from synthetic wheat have the potential to significantly improve grain yield across environments. QTL mapping techniques are a hope of the future with regard to improved abiotic stress tolerance and drought stress in particular. The QTL locations are now easy to compare across species, as demonstrated through the comparison of QTL of root characters in maize and rice (Lebreton et al. 1995, Buckler et al. 2009). A decade ago, a marker-assisted selection using random-amplified polymorphic DNA (RAPDs) was demonstrated to improve the yield performance in common bean in stress conditions (Schneider et al. 1997). In rice, Li et al. (2005) suggested that the high yield potential and drought tolerance/water use efficiency (DT/WUE) can be combined more efficiently by the designed QTL pyramiding (DQP) strategy. Though molecular genetics might prove important, the identification of stress-responsive genes or even their cloning and insertions seem to be beyond practical application, unless their function and value within the ideotype can be demonstrated (Blum 1996). There are a few reports of the transfer of alien DNA into crop species specifically to improve drought responses, but an extensive introgression programme is in progress to transfer useful traits from Festuca into Lolium (Thomas et al. 1994). In some of the introgression lines with Festuca arundinacea in Lolium multiflorum × F. arundinacea crosses, drought resistance was equivalent to the Festuca donor (Thomas et al. 1994). This single chromosome addition line of F. arundinacea on to L. multiflorum also showed improved drought resistance (Quarrie et al. 1997). Hence, this work looks promising in future to improve drought responses of other graminaceous crops (Quarrie et al. 1997). The introgression of the drought-tolerant mechanism present in wild species in cultivated plants is in course in several laboratories. Another strategy for the genetic improvement of plants under drought has been to identify gene(s) of desiccation tolerance, for example, in desert plants or wild species (Bartels and Nelson 1994, Bohnert et al. 1995), and transfer them to agronomic crops (Tardieu 1996). A transgenic rice plant having tolerance to water deficit and osmotic stress has been reported (Nguyen et al. 1997). In wheat, transgenic approaches for incorporating stress-inducible regulatory genes that encode proteins such as TFs (e.g., DREB1A) into the wheat cultigens pool are also being pursued (Hoisington and Ortiz 2008).

32.3  Submergence Tolerance Submergence tolerance is defined as the ability of crop plants to survive and continue growing after being completely submerged in water for several days. Tolerance to water logging or submergence tolerance is associated either with crops grown in high rainfall areas of the world or in lowland areas that are kept flooded by natural rain, flood, or canal irrigation. Most of our knowledge regarding submergence tolerance is obtained from the studies on rice crop, 16% of whose total world area is affected by this problem (Khush 1984). Compared to other parts of the world, flooding is a serious, naturally occurring problem for rice production in the rainfed lowlands of south and southeast Asia during the monsoon season. It is estimated that around 50% of the rice-growing area in this ecosystem is affected by flash flooding at various stages of growth (Dey and Upadhyaya 1996). Limited gas diffusion in water is considered the principal cause for the adverse effects of submergence (Setter et al. 1995). Therefore, tolerance to flooding is associated to cope up with problems associated with submergence, such as anaerobiosis, lower carbon assimilation due to less CO2 and radiation (Setter et al. 1987, 1989), and high ethylene. This is partly achieved by avoidance— through maintenance of growth processes leading to the elongation of plants to maintain their foliage above water (Mohanty and Choudhary 1986).

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32.3.1  Morphophysiological Traits: Genetic Basis Several morphophysiological traits have been reported to be associated with submergence tolerance (Setter et al. 1997). Setter et al. (1997) listed 17 morphophysiological traits as part of the mechanism explaining submergence tolerance in rice. These traits were classified into presubmergence, submergence, and postsubmergence traits. Among these, the three important traits are (1) carbohydrate concentration, (2) alcoholic fermentation, and (3) elongation of the stem. Favorable effects of high carbohydrate concentration (Palada and Vergara 1972, Setter et al. 1987, Mallik et al. 1995) and high alcoholic fermentation (Waters et al. 1991, Ricard et al. 1994) are well documented. Stem elongation does favor through avoidance, but, on the other hand, there is a strong negative correlation between elongation growth and percent survival of seedlings during submergence (Setter et al. 1994), because elongation growth competes for energy and carbohydrates required for maintenance processes for survival (Setter et al. 1997). The elongation mechanism is effective only when the water level remains high for a considerable period, as in deep water rice culture. It is not desirable under flash flood conditions, because when the water recedes, the plants tend to lodge (Seshu and Akbar 1995). The capacity to survive submergence depends not only on specific environmental factors, but also on the strategy that plants have evolved for adoption to particular flood-prone environments (Ram et al. 2002). For instance, in rice, the two main strategies are to elongate and escape, or not to elongate and conserve resources. For rainfed lowland rice exposed to flash flooding, the elongation growth during complete submergence has major adverse effects on survival, presumably since this competes with maintenance processes that require carbohydrates and energy. Therefore, selection for minimal elongation during submergence is currently being exploited as a trait for submergence tolerance by rainfed lowland rice breeders in south and southeast Asia (Ram et al. 2002). Recently, Erythrina speciosa was shown to display 100% survival until the 60th day of flooding and was able to recover its metabolism (Medina et al. 2009). The recovery during soil flooding appeared to be associated with morphological alterations, such as the development of hypertrophic lenticels, adventitious roots, and aerenchyma tissue, and with the maintenance of neutral amino acids in roots under long-term exposure to root-zone O2 deprivation (Medina et al. 2009).

32.3.2  Genetics Submergence tolerance is a rare, genetically determined trait with relatively high heritability that is controlled by one or a few genes with major effects, and minor modifiers (Suprihatno and Coffman 1981, Mohanty and Khush 1985, Sinha and Saran 1988, Haque et al. 1989). Varietal differences in the degree of submergence tolerance have been noted many times (Ramiah and Rao 1953, Palada and Vergara 1972, IRRI 1978), and this genetic resource has been used in several conventional breeding programmes (Mohanty and Khush 1985). A 10 × 10 half-diallel analysis (Mohanty and Khush 1985) showed significant additive and nonadditive gene actions for submergence tolerance, but additive effects were more important. Tolerance was dominant over nontolerance and the average dominance was within the range of incomplete dominance; the Wr/Vr graphic analysis also suggested the involvement of both major and minor genes (Mohanty and Khush 1985). In an earlier study (Suprihatno and Coffman 1985), it has been reported that at least three submergence tolerance genes are present in the four most tolerant rice varieties, namely, FR13A, Thavalu, Kurkaruppan, and Goda Heenati. The analyses of F2 and backcross generations of the above four rice varieties and two susceptible (IR 42 and Nona Bokra) lines indicated the presence of a single dominant gene for submergence tolerance (Setter et al. 1997). It was also found that the first three tolerant lines possessed the same gene for submergence, but a different one in the cultivar Goda Heenati. This finding was supported by the bimodal distribution of rice lines for submergence in Thailand (IRRI 1996), but was contradicted by normal distribution noted in Philippines (IRRI 1996). The finding of the three most tolerant rice lines having the same gene for submergence

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tolerance suggested that a common factor related to the tolerance of limited gas diffusion (e.g., one of the enzymes of alcoholic fermentation) may be responsible for genotypic differences in the submergence tolerance of rice (Setter et al. 1997). It also suggests that a gene for TF is involved in the expression of a multiple gene cascade that confers submergence tolerance (Setter et al. 1997). A visual observation of symptoms is useful in attempts to identify genes or QTLs associated with tolerance, because many lines can be screened in a reasonably short time (Toojnida et al. 2003). Using this approach, QTLs for submergence tolerance have been reported on chromosomes 6, 7, 9, 11, and 12 (Xu and Mackill 1996, Nandi et al. 1997). Xu and Mackill (1996) mapped the major QTL for submergence tolerance in rice on chromosome 9. They used RAPD and RFLP markers to map submergence tolerance using a cross between a tolerant indica rice line, IR4093 I-26, and a susceptible japonica line, PI54385 1. A submergence tolerance QTL, designated as Sub1, was located ca. 4 cm from the RFLP marker C 1232 and accounted for 69% of the phenotypic variance for the trait (Xu and Mackill 1996). In recent years, more than 1000 DNA markers have been mapped onto the rice genome based on RFLPs, simple sequence length polymorphisms (SSLPs), and amplified fragment length polymorphisms (AFLPs) (McCouch et al. 1988, Kurata et al. 1994, Toojnida et al. 2003). Perata and Voesenek (2006) reported that in rice, a large portion of the variation in submergence tolerance can be explained by one locus (Sub1, an ethylene-response-factor-like gene) on chromosome 9. This gene controls physiological and developmental processes that determine the rate of elongation when submerged (Perata and Voesenek 2006).

32.3.3  Gene Expression The gene expression for anaerobic (submergence) stress is better known than other abiotic stresses (Sachs et al. 1980, Dennis et al. 1992, Setter et al. 1997). The knowledge of gene expression during anaerobiosis is due to the finding in maize that the alcohol dehydrogenase (ADH) activity increases due to flooding (Hegeman and Flesher 1960, Freeling and Birchler 1981, Freeling and Bennet 1985). The ADH activity that allows maize to survive in flooding reflects a simultaneous expression of two unlinked genes, Adh1 and Adh2 (Freeling 1973). The Adh1 and Adh2 cloned cDNA families have been identified and analyzed extensively (Dennis et al. 1984, 1985). The regions of Adh1 and Adh2 genes upstream from the site of transcription initiation show homology with respect to an 11 bp homologous region that includes the TATA box and three other segments of 8 bp size (Sachs and Ho 1986). The other important enzyme is pyruvate decarboxylase (PDC) (Reggiani et al. 1986), which is governed by three Pdc genes in rice (Reggiani et al. 1986). During anaerobiosis, a shift in protein synthesis was reported (Sachs and Ho 1986), where it was found that there is a repression of the preexisting protein synthesis followed by a de novo synthesis of a new set of proteins. This has been reported in several crops, for instance, soybean (Lin and Key 1967), maize (Cooper and Ho 1983, Sachs and Ho 1986), rice (Bertani et al. 1981), sorghum, barley, pea, and carrot (Setter et al. 1997). The shift in protein synthesis is quite fast with a transition period of few hours. The polypeptides (33 kDa) formed during the transition period are referred to as transition polypeptides (Tps). Another group of 20 polypeptides (anaerobic polypeptides [ANPs]) is also induced after a 90 min gap, which includes isozymes of ADH (Ferl et al. 1979), glucose-6phosphate isomerase (Kelley and Freeling 1984a), fructose-1,6-diphosphate aldolase (Kelley and Freeling 1984b), and sucrose synthetase (Setter et al. 1997). In rice, the two Adh genes (Xie and Wu 1989) and three Pdc genes have been cloned and characterized (Reggiani et al. 1986, Hossain et al. 1994, Hossain et al. 1996). Genes of these two enzymes are now being used for over- and underexpression studies in rice (Setter et al. 1997) by using the coding regions of Adh and Pdc genes through two types of promoters: constitute (CaMV35S and Act1) (McElroy et al. 1991) and inducible (6 XARE) (Last et al. 1991). Submergence tolerance being a complex character, it is likely that a putative single gene for submergence tolerance either (a) encodes a TF (trans-acting factor), that is, a protein or an RNA that

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binds to specific regulatory DNA sequences (Singer and Berg 1991) or (b) affects the signal transduction pathway, that is, the number of steps after the submergence treatment by which the plant activates the set of genes required for survival (Dolferus et al. 1994). Around a dozen major genes known to be induced under anaerobic conditions in maize (Freeling and Bennett 1985, BaileySerres et al. 1988) and rice (Ricard and Pradet 1989, Rivoral et al. 1989) possess a similar sequence in their promoters (Dennis et al. 1987), and therefore suggest the involvement of a common TF (Setter et al. 1997). Xu et al. (2006) reported a cluster of three genes at the Sub1 locus, encoding putative ethylene response factors. Two of these genes, Sub1B and Sub1C, are invariably present in the Sub1 region of all rice accessions analyzed. In contrast, the presence of Sub1A is variable. Further work identified two alleles within indica varieties carrying this gene: a tolerance-specific allele named Sub1A-1 and an intolerance-specific allele named Sub1A-2 (Xu et al. 2006). The phylogeny of the Sub1 genes of the domesticated and wild rice suggests that Sub1A arose from the duplication of Sub1B, and the variation in Sub1B alleles is correlated with the absence or presence of Sub1A (Fukao et al. 2009). Fukao et al. 2009 concluded that genetic variation at the Sub1 locus is due to gene duplication and divergence that occurred both prior to and after rice domestication.

32.3.4  Breeding The majority of loss caused by submergence is reported in the rice crop. Most O. sativa cultivars die within a week of complete submergence—a major constraint to rice production in south and southeast Asia that causes annual losses of over $1 billion and affects disproportionately the poorest farmers in the world (Xu et al. 2006). Rice farming in the intensively farmed irrigated areas has become increasingly vulnerable to flooding because of the popularity of semidwarf cultivars. Therefore, breeding for submergence tolerance has been an important objective for rice breeders, and it has been felt for long that there is also a need for irrigated rice cultivars that can survive a medium depth of water (75 cm) (Toojinda et al. 2003). For these reasons, a high priority has long been set for breeding submergence-tolerant rice in the tropics utilizing the inherent variability in tolerance known to be present in the available landraces (Mackill et al. 1993). Systemic screening at IRRI, Philippines, has resulted in the identification of flood-resistant rice cultivars, such as FR13A and FR43B from India, and Kurkaruppan and Goda Heenati from Sri Lanka (Mohanty and Khush 1985). Another submergence-tolerant elite line of IRRI, IR 49830 (-7-1-2-2), whose ancestry includes FR13A, IR42, and IR48 (Mackill et al. 1993), has been reported to have a 4–5 times higher yield than FR13A (Setter et al. 1997). Shuttle-breeding programmes between IIRI and national agricultural research institutes of several countries now provide promising advance lines as well as segregating populations for use in their target environments (Setter et al. 1997). However, the success achieved in submergence tolerance all over the world is far below our need and expectations. A probable reason for the absence of a significant introduction of improved submergence-tolerant rice cultivars during the last two decades has been the confusion between genuine submergence tolerance and shoot elongation (Maurya et al. 1988). Another reason for this fact might be the intolerance to other common stresses, like phosphorus and zinc deficiency and presubmergence drought (Neue et al. 1990). Around a decade, it was suggested (Setter et al. 1997) that although there is limited knowledge about the physiological and molecular bases of submergence stress, the association of submergence tolerance in traditional tolerant genotypes with poor agronomic traits suggests that molecular tools need to be used to enhance the submergence tolerance of rice. Since then, our knowledge has grown and the genes for submergence tolerance have been mapped in rice. There are cultivars, such as the O. sativa ssp. indica cultivar FR13A, that are highly tolerant and survive up to 2 weeks of complete submergence owing to a major QTL designated Submergence 1 (Sub1) near the centromere of chromosome 9 (Jackson and Ram 2003, Toojinda et al. 2003). The overexpression of Sub1A-1 in a submergence-intolerant O. sativa ssp. japonica conferred an enhanced tolerance to the plants, the downregulation of Sub1C, and the upregulation of

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Alcohol dehydrogenase 1 (Adh1), indicating that Sub1A-1 is a primary determinant of submergence tolerance (Xu et al. 2006). In addition, the FR13A Sub1 locus, the new variety, when introgressed into a widely grown Asian rice cultivar (Swarna) using MAS, displayed high yield and other agronomic properties of the recurrent parent and was tolerant to submergence (Xu et al. 2006).

32.4  Heat Tolerance Heat stress, especially during reproductive development, causes severe yield reductions in different crops. It is an important problem in tropical and subtropical environments and is believed to increase in future due to global warming (Schneider 1989, IPCC 2007, Battisti et al. 2009). Transitory or constantly high temperatures cause an array of morpho-anatomical, physiological, and biochemical changes in plants, which affect plant growth and development and may lead to a drastic reduction in the economic yield (Wahid et al. 2007). In dryland areas, heat stress may occur in association with radiation and drought stress (Loss and Siddique 1994). Plant responses to heat stress are diverse, but photosynthesis is considered the most heat-prone plant process (Bjorkman et al. 1980). Photosynthesis and respiration are more sensitive to heat stress in cool season species, such as wheat, than warm season plants (Bjorkman et al. 1980). The thermal stability of warm season species is associated with the properties of the photosynthetic system, including key enzymes and the thylakoid membrane, with the thylakoid membrane being more heat sensitive than the cell membrane (Bjorkman et al. 1980). Other reports (Bhullar and Jenner 1986, Rijven 1986) suggest that high temperature retards the conversion of sucrose to starch in developing grains (e.g., in wheat). Thus, any of a number of important metabolic functions may be disrupted due to heat stress, but a cell membrane system that remains functional during heat stress appears central to the adaptations of plants to high temperature (Raison et al. 1980). It has been suggested that the incorporation of terminal heat tolerance into high-yielding cultivars will have an energetic cost, and would require additional carbon assimilates and N inputs (Mitra and Bhatia 2008). Understanding the molecular and physiological bases of heat tolerance in higher plants has proved difficult owing to its complexity (Blum 1988). However, other nonconventional approaches, namely, genetic engineering, are being searched to enhance the heat tolerance in plants. Hightemperature tolerance has been genetically engineered in plants mainly by overexpressing the heat shock protein (HSP) genes or indirectly by altering the levels of heat shock TF proteins (Singh and Grover 2008). Apart from HSPs, thermotolerance has also been altered by elevating levels of osmolytes, increasing levels of cell detoxification enzymes, and altering membrane fluidity (Singh and Grover 2008).

32.4.1  Genetic Basis Knowledge regarding traits conferring high-temperature tolerance and their genetics is essential for the creation of genotypes capable of giving high yields under high-temperature environments all over the world (Reynolds et al. 2007). However, no single trait can be said to be directly responsible for heat stress tolerance, though several traits are found to be associated with this mechanism. Therefore, high-temperature tolerance is characterized by measuring whole plant productivity traits (e.g., yield traits) or by utilizing bioassays in different crops (Porter et al. 1995). The traits utilized in such studies are flower bud abortion and reduction in pod fill in common bean (Shonnard and Gepts 1994); electrolyte leakage (Chen et al. 1982, Shanahan 1990), membrane thermostability (Saadalla et al. 1990), reduction of 235 triphenyl tetrazolium chloride (Towill and Mazur 1975, Porter et al. 1994), and chlorophyll fluorescence in wheat (Moffat et al. 1990); electrolyte leakage in soybean (Sullivan and Ross 1979) and common bean (Marsh et al. 1985); HSPs in sorghum (Jorgensen et al. 1993), cotton (Fender and O’Connell 1989.), wheat (Vierling and Nguyen 1990), maize (Frova et al. 1980, Jorgensen et al. 1992), and pigeon pea (Devi et al. 1994); etc. Most of these traits are the measure of

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the effect of the heat stress rather than the cause of heat stress tolerance. Therefore, genetics of traits causing heat stress tolerance and the heat tolerance itself are difficult to be separated. Hence, all traits are discussed below for understanding the genetics of heat stress tolerance. A substantial genotypic variation for heat tolerance was found in groundnut, soybean, pigeon pea, and chickpea, and they were ranked from heat tolerant to heat sensitive in the same order (Srinivasan et al. 1996). Quantitative inheritance with a large environmental effect was reported for heat tolerance at pod and seed set in snap bean (Dickson and Petzoldt 1989). In another study, a single dominant gene in one snap bean accession and two genes with an epistatic effect in another were reported (Bouwkamp and Summers 1982). In a study involving two resistant and two susceptible genotypes of common bean (Phaseolus vulgaris) crossed in all possible combinations including reciprocals, quantitative inheritance was noted for heat tolerance (Shonnard and Gepts 1994). Additive effects were significant for two heat tolerance traits (flower bud formation and pod filling) in common bean. Cytoplasmic effects including the interaction of cytoplasmic and nuclear genes were also recorded (Shonnard and Gepts 1994). In cowpea, tolerance to inhibition of flower bud development under high temperature and for a long day was due to a recessive gene (Hall 1992), while tolerance during pollen formation was under the control of a single dominant gene (Marfo and Hall 1992). In tomato (Lycopersicon esculentum), it was reported that heat tolerance during fruit set was conferred by a few partially dominant genes, but narrow sense heritability was very low (8%) due to large environmental effects (Shelby et al. 1978). Genetic effects on membrane thermostability in wheat in 90 F2 derived lines of heat-tolerant and susceptible lines showed that heat tolerance is not simply inherited (Saadalla et al. 1990). Genetic differences in membrane thermostability were noted in soybean (Glycine max L. Merr.) (Wallner et al. 1982). Marsh et al. (1985) examined inheritance of membrane stability in common bean and found that heat tolerance was under the control of few genes. They also found low additive effects along with epistasis. In a diallel including a reciprocal of six wheat genotypes, significant general combining ability effects and maternal effects were noted (Moffat et al. 1990). On the basis of the relation of heat stress effect and the reduction of 235 triphenyl tetrazolium chloride, which produces a red formazen, significant differences in acquired high-temperature tolerance were reported in wheat (Porter et al. 1994). Using 20 F1 progenies, produced through a complete 5 × 5 diallel mating design of tolerant and susceptible genotypes, showed that only the general combining ability effect was highly significant accounting for 67% of the total genetic variation (Porter et al. 1995). There is extensive evidence of both qualitative and quantitative intraspecific genetic variability for low-molecular-weight (LMW) HSPs in crops, for example, sorghum (Jorgensen et al. 1993), cotton (Fender and Connell 1989), wheat (Towill and Mazur 1975, Vierling and Nguyen 1990), and maize (Jorgensen et al. 1992). Very few reports are available regarding HSP gene transmission in plants. Additive inheritance for some HSPs has been reported in barley, where the presence of hybrid-specific HSPs indicated the activation of genes that were suppressed in one parent (Marmiroli et al. 1989). Both additive and nonadditive inheritance was demonstrated in the F1 hybrids of maize (Frova et al. 1988). Intraspecific qualitative polymorphism in LMW synthesis is extremely rare (Ottaviano et al. 1991), and quantitative variation in HSP synthesis may determine relative thermal tolerance levels (Frova and Gorla 1993). A genetic analysis of HSPs in maize, HSP synthesis, revealed both qualitative and quantitative polymorphism implicative of differences in HSP structural genes and regulatory factors (Jorgensen and Rosenow 1995). The F1 hybrid HSP profile indicated that the synthesis of all parental HSPs conformed to dominant inheritance patterns, including complete dominance, overdominance, and codominance; there was evidence for unlinked loci of four different HSP gene pairs, but data for three other HSP gene pairs were inconclusive (Jorgensen and Rosenow 1995).

32.4.2  Gene Expression Heat stress is known to induce a set of proteins called heat shock proteins (HSPs) (Key et al. 1981). HSPs are known to be associated with acquired thermal tolerance in many species, including bacteria (Sanchez and Lindquist 1990); mammalian fibroblasts (Riabowol et al. 1988); and higher plants

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such as soybean (Lin et al. 1984, Kimpel and Key 1985a), wheat (Krishnan et al. 1989), cotton (Burke et al. 1985), and maize (Jorgensen et al. 1992). In plants, a heat shock of 8°C–10°C above the normal growing temperature induces the synthesis of both high (65–110 kDa) and low (15–27 kDa) molecular HSPs, with the LMW proteins being the most prevalent (Kimpel and Key 1985b, Mansfield and Key 1987). A subset of the LMW group, 15–18 kDa, is unique to higher plants (Sachs and Ho 1986). The LMW HSPs are a complex group, with as many as 30 members present in soybean (Nagao et al. 1985). The induction of LMW HSPs has been well documented (McElwain and Spiker 1992) and their number is known in some monocot species (Mansfield and Key 1987). The detection of low- and high-molecular-weight HSPs synthesized in seedlings and flag leaves in flowering plants suggest that HSPs are synthesized before leaf temperatures reach levels that are considered injurious to growth and development (Hendershot et al. 1992). HSPs are among the fastest known gene expressions in plants (Ougham 1987). Heat shock response may be the accumulation of damaged proteins (Munro and Pelham 1985). This is supported by the fact that the small protein ubiquitin, which has a role in the ATP-dependent breakdown of abnormal proteins (Hershko and Ciechanover 1982), is itself an HSP (Bond and Schlesinger 1985). Limited information is available about structural relationships among the HSP genes and the molecular regulation of their transcription and translation to protein (Frova et al. 1988, Leonardi et al. 1988, Jorgensen and Rosenow 1995). On the basis of nucleotide and amino acid similarities and protein localization, there are four families of structural LMW HSP genes known in plants (Vierling 1991); one each encoding plastid localization and endomembrane proteins, and two that encode cytopalsmic proteins (classes I and II). The HSPs of 17–18 kDa comprise classes I and II. There are several class II gene sequences (Dietrich et al. 1991, Goping et al. 1991) as well as class I cDNA HSPs (Jorgensen and Nguyen 1994).

32.4.3  Breeding Increasing productivity under heat stress conditions requires the development of heat-tolerant genotypes in all crops. The improvement of heat stress tolerance can contribute to sustainability and provides a way to extend the area under cultivation (Srinivasan et al. 1996). Limited progress has been made with regard to breeding heat-tolerant genotypes, probably because yield losses due to heat are more subtle than biotic stresses (Summerfield et al. 1984). The two most important hurdles are the absence of substantial information on the genetic diversity for heat tolerance traits and effective screening techniques (Wery et al. 1994). Both in vivo and in vitro methods are used for screening, but in vitro methods are advantageous in that they are plant conserving, and this feature is important in plant breeding for heat tolerance (Srinivasan et al. 1996). The genetic control of heat tolerance is poorly understood (Ortiz et al. 2008a,b). However, the level of tolerance to high temperature varies among genotypes (Blum 1986, Shipler and Blum 1986, Moffat et al. 1990, Pfeiffer et al. 2005), suggesting that the trait can be improved (Moffat et al. 1990). The indication of a larger additive genetic variation with regard to some traits believed to be associated with heat stress also indicates that gains from selection for improved heat tolerance should be possible. Resistance to high temperature involves complex tolerance and avoidance mechanisms. However, the cell membrane is believed to be a site of primary physiological injury by heat (Blum 1988). The damage to a membrane can be measured by estimating solute leakage from the cell. Since membrane thermostability is reasonably heritable (Fokar et al. 1998) and shows a high genetic correlation with the yield, it has potential application in breeding, but does require a laboratory methodology to make measurements (Reynolds et al. 2001a,b). Breeding for heat tolerance in cowpea has involved a pedigree breeding approach with selection beginning in the F2 generation (Hall 1990) to incorporate major recessive genes conferring heat tolerance during early floral bud and seed coat development (Patel and Hall 1988). It has been

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suggested that to overcome difficulties caused by environmentally induced variation, incorporating heat tolerance pod set into other genetic backgrounds will require family selection in advanced generations to ensure that trait is fixed (Marfo and Hall 1994). Recurrent selection has been suggested to accumulate genes favoring high-temperature tolerance based on chlorophyll fluorescence measurements in wheat (Moffat et al. 1990). In many countries and environments, late planting can expose the crop and breeding nurseries to high temperatures from flowering onward, giving wheat breeders the opportunity to select lines with high levels of heat tolerance (Ortiz et al. 2008a,b). At CIMMYT, lines are selected during the segregating phase for adaptation to heat by planting late, and a gravity table is used to separate bulk populations into those that can maintain seed weight under high temperature; the derived lines are then tested under heat stress in yield trials (Ortiz et al. 2008a,b). Physiological tools, such as the infrared thermometer that measures canopy temperature depression (CTD), are also available to assist the plant breeder in discriminating among progenies (Reynolds et al. 1998). Heat avoidance or early maturity is an extremely important trait to circumvent the effects of high temperature at grain filling (Ortiz et al. 2008a,b). All currently grown popular cultivars in the eastern Gangetic Plains are earlier-maturing than cultivars popular in the northwestern Gangetic Plains. A simultaneous improvement of heat tolerance and yield potential of earlier-maturing germplasm is the best option to increase production in heat stress environments, and is being practiced (Joshi et al. 2007a–e). One of the most popular wheat varieties of India, HUW234, which currently occupies more than three million hectares of area, possesses unique features of both avoidance and tolerance of heat stress of the northeastern plains zone of the country. Its early maturity, profuse tillering, high grain number per spike, and fast ripening provides it a clear superiority over other varieties under late- to verylate-sown conditions when hot winds of early summer cause serious yield losses.

32.5  Cold Tolerance Low temperature, especially in the northern region of the temperate climatic zone, presents substantial obstacles to the survival of plants throughout the winter (Andrews 1997) and is one of the most severe stresses that limits crop growth and productivity (Boyer 1982). Reduction in grain yield is incurred not only as a direct result of winter damage but also as a result of limiting the areas where such crops can be sown (Steponkus 1978). Although low-temperature (LT) stresses are usually of two types, chilling above 0° temperature and freezing at subzero temperature (Welin et al. 1996), the winter may expose young seedlings to many kinds of stresses (as in wheat), such as direct frost effect, cold winds, snow cover, intense freezing and glaciation of the soil, and frost lifting in spring. In grasses and wheat, there are two different mechanisms of tolerance to ice encasement, that is, rapid (wheat) and slower (grasses) glycolysis (Andrews 1997). Following cold acclimation, a number of forage species are highly tolerant to extreme cold conditions of ice encasement (Gudleifsson et al. 1986), even greater than winter wheat (Andrews 1997). In grasses, berrings hairgrass has recently been shown to have an extremely high tolerance to anoxia (Crawford and Braendle 1996), a property that is common to many arctic plants (Andrews 1997). The physiological and biological processes that lead to cold tolerance or the adaptation of plants to low temperature are extremely complex (Paldi et al. 1996a).

32.5.1  Morphophysiological Traits: Genetic Basis Like other abiotic stresses, tolerance to low temperature is also due to the joint action of several traits of plants. Significant correlations were established between cold hardiness and day to heading (Fowler and Carles 1979), and the growth habit (Hayes and Aamodt 1927, Quisenberry 1931, BruleBabel and Fowler 1988) in wheat. In general, spring wheat lines are less hardy than winter lines and spring growth habit is dominant over winter growth habit. Winter growth habit of wheat is possibly inherited by a Vrn (vernalization requirement) gene (Brule-Babel and Fowler 1988). Chahalan

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and Law (1979) found no evidence of genetic linkage between cold hardiness and vernalization requirement in wheat even though chromosomes in the homologus group 5 were implicated in the control of both of these characters. Studies in grass species (e.g., clover) also do not show correlation between a single trait and cold tolerance (Smith 1949, Ronningen 1953, Annicchiarico and Piano 1995). Photoperiodism, an important trait for adaptability in cold climate, is governed by genes present in the group 2 chromosome of wheat (Welsh et al. 1973). At least three genes Ppd1, Ppd2, and Ppd3 governing photoperiodism are known in chromosomes 2D, 2B, and 2A, respectively (Worland et al. 1987). Low temperature has been found to enhance the anthocynanin synthesis in plants such as sorghum, cabbage, maize, Arabidopsis, apple, roses, and petunia (Shvarts et al. 1997).

32.5.2  Genetics Winter hardiness is a genetically programmed integrated process (Weiser 1970, Sutka and Veisz 1988). The two major components of freezing stress resistance are freezing tolerance in the nonacclimated state (normal growing condition) and the capacity to cold-acclimate (increase in freezing tolerance) upon exposure to chilling temperatures (Palta and Simon 1993). Genetics of winter hardiness was attempted as early as 1912 when Nilsson Ehle (Nilsson-Ehle 1912) of Sweden investigated winter hardiness in wheat, and on the basis of the appearance of transgressive segregants in a cross of two cultivars intermediate in winter hardiness, he reported that it is a quantitative trait under the control of a polygenic system (Nilsson-Ehle 1912). Since then, no general opinion has arrived on this issue. It has been reported to be recessive (Rosenquist 1933), intermediate (Lyfenko 1979, Erickson 1980), dominant (Rosenquist 1933), or overdominant (Rosenquist 1933, Kir’yan and Barashkova 1981). It has been reported that winter hardiness is under the control of dominant genes in mild cold, while under severe cold it is governed by recessive genes (Quisenberry 1931, Gullord et al. 1975, Sutka 1981). Winter hardiness behaved as a recessive factor as early as 1923 in a cross made by Schafer (1923). A majority of studies indicate a polygenic control of cold tolerance (Hayes and Aamodt 1927, Worzella 1935, Parodi et al. 1983, Limin and Fowler 1988, Norell et al. 1986). The quantitative nature of winter hardiness is also supported by the absence of a drastic improvement in the winter hardiness of different crops (Quamme et al. 1972), the appearance of transgressive segregants (Smith 1949), and a complex of factors influencing winter hardiness (Levitt 1972). However, all the genes do not work together and different genes affect tolerance at different levels of stress (Gullord et al. 1975). This is evidenced by studies showing that winter hardiness genes may act as dominant or recessive depending on the type of environment (Quisenberry 1931, Worzella 1935, Muehlbauer et al. 1970, Sutka and Veisz 1988). In wheat, 11 chromosomes carry genes for cold tolerance with chromosome number 5 being the most important (Thomashow 1990). Some studies have implicated 15 of 21 chromosome pairs of wheat to be associated with cold tolerance (Sutka 1981) with chromosome 5A (Cahalan and Law 1979, Sutka and Kovacs 1985), 7A, 4D, and 5D (Law and Jenkins 1970, Sutka 1981) most commonly mentioned. In barley, a major QTL was found associated with the 7th chromosome (Hayes et al. 1993, Pan et al. 1994). In Solanum species, the nonacclimated freezing tolerance and acclimation capacity were found to be separate heritable traits controlled by few genes (Stone et al. 1993). In rye (Secale cereale), cold hardiness is controlled by genes mainly with additive effects (Shanahan et al. 1990). The genetics of frost tolerance studied in winter wheat by using a complete diallel (Gullord et al. 1975, Briggle, Sutka 1994) showed that frost tolerance is controlled by an additive dominance system (Puchkov and Zhirov 1978, Sutka 1981, 1984). Several studies (Sutka 1984, Sutka and Veisz 1988, Sutka 1989, Sutka 1994) have shown that frost tolerance is a complex character controlled by at least 10 of the 21 pairs of chromosomes (Sutka and Veisz 1988); chromosomes 5A and 5D have been implicated most frequently and they appear to carry major genes (Sutka 1994). The gene for frost resistance (Fr1) was located on the long arm of chromosome 5A (Roberts 1990, Sutka 1994), and there might be close genetic linkage between Vrn1 and Fr1

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(Roberts 1990). Studies done so far indicate the presence of four major genes for vernalization requirement: Vrn1, Vrn2, Vrn3, (Pugsley 1972, 1973), and Vrn4 (Pugsley 1973). Another gene Vrn5 was also reported (Law 1966). Cold tolerance is under the control of both additive and nonadditive gene effects in chickpea (Malhotra and Singh 1990) and pea (Markarian and Anderson 1966, Auld et al. 1983). Genetic interactions also play an important role in cold tolerance (Malhotra and Singh 1990). Three additive genes or linkage groups are reported to control winter hardiness in pea (Liesenfeld et al. 1986). The expression of LT tolerance has been found to be under the control of same genetic factors in the sporophyte and gametophyte of potato (Zamir et al. 1981, 1982, Zamir and Vallejos 1983). Highheritability estimates for cold hardiness have been reported in wheat (Sutka 1981, Sutka 1984), barley (Rohde and Pulham 1960, Eunus et al. 1962), and oats (Muehlbauer et al. 1970). One of the important conclusions from a large number of studies was that cold acclimation includes the expression of certain cold-induced genes that function to stabilize membranes against freeze-induced injury (Thomashow 1999). In addition, a family of Arabidopsis TFs, the CBF/DREB1 proteins, was identified that controls the expression of a cold-induced gene that increases plant freezing tolerance (Thomashow 1999). In potato, Stone et al. (1993) demonstrated that freezing tolerance and the ability to cold-acclimate are under independent genetic control. This was later confirmed in other plant species (Teutonico et al. 1995, Arora et al. 1998). These results have important implications for the improvement of cold tolerance of cultivated potatoes. For a successful improvement of frost hardiness, both components must be transferred to the cultivated potatoes (Palta and Simon 1993).

32.5.3  Gene Expression The molecular mechanism that regulates cold tolerance is not sufficiently well known (Crawford and Braendle 1996). Weiser, in 1970 (Weiser 1970), suggested that cold acclimation might involve changes in the gene expression. Since then, however, more and more information has been reported in this field (Guy et al. 1985, Houlde et al. 1972, Paldi et al. 1996b). The realization that cold acclimation requires an altered expression of tolerance-related genes not seen under nonacclimating conditions is the basis for the isolation and characterization of cold-induced genes (Paldi et al. 1996b). Several studies have demonstrated that plants synthesize a new set of proteins when exposed to a cooler environment (Guy 1990). The existence of partially different mRNA populations in nonacclimated and acclimated plants has allowed the isolation of cDNAs corresponding to acclimationspecific mRNAs by differential screening as in alfalfa (Mohapatra et al. 1987, Mohapatra et al. 1989), Arabidopsis (Kurkela and Frank 1990), and barley (Cattivelli and Bartels 1990, Berkel et al. 1994, Welin et al. 1996). The temporal pattern of LT-induced gene activation varies between different plant species ranging from few hours (A. thaliana) (Hajela et al. 1990, Nordin et al. 1991, Yamaguchi-Shinozaki and Shinozaki 1993, Welin et al. 1994) to several days (Mohapatra et al. 1987, Monroy et al. 1993, Wolfraim et al. 1993). LT-responsive genes are transcriptionally regulated through sequence-specific TFs that bind to their target sequence on the corresponding promoters (Welin et al. 1996). LT-induced genes contain certain sequence elements that resemble ABA-responsive (Palva et al. 1994) and drought-responsive elements (120). Homologous regions are also present in the promoters of LT-induced genes: cor15a (Baker et al. 1994), rab18, kin1, kin2 (Palva et al. 1994), lti29, and cor47 (Welin et al. 1996). The identification of an mRNA group that only functions during a cold effect and codes proteins found only in frost-resistant wheat varieties (Perras and Sarhan 1989) suggests a positive correlation between the quantity of proteins synthesized during the cold effect and the frost resistance of the varieties (Paldi et al. 1996b). Some mRNAs decline during exposure to low temperature, as in Brassica (Anderson et al. 1994), rice (Hahn and Walbot 1989), and spinach (Guy 1990).

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The cold-induced rRNA synthesis in wheat takes place in seedlings as a result of low temperature during the first few days of cold treatment (Paldy and Devay 1977, Devay and Paldy 1990), and the cold-induced rRNA synthesis is closely correlated to the rRNA cistron number (Devay and Paldy 1977). Quantitative and qualitative changes have been noted in the rRNA maturation processes due to low temperature in the weak frost resistant line of wheat, as a consequence of which there is an increase in the last precursors (1.4 and 0.9 MD) of the two stable cytoplasmic rRNAs (Paldi et al. 1996b). Freezing tolerance includes tolerance to freeze-induced dehydration (Guy 1990, Levitt 1980). This is further substantiated by the fact that several of the LT-induced proteins are similar to proteins induced in response to water stress (dehydrins) (Baker et al. 1988, Mundy and Chua 1988). Certain proteins having the putative protein-stabilizing function (Bip, Hsp70, and Hsp90) have been identified among LT-induced proteins (Anderson et al. 1994, Kishor et al. 1995). Cryoprotective polypeptides capable of protecting the plant thylakoid membrane in vitro against a freeze-thaw cycle have also been reported in cabbage and spinach (Hincha et al. 1989, 1990). The structural similarity of the gene product of kin1 from A. thaliana with an antifreeze protein of swinter flounder (Kurkela and Frank 1990) led to the speculation of the presence of the antifreeze protein, to be contradicted by Thomashow (1993). However, there are reports of the presence of an antifreeze protein in winter rye (Griffith et al. 1992, Urrutia et al. 1992). In A. thaliana leaves, pigment accumulation in response to low temperature results from the activation of phenylalanine ammonia-lyase (pal) and chalcone synthase (chs) gene transcription in a light-dependent manner (Leyva et al. 1995). It was suggested that light dependency is a general feature of the cold-induced gene expression. However, in Petunis corolas, cold activation of the chs expression was not light dependent (Shvarts et al. 1997). The effect on the chs expression was not always correlated with that on the anthocyanin content, suggesting a posttranslational effect (Shvarts et al. 1997). Earlier, Christie et al. (1994) suggested that the effect of temperature is associated with transcription, transcript stability, translation, and enzyme activity. Low temperatures do not simply create conditions that facilitate the developmental activation of the chs expression; they act as a separate inducing signal (Christie et al. 1994, Shvarts et al. 1997). The transduction of an LT signal (2°C–5°C) for the activation of a cold-acclimation-specific (cas) gene has also been studied, which probably does not belong to the chs group (Shvarts et al. 1997). The transcripts of enzymes of the fermentation pathway, ADH and PDC, have been found to increase as a result of the hypoxic acclimation in wheat (Waters et al. 1991) and maize (Andrews et al. 1994). However, more tolerant forage grasses, timothy (Phleum pratense) and berrings hairgrass (Deschampsia berengensis), show lesser activity of ADH and PDC (Andrews 1997). Desiccation often accompanies cold acclimation and freezing stress (Shvarts et al. 1997); therefore, at the molecular level, genes induced by water stress and ABA are also induced by cold stress in barley, rice, and spinach (Hahn and Walbot 1989). In contrast, genes induced by cold temperature can respond to water stress and ABA (Sutka 1981, Frank 1990, Berkel et al. 1994). The homology between HSPs and cold-induced proteins has been reported in potato (Berkel et al. 1994).

32.5.4  Breeding Conventional breeding has been utilized by breeders all over the world to enhance cold tolerance in different crops. Handling a complex trait, such as winter hardiness, in a breeding program is a difficult task, due to the large number of genes involved and the numerous interactions with the environment (Sãulescu and Braun 2001). But the main difficulty in breeding a cold-tolerant line in crops such as wheat is that high freezing tolerance is generally associated with lower yields and, later, maturity (Sãulescu and Braun 2001). Since many traits are associated with the freezing tolerance and every additional breeding objective is expected to slow down the genetic progress for all other traits of interest, the breeding objective should not be to maximize winter hardiness, but to develop cultivars with the minimum winter hardiness necessary for a given target area (Sãulescu

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and Braun 2001). This is supported by the fact that, in general, the most successful winter wheat cultivars have only marginally greater winter hardiness than the minimum required for the area in which they are grown (Fowler et al. 1981). Sutka (1994) suggested three ways to improve genetic variation for frost tolerance in wheat: interspecific crossing, chromosome manipulation, and the induction of somaclonal variation. Wild species related to cultivated wheat, for instance, Ae. cylindrica, Agropyron glaucum (intermedium), and Agropyron elongatum, are an extremely promising source of increased genetic variation (Sutka 1994) for cold tolerance. Disomic additions of A. glaucum were able to survive freezing to a temperature as low as −18°C. A somaclone of wheat was significantly better than control for cold tolerance, and thus, of practical importance (Hincha et al. 1989). It has been suggested that in chickpea, selection would be more effective if dominance and epistatic effects were reduced after a few generations of selfing (Malhotra and Singh 1990). The in vitro selection through the anther culture has been suggested as a useful tool for breeding LT tolerance in crops (Zamir et al. 1981, 1982, Zamir and Vallejos 1983) on the ground in that there is genetic overlap between the sporophyte and the gametophyte (Mulcahy 1979). Gametophytic selection for LT tolerance has been successfully demonstrated in tomato (Zamir and Gadish 1987), maize (Kovacs and Barnabas 1992, Krisjansdottir 1993, Lyakh and Soroka 1993), and potato (Lynch and Steponkus 1987). Despite a great progress in understanding the molecular basis for plant cold acclimation, the complexity of the system hampers the genetic engineering of plants having freezing tolerance (Welin et al. 1996). Among the possible approaches to enhance cold tolerance in plants, the ways that hold promise are (Welin et al. 1996) (1) increasing the freezing resistance of plant plasma membrane by increasing the amount of phospolipid (Lynch and Steponkus 1987, Palta and Weiss 1993), as showed successfully in tobacco (Murata et al. 1992, Kodama et al. 1994); (2) metabolic alterations alleviating the detrimental effects of desiccation stress, for example, OA through osmoprotectants (Welin et al. 1996); (3) exploiting cryoprotective and antifreeze proteins, for example, fish antifreeze proteins (Hightower et al. 1991); and (4) manipulation of signal pathways leading to the expression of tolerance genes. For several years, it has been felt that limited success has been achieved using traditional plant-breeding methods to improve the freezing stress resistance in crops (Marshall 1982, Palta and Simon 1993, Vega et al. 2003). Even crops that have undergone extensive breeding, such as winter wheat, have not had significant improvements, and cultivars that were released more than 50 years ago remain among the most cold-hardy today (Limin and Fowler 1991). Recent progress in plant breeding has focused on the use of molecular marker techniques to facilitate cloning and efficient introgression of favorable genes through marker-assisted selection (Lande and Thompson 1990, Dudley 1993). Genomic regions with a significant effect on the freezing tolerance have been detected in several crops: Citrus (Cai et al. 1994), Brassica (Teutonico et al. 1995, Kole et al. 2002), Triticum (wheat) (Sutka 1994), Eucaliptus nitens (Byrne et al. 1997), Lycopersicon (tomato) (Foolad et al. 1998), Vaccinium (blueberry) (Rowland et al. 1999), and O. sativa (rice) (Saito et al. 2001).

32.6  Salinity Resistance Soil salinity is a major agricultural problem, particularly in irrigated agriculture. Around 10% of the world’s arable land is affected by salinity (Tanji 1990, Shanon 1997), while it is a serious problem in around 20% of irrigated agricultural land (Flowers and Yeo 1995). Another report (http://www. plantstress.com/Articles/index.asp- October 22, 2007) suggests that more than 6% of the world’s land (19.5% of irrigated land plus 2.1% of dryland agriculture) is now affected by salinity of various magnitudes (Kader and Lindberg 2008). One-third of the land in Australia is salt affected (Northcote and Skene 1972), while in India and Pakistan, such areas constitute around 5% of their total cultivable land. In irrigated areas, the percentage of salt-affected land is much higher. In the

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United States, 23% of irrigated land is under salt stress (Shanon 1997). With as much as half of the world’s existing irrigation systems under the influence of secondary salinization, alkalization, and water logging (Szabolcs 1987), the coexistence of irrigation and salinization threatens current agricultural productivity (Flowers and Yeo 1995). Despite such importance of salt-affected areas in world agriculture, too little progress has been made in improving the salt tolerance of crops (Flowers and Yeo 1995, Shanon 1997). The problem is expected to increase in the coming future, and an integrated approach seems to be the only answer. Genetic engineering is also being seen as a potent tool to handle this problem through the overexpression of Na+/H+ antiporters in plants (Zhang et al. 2004).

32.6.1  Morphophysiological Traits: Genetic Basis Traits associated with salt tolerance have been investigated by several workers (Yeo and Flowers 1989, Noble and Shannon 1990, Tanji 1990, Yeo et al. 1990, Foodlad 1997, Shanon 1997), but exact traits are still to be identified (Tanji 1990). The differential response of plants to salt stress at different growth stages has added further problems in this direction. In wheat and sorghum, salt tolerance is associated with seed size, with larger seed having greater tolerance (Amthor 1983, Grieve and Francois 1992). Seedling survival in saline solution is also an indicator of salt tolerance of crops, and has been studied in Medicago (Allen et al. 1985, Al-Khatib et al. 1993), forage crops (Ashraf et al. 1987), and potato (Jefferies 1994). The results showed that characters underlying short-term tolerance may contribute to long-term tolerance, but did not themselves confer long-term tolerance (Jefferies 1996). Two broad physiological mechanisms by which plants respond to salt stress (Foodlad 1997) are (1) the inclusion and use of inorganic ions as osmotica to maintain a favorable water balance (halophytic response), and (2) a partial exclusion of ions and the synthesis of organic solutes for OA (glycolytic response) (Greenway and Munns 1980, Curatero 1992). Salt tolerance is associated with an increased capacity of ion regulation through compartmentation and transport of toxic ions, OA, and maintenance of membrane integrity (Yeo 1983). Most of the crops respond to salt stress by excluding ions from the shoot, and genetic variation exists for the threshold level at which the exclusion mechanism fails (Foodlad 1997). Physiological and genetic factors that contribute to the growth of glycophytes at a very high salt concentration are related to the survival more than the yield potential, and hence, are of little interest to growers except those engaged in subsistence agriculture (Shannon and Noble 1990). Low sodium transport has been suggested as an important heritable trait for salt tolerance in rice (Yeo 1992, Garcia et al. 1995). Cl− exclusion was responsible for the genetic difference for salt tolerance in soybean (Abel and Mackenzie 1963), and was found a heritable character in white clover (Trifolium repens) and lucern (Medicago sativa) (Noble and Shannon 1990). Under salt stress, the tolerant tomato genotype accumulated significantly less Na+ and Cl− and more Ca2+ than the leaves of the sensitive genotype (Foodlad 1997). A generation mean analysis indicated that under salt stress, both the absolute and relative growth and the Na+ and Ca2+ accumulation in the leaf were genetically controlled with additivity being the major genetic component (Foodlad 1997); a moderate estimate of narrow sense heritability (0.49 + 0.09) was obtained for the shoot dry weight under salt stress treatment (Foolad 1996). K+ selectivity was identified to be a principal adaptive mechanism to salt stress in legumes and cereals (Lauchli 1984, Dvorak and Gorham 1992). Under saline conditions, the ability to keep a low cytosolic Na+-concentration appears to be an important trait of salt-tolerant plants (Flowers and Hajibagheri 2001, Maathuis and Sanders 2001, Carden et al. 2003, Golldack et al. 2003, Kader and Lindberg 2005, Anil et al. 2007). Plant cells can maintain a low cytosolic Na+-concentration, either by restricting Na+ influx into the cell, or by extruding cytosolic Na+ into the apoplast or vacuole, or by both (Kader and Lindberg 2008). Another report (Anil et al. 2007) suggests lesser permeability of the plasma membrane to Na+ in the salt-tolerant rice cv. Pokkali compared to that in the salt-sensitive rice cv. Jaya.

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32.6.2  Genetics The degree to which different plants can tolerate high concentrations of salt in their rooting medium is under genetic control (Epistein and Jefferies 1964, Allen et al. 1985, McNeilly 1990, Shanon 1990). Genetics of salt tolerance has been investigated in several crops, and the results so far indicate monogenic to polygenic control. Salt tolerance was recorded as a heritable trait in Agropyron intermedium (Hunt 1965) and barley (Norlyn 1980). In sorghum, genetic variation for OA was studied in 10 inbred lines and variation was noted due to more than a single gene, and both general and specific combining ability effects were found significant (Basnayake et al. 1994). The greater tolerance of wild sunflower Helianthus paradoxus is due to a single dominant gene Sal, but a modifier may also be present (Miller 1995). Salt tolerance in wheat grass showed that tolerance behaves in an additive fashion (Weimberg and Shanon 1988). In tomato, a stage-specific polygenic control was suggested to control salt tolerance (Shanon 1982, Jones and Qualset 1984, Jones 1987). In another study, a generation mean analysis showed that the additive gene action was the predominant component for salt tolerance in tomato; the narrow sense heritability was estimated to be moderately high (Foolad and Jones 1991). Six marker loci in tomato have shown association with QTLs involved in yield under salinity (Breto et al. 1994). The Gpert (“Golden Promise” erectoides) mutation, produced by gamma-ray irradiation in the barley variety Maythorpe in the late 1950s, which is allelic to ari-e mutants (short awned, breviaristatum), has a significant effect on salt tolerance (Froster et al. 1994). Gpert performs similar to other ari-e mutants (ari-e.1, ari-e.119, ari-e.156, and ari-e.228) and possesses a relatively low shoot Na+ content and a higher salt tolerance index (Pakniyat et al. 1997a) in comparison to nonmutants. These mutants show greater tolerance than denso (sdw) or ert-k32 dwarfing mutants (Froster et al. 1994, Pakniyat et al. 1997a). The Gpert mutation is modified by a genetic background (Pakniyat et al. 1997b) and is also associated with drought tolerance characters. In saline environments, bread wheat, T. aestivum (genomes AABBDD), accumulates less Na+ and more K+ on expanding and young leaves than durum wheat, T. turgidum (genomes AABB) (Dubcovsky et al. 1996). Chromosome 4D accounts for 50%–60% of the difference between bread wheat and durum wheat for this trait (Gorham et al. 1987, Dvorak and Gorham 1992). Dvorak and Gorham (1992) recombined chromosome 4D with durum wheat chromosome 4B by using the ph1c mutant of durum wheat and found that K+/Na+ discrimination is controlled by a single locus on the long arm of chromosome 4D, which was designated Knal. The Knal locus was mapped on a short region in the 4DL arm and was completely linked to Xwg199, Xabc305, Xbcd402, Xpsr567, and Xpsr375 (Dubcovsky et al. 1996). The 5J chromosome of Agropyron junceum carries a major dominant gene(s) conferring tolerance to salt (Forster et al. 1988). Stølen and Andersen (1978) found that tolerance to high soil acidity is controlled by a single dominant gene, designated Pht, on chromosome 4. Subsequently, genes with positive effects for salt tolerance were located to chromosomes 4H and 5H of Hordeum vulgare and 1Hch, 4Hch, and 5Hch of Hordeum chilense (Forster et al. 1990). In a study, Minella and Sorrells (1997) reported that the aluminum tolerance gene (Alp) is distally located from the centromere on chromosome 4, suggesting that tolerance to low pH (Pht) and aluminum tolerance are controlled by the same locus. Salt tolerance at the cellular level involves several hundreds of stress-responsive genes for ionic homeostasis as well as osmotic homeostasis (Bartels and Sunkar 2005, Chen and Zhu 2005, Sreenivasulu et al. 2007). Recently, Huang et al. (2009) cloned and characterized DST (drought and salt tolerance) in rice crop. This was a previously unknown zinc finger TF that negatively regulates stomatal closure by a direct modulation of genes related to H2O2 homeostasis, and identifies a novel pathway for the signal transduction of DST-mediated H2O2-induced stomatal closure (Huang et al. 2009). Loss of the DST function increases stomatal closure and reduces stomatal density, consequently resulting in enhanced DST in rice (Huang et al. 2009). This provides a new opening to understand the mechanism of stomata-regulated abiotic stress tolerance, and therefore, could lead to an important genetic engineering approach for improving salt tolerance.

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32.6.3  Gene Expression The adaptation of plants to a saline environment must be due to some salt-related changes in the pattern of gene(s) expression (Foodlad 1997). More than 100 genes were estimated to be expressed when subjected to salt stress (Meyer et al. 1990). There are several reports of alterations in protein accumulation due to salinity (Meyer et al. 1990, Yeo et al. 1990, Jain et al. 1993). One of the most characterized genes associated with salt tolerance is the gene encoding a 26 kDa protein, called osmotin, which is responsive to several environmental and hormonal signals, including osmotic and pathogen stress (Singh et al. 1985, Singh et al. 1986, Singh et al. 1987, King et al. 1988, LaRosa et al. 1989). Osmotin gene expression and protein accumulation were elicited vegetative tissues of tomato in response to short- or long-term exposure to NaCl as well after severe water loss (Grillo et al. 1995). This gene is also stimulated by ABA (Singh et al. 1987, Skriver and Mundy 1990). Although NaCl can induce the osmotin gene through changes in ABA levels, this signal can also regulate osmotin mRNA accumulation by ABA-independent signal transduction pathways, as suggested for the Em gene in rice (Bostock and Quatrano 1992), and other ABA-inducible mRNAs from wheat (Morris et al. 1990) and rapeseed (Finkelstein and Crouch 1986). A cis-deletion analysis of the osmotin promoter indicated that the induction by NaCl, ABA, and ethylene is associated with the same region of the promoter (Raghothama et al. 1993). Many molecular responses to salt stress in a common ice plant (Mesembryanthemum crystallinum) are elicited primarily by the transcriptional induction of specific genes (Cushman et al. 1989, Andolfatto et al. 1994). Several classes of transporters are reported to be required in regulating sodium homeostasis under salt stress (Zhang et al. 2004). Arabidopsis has played a vital role in many investigations of the basic processes that occur during salt stress (Kader and Lindberg 2008). High-affinity potassium transporters (HKTs) are suggested to mediate a substantial Na+ influx in many species (Uozumi et al. 2000, Horie et al. 2001, Golldack et al. 2002, Mäser et al. 2002, Gárciadeblás et al. 2003). In rice, nine HKT homologues are identified (Gárciadeblás et al. 2003), out of which eight encode proteins with distinct transport activities, which might be expressed in various tissues and/or organs (Kader and Lindberg 2008).

32.6.4  Breeding Like all other stresses, breeding of tolerant cultivars is crucial to fight the ill effects caused by high salt concentration. Both breeding and screening germplasm for salt tolerance encounter the following limitations: (a) different phenotypic responses of plants at different growth stages, (b) different physiological mechanisms, (c) complicated genotype × environment interactions, and (d) variability of the salt-affected field in its chemical and physical soil composition (Arzani 2008). In general, two main approaches are being used to improve salt tolerance: (1) the exploitation of natural genetic variations, either through direct selection in stressful environments or through mapping QTLs and subsequent marker-assisted selection; and (2) the generation of transgenic plants to introduce novel genes or to alter expression levels of the existing genes to affect the degree of salt stress tolerance (Yamaguchi and Blumwald 2005). Breeding for salt tolerance has been proposed to the extent of possible crop production in sea water (Boyko 1966, Epstein and Norlyn 1977). Of various ways to tackle the salinity problem, exploitation of genetic mechanism is the most important strategy. Deliberate exploitation of genetic mechanism is mainly possible through (1) the direct use of halophytes (Malcolm 1969, Malcolm and Allen 1981, O’Leary 1994.) or choosing salt-tolerant crops as per the problem; (2) the introgression of tolerant genes from salt-tolerant genotypes (related or distant) (Flowers and Yeo 1995), and (3) the use of nonconventional approaches, such as tissue culture and molecular biology. Despite having knowledge about a long list of halophytes and their economic potential as fodder, fuel (Malcolm 1969, Malcolm and Allen 1981, Flowers and Yeo 1995), and oilseed (O’Leary 1994), their direct use as an economic crop is still under dormancy. Among these, jojoba (though

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not too salt tolerant) is a suitable crop for such areas (Shanon 1997). On the basis of our knowledge about the sensitivity of crop plants to high salt concentration, appropriate crops can be grown as per the intensity of the salt concentration in the soil. Cultivated crop plants can be classified into tolerant, intermediate, and sensitive types. Shanon (1997) has presented an elaborate review on the genetic variability of domesticated crop plants to salt stress. Barley is one of the most salt-tolerant crops; other tolerant crops are sugarbeet, cotton, canola (Brassica spp.), asparagus, red beet, zucchini squash, date palm, pomegranate, grape, wheat grass, bermuda grass, etc. Crop plants having an intermediate tolerance are sorghum, sunflower, safflower, sugarcane, potato, alfalfa, faba bean, almond, plum, orange, grape fruit, pea nut, chrysanthemum, carnation, etc., while the salt-sensitive group includes rice, corn, wheat, legumes, linseed, cowpea, lentil, chickpea, citrus, avocado, stone fruits, apricot, peach, blackberry, strawberry, aster, poinsettia, gladiolus, azalea, gardenia, gerbera, amarylis, African violet, etc. The introgression of salinity tolerance has been attempted from related genera and species in some crops, such as wheat and tomato, but without success due to the absence of the right knowledge about the exact kind of traits, their genetics (Chaubey and Senadhira 1994, Winicov 1994), and difficulties in recovering the traits of agronomic value. However, intervarietal crossing has yielded successful salt-tolerant genotypes in some crops. For example, few salt-tolerant wheat varieties (KRL 1-4, Raj 3077, WH 157, and JOB 66) have been developed in India during the past few years, and are being successfully grown in salt-affected areas. Based on the problems associated with the breeding of salt-tolerant genotypes in crops, it has been suggested that it is better to select for yield rather than for salt tolerance (Richards 1992, Jain et al. 1993). Rosielle and Hamblin (1981) also suggested that selection for productivity will increase yields in both stressed and nonstressed environments. However, this strategy may not work in all agro-eco environments, for instance, in a water-logged condition, where salt-tolerant rice is the only alternative (Flowers and Yeo 1995). Therefore, the use of physiological parameters might prove a useful component of breeding through pyramiding component physiological traits at least in sensitive species (Flowers and Yeo 1995). According to Flowers and Yeo (1995), salt-tolerant genotypes can be developed through a crossing programme that maximizes recombination, followed by single seed descent and selection for resistance along with agronomic characters. Among the novel ways of enhancing the salt tolerance of crops, the important ones proposed are the use of undifferentiated cells in tissue culture, and gene manipulation through molecular biology. Although difficulties are still present in both these methods, some success has been obtained in crops such as alfalfa (Winicov 1991), bent grass (Kuo et al. 1994), potato (Naik and Widholm 1993), and citrus (Kochba et al. 1982, Ben-Hayyim and Goffer 1989). The major problem in breeding for salinity tolerance continues to be the quantitative nature of stress tolerance and the problems associated with developing proper replicable testing environments (Arzani 2008).

32.7  Acid Soil Tolerance Similar to other abnormal environments, a low pH of soil also retards plant growth and development, thereby causing yield reductions. Soil acidity is a function of H+ activity in soil solution (Johonson 1988) and shows both chronological and spatial (horizontal and vertical) variation (Carver and Ownby 1995). Acid soils are phytotoxic due to a complex of nutritional disorders, which includes both deficiency (Ca, Mg, Mo) and excess (Al, Mn, H) of different nutrients (Adams 1984, Robson 1989). In this complex situation, the most damaging is the Al toxicity (Foy et al. 1978), which causes a number of disorders and may also influence water stress tolerance of crops (Goldman et al. 1989, Carver and Ownby 1995). Mn toxicity, not as important as Al toxicity, has also received attention during recent years (Foy et al. 1988, Mukhopadhyay and Sharma 1991). Acid soils are scattered all over the world in patches with a greater proportion in tropical regions. They significantly limit crop production worldwide, because approximately 50% of the world’s potentially arable soils are acidic (Kochian 2004). Hence, this is of concern to a vast population of growers. The increased awareness about the soil acidity problem and consequent yield reductions

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has attracted researchers to unravel the mechanism of resistance against the acid pH of soils (Foy et al. 1988, Haug and Shi 1991, Taylor 1991, Rao et al. 1993, Carver and Ownby 1995). Aluminum toxicity, being most crucial, has attained most attention in our venture to understand the tolerance to soil acidity. Mechanisms involved in Al tolerance are complex and could differ among species (Hanson 1991). Plants tolerate Al toxicity in two ways: (1) Al exclusion from plant tissues, especially the symplastic portion of root meristems (e.g., by chelation of Al by organic acids); and (2) internal Al detoxification, by converting Al into a harmless form (Hanson 1991, Carver and Ownby 1995, Delhaize et al. 2007). In contrast to Al, Mn tolerance seems to be largely based on an internal mechanism only. The probable reasons for this are the role of Mn as an essential element, and the biological and chemical similarities between Mn and Mg (Carver and Ownby 1995). However, recent evidence has shown that organic acids also play an important role in detoxifying Al internally and externally (Ma 2000, Ma et al. 2001, Ryan et al. 2001, Ma and Furukawa 2003). Since, both kinds and the amount of exuded OA anions contribute to the Al-detoxification capacity, the OAs have been classified as strongly (citrate, oxalate, and tartrate), moderately (malate, malonate, and salicylate), and weakly (succinate, lactate, formate, and acetate) Al-detoxifying compounds according to the stability of the Al complex (Hue et al. 1986). The enhanced exudation of citrate in response to Al stress has been reported in a number of crops, namely, common bean (Shen et al. 2002, Stass et al. 2007), maize (Pellet et al. 1995, Kollmeier et al. 2001), and soybean (Yang et al. 2000, 2001, Silva et al. 2001); the exudation of malate in wheat (T. aestivum L. (Delhaize et al. 1993, Pellet et al. 1997); a combination of both (citrate and malate) in S. cereale L. (Li et al. 2002) and triticale (x Triticosecale Wittmack) (Ma et al. 2000, Hayes and Ma 2003); and oxalate in buckwheat (Fagopyrum esculentum Moench) (Zheng et al. 1998) and taro (Colocasia esculenta L. Schott.) (Ma and Miyasaka 1998). These OAs are thought to complex Al within the apoplast of the root apex (Kinraide et al. 2005). Recently, it has been reported that aluminum resistance in common bean (P. vulgaris) involves the induction and maintenance of citrate exudation from root apexes (Rangel et al. 2009).

32.7.1  Morphophysiological Traits: Genetic Basis There is strong correlation between soil acidity and the root growth of plants (Khatiwada et al. 1916, Hanson 1991, Bona et al. 1994). The inheritance of root length under acid soils in wheat showed polygenic control with a wide range of the degree of dominance (Bona et al. 1994). In rice, the relative root length under acidic pH showed both additive and dominance effects with a preponderance of the additive effect; the trait was partially dominant with high heritability, and one group of genes was detected (Khatiwada et al. 1916). In soybean, along with thicker roots, increased seed weight was also found as an associated response with selection for seedling tolerance to acidity (Hanson and Kamprath 1979, Hanson 1991). For Mn, a common gene system in both the root and shoot of wheat has been suggested (Burke et al. 1990). Among physiological mechanisms countering low pH effect, the exudation of organic acids by plant roots is the most acceptable mechanism of external tolerance to Al toxicity (Delhaize et al. 1993). Al tolerance is also reported to be associated with greater efficiency of phosphate uptake (Foy et al. 1978, de Miranda and Rowell 1990) and cation exchange capacity (Blamey et al. 1990). Though there are indications of variation for these traits (Blamey et al. 1990, de Miranda and Rowell 1990, Delhaize et al. 1993), genetics of these traits has not been elucidated.

32.7.2  Genetics Substantial genetic variation for tolerance to acid soil has been reported in different crops, such as wheat (Foy et al. 1965, Lafever et al. 1977, Berzonsky 1992, Gustafson and Ross 1990, Carver and Ownby 1995), rice (Sivaguru et al. 1992), maize (Rhue et al. 1978, Miranda et al. 1984, Kasim et al. 1990), soybean (Hanson 1991), etc. Genetic studies on acid tolerance in crops indicate both

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qualitative and quantitative inheritance. Monogenic control was reported in wheat (Foy et al. 1965, Kerridge and Kronstad 1968, Lafever et al. 1977, Larkin 1987, Wheeler et al. 1992) and maize (Rhue et al. 1978, Miranda et al. 1984). Two dominant genes have been shown to be responsible for Al tolerance in the wheat variety Atlas 66 (Camargo 1981), while several genes were found in a relatively less tolerant line Chinese Spring (Carver and Ownby 1995). Resistance to Al toxicity may be different at seedling and adult plant stages (Carver and Ownby 1995). Quantitative inheritance for tolerance in acid soils is reported in wheat (Aniol 1984, Bona et al. 1994), rice (Khatiwada et al. 1916), maize (Magnavaca et al. 1987, Sawazaki and Furlani 1987, Lima et al. 1992, Pandey and Gardner 1992, Duque-Vargas et al. 1994), and soybean (Hanson 1991). Al tolerance was dominant over sensitivity, but, at the same time, it showed a greater role of the additive gene effect (Aniol 1984, Bona et al. 1994). Allelic variation for genes controlling Al tolerance has been noted in wheat (Lafever and Campbell 1978), barley (Minella and Sorrells 1992), and maize (Rhue et al. 1978). The change in the direction of dominance with a change in Al toxicity has been reported in wheat (Camargo 1981, Campbell and Lafever 1981, Bona et al. 1994) and barley (Minella and Sorrells 1992), which might be due to the differential expression of tolerant genes at varying levels of Al concentrations. In rice, both general combining ability (GCA) and specific combining ability (SCA) were important for Al toxicity (Khatiwada et al. 1916), but GCA was more prevalent. Reciprocal effects were also noticed (Khatiwada et al. 1916). Similarly, in maize, both additive and dominance genetic variations were reported for yield under acid soils (Magnavaca et al. 1987, Sawazaki and Furlani 1987, Lima et al. 1992, Pandey and Gardner 1992, Duque-Vargas et al. 1994). In wheat, hexaploid (AABBDD) wheat is more tolerant than tetraploid or diploid. The tolerance of the D genome is maximum followed by A and B genomes, respectively. The R genome of rye (S. cereale) possesses even greater tolerance than the D genome of wheat. The N genome of wheat (T. ventricosum) also possesses acid soil tolerance. Although the genes associated with Al tolerance are present in all the three genomes of wheat, the most important locations are 2DL and 4DL (Takagi et al. 1983, Aniol and Gustafson 1984, Aniol 1990). In rye, Al tolerance genes are located on chromosomes 3R, 4R, and 6R (Aniol and Gustafson 1984), but show reduced tolerance when transferred to wheat, probably due to their suppression by unknown genes of wheat (Aniol and Gustafson 1984). A wide genetic variation for Mn tolerance has also been recorded in wheat (Foy et al. 1988, Macfie et al. 1989) with the suggestion that only few genes are involved in the Mn tolerance (Foy et al. 1988). It has been suggested that the inheritance of tolerance to Al and that to Mn are independent and different genes may be involved (Neenan 1960, Foy et al. 1973, Burke et al. 1990, Fisher and Scott 1993), but there might be genes for the coregulation of their inheritance (Carver and Ownby 1995).

32.7.3  Gene Expression Plants’ ability to tolerate soil acidity is associated with a syndrome of cellular and molecular activities. A large number of genes take part in the whole operation, synthesizing different proteins. The idea that plants develop Al tolerance through the synthesis of proteins capable of inactivating Al (Aniol 1984) has now grown to a near reality with the identification of several proteins showing increased synthesis in response to Al (Slaski 1990, Horst et al. 1991, Cruz-Ortega and Ownby 1993, Snowden and Gardner 1993). A protein named RMP51 (51 kDa) has been reported to occur in the roots of an Al-tolerant cultivar of wheat; this protein has shown insensitivity to Mn, Cu, and heat stress (Basu et al. 1994). However, none of the known proteins can be said to be the product of a gene conferring Al tolerance (Carver and Ownby 1995).

32.7.4  Breeding In view of the increasing food demand and shrinking land resources, the need for a genetic improvement of crops for their tolerance to acid soils is beyond question. This might be a case of necessity

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rather than economics, though might prove useful in the long run. It is true that genetic tolerance does not correct the problem of soil acidity and only postpones the need to take corrective actions (Carver and Ownby 1995), but a judicious crop cultivation may have a beneficial effect on the soil in a variety of ways. Tolerance to acid soils varies from crop to crop and genotype to genotype. For example, rye is more tolerant than common wheat, and common wheat is more tolerant than barley. In wheat, a number of Al-tolerant genotypes have been identified, for example, Atlas 66. Genotypes of Brazilian origin show high tolerance to Al toxicity (Carver and Ownby 1995). Breeding for acid soil tolerance has gained momentum with the development of reliable screening techniques. Both laboratory and field screening methods are used. The most common screening medium for Al and Mn tolerance is solution culture, which is a nondestructive measurement of tolerance. During screening, the tolerance is generally measured based on the damage to root/shoot (Konzak et al. 1997) and the degree of severity following exposure to Al (Aniol 1984). The presence of dominance for acid soil tolerance has enabled breeders to use the backcross method of breeding for improving acid soil tolerance. A successful example is the transfer of a major gene for Al tolerance from the Carazinho variety of wheat to Egret (Fisher and Scott 1987) in Australia. It has been suggested that an early generation selection may be beneficial in breeding for Al tolerance in rice, and the pedigree method may also be used (Khatiwada et al. 1916). Recurrent selection has also been suggested as an alternative method to exploit the additive gene action related to Al tolerance in wheat (Carver et al. 1988, Minella and Sorrells 1992) and maize (Magnavaca et al. 1987, Lima et al. 1992, Granados et al. 1993). In the CIMMYT breeding programme, which utilizes a shuttle-breeding program, selection for acid tolerance is generally done following the evaluation of genotypes for yield and quality (Rajaram et al. 1986). Interspecific hybridization is also a possible way of improving acid soil tolerance in crops. For example, tetraploid hybrids of T. aestivum × T. ventricosum (Maan 1987) were developed for meeting the same objective. Molecular biology, though under investigation (Putrill et al. 1991, Snowden and Gardner 1993, Basu et al. 1994), is still to play a role in breeding for tolerance to acid soils.

32.8  Conclusions The increasing pressure on our natural resources including land is being realized by all. As our land area cannot be increased, the only hope of extending the cultivable area is through a greater utilization of the so-called “unfit areas,” which suffer from one or the other problem. A big chunk of this area suffers from severe abiotic stresses. However, this does not mean that abiotic stresses do not occur in other areas. In fact, abiotic stresses are prevalent in almost all cultivable lands, though in variable intensities and durations. It is common to see our crops suffering or fighting with abiotic stresses such as abnormal levels of water, temperature, and soil pH. When our crop plants suffer, we also suffer, directly or indirectly. Despite abiotic stresses being so vivid and important for crop production, tolerance mechanisms are not understood the way they should be and efforts to enhance the tolerance of crops are still far from satisfactory. Of various reasons for this slow progress, the most important ones are the absence of clear-cut traits conferring tolerance and the complexity caused by the simultaneous occurrence of more than one stress in variable intensities and durations. However, efforts of various researchers have succeeded in correlating some morphophysiological traits with concerned stresses along with the genetic mechanism involved. Molecular genetics is also contributing in a slow but sure manner to expand our knowledge in this direction. Several varieties have been released in different countries for meeting the challenge posed by abiotic stresses. Even a man-made crop like triticale was created to occupy marginal lands with a high stress pressure. Though abiotic stresses can be tackled better by an integrated approach, genetic manipulation should remain on top of this strategy. Growing knowledge of molecular genetics appears to be the key of the future.

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Acknowledgment The help rendered by Dr. B. Arun, Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, India, is gratefully acknowledged.

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Setter, T. L., I. Waters, B. J. Atwell, T. Kupkanchanckul, and H. Greenway. 1987b. In Plant Life in Acquatic and Amphibious Habitats. Special Publication No 5. ed. R. M. M. Crawford, pp. 411–433. British Ecological Society, Oxford, U.K.: Blackwell Scientific Publications. Setter, T. L., I. Waters, I. Wallace, P. Bhekasut, and H. Greenway. 1989. Submergence of rice. I. Growth and photosynthetic response to CO2 enrichment of floodwater. Aust J Plant Physiol 16:251–262. Setter, T. L., M. Ellis, E. V. Laureles, E. S. Ella, D. Senadhira, S. B. Mishra, S. Sarkarung, and S. Dutta. 1997. Physiology and genetics of submergence tolerance in rice. Ann Bot 79(supplement A):67–77. Shanahan, J. F., I. B. Edwards, J. S. Quick, and J. R. Fenwick. 1990. Membrane thermo-stability and heat tolerance in spring wheat. Crop Sci 30:247–251. Shannon, M. C. 1982. Genetics of salt tolerance: New challenges. In Biosaline Research: A Look in to the Future, ed. A. San Pietro, pp. 271–282. New York: Plenum Press. Shannon, M. C. 1997. Adaptation of plants to salinity. Adv Agron 60:75–120. Shannon, M. C. and C. L. Noble. 1990. Genetic approaches for developing economic salt tolerant crops. In Agricultural Salinity Assessment and Management. ASCE manuals and reports on Engineering Practice No. 71, ed. K. K. Tanji, pp. 161–185. New York: ACSE. Shelby, R. A., W. H. Greenleaf, and C. M. Paterson. 1978. Comparative floral fertility in heat tolerant and heat sensitive tomatoes. J Am Soc Hort Sci 103:778–780. Shonnard, G. and P. Gepts. 1994. Genetic of heat tolerance during reproductive development in common bean. Crop Sci 34:1168–1175. Shpiler, L. and A. Blum. 1986. Differential reaction of wheat cultivars to hot environments. Euphytica 35:483–492. Shvarts, M., A. Borochov, and D. Weiss. 1997. Low temperature enhances petunia flower pigmentation and induces chalcone synthase gene expression. Physiol Planatarum 99:67–72. Siddique, K. H. M., R. K. Belford, M. W. Perry, and D. Tennant. 1989. Ear: Stem ratio in old and modern wheat varieties; relationship with improvement in number of grain per ear and yield. Aust J Agric Res 40:473–487. Singer, M. and P. Berg. 1991. Genes and Genomes, A Changing Perspective. Oxford, U.K.: Blackwell Scientific Publications. Singh, A. and A. Grover. 2008. Genetic engineering for heat tolerance in plants. Physiol Mol Biol Plants 14(1&2):155–166. Singh, N. K., A. K. Handa, P. M. Hasegawa, and R. A. Bressan. 1985. Proteins associated with adaptation of cultured tobacco cells to NaCl. Plant Physiol 79:126–137. Singh, N. K., A. K. Handa, P. M. Hasegawa, and R. A. Bressan. 1986. Hormonal regulation of protein synthesis associated with NaCl adaptation in plant cells. Proc Natl Acad Sci USA 84:739–743. Singh, N. K., C. E. Bracker, P. M. Hasegawa, A. K. Handa, S. Buchel, M. A. Herodson, E. Pfankoch, F. E. Regnier, and R. A. Bressan. 1987. Characterization of osmotin: A thaumatin-like protein associated with osmotic adaptation in plant cells. Plant Physiol 85:529–536. Sinha, S. K. and R. Khanna. 1975. Physiological, biochemical, and genetic basis of heterosis. Adv Agron 27:123–174. Sinha, M. M. and S. Saran. 1988. Inheritance of submergence tolerance in lowland rice. Oryza 25:351–354. Sivaguru, M., M. R. James, P. R. Unbudurai, and R. Balkumar. 1992. Characterization of differential aluminum tolerance among rice genotypes cultivated in South India. J Plant Nutr 15:233–246. Skriver, K. and J. Mundy. 1990. Gene expression in response to abscisic acid and osmotic stress. Plant Cell 2:503–512. Slaski, J. J. 1990. Response of calmodulin-independent NAD kinase to aluminum in root tips from various cultivated plants. J Plant Physiol 136:40–44. Smith, D. 1949. Differential survival of ladino and common white clover encased in ice. J Agron 41:230–234. Snowden, K. C. and R. C. Gardner. 1993. Five genes induced by alumninum in wheat (Triticum aestivum L.) roots. Plant Physiol 103:855–861. Somvanshi, V. S. 2009. Patenting drought tolerance in organisms. Recent Pat DNA Gene Seq 3(1):16–25. Specht, J. E., K. Chase, M. Macrander, G. L. Graef, J. Chung, J. P. Markwell, M. Germann, J. H. Orf, and K. G. Lark. 2001. A QTL analysis of drought tolerance. Crop Sci 41:493–509. Srinivasan, A., H. Takeda, and T. Senboku. 1996. Heat tolerance in food legumes as evaluated by cell membrane thermostability and chlorophyll fluorescence techniques. Euphytica 88:35–45. Steponkus, P. L. 1978. Cold hardiness and freezing injury of agronomic crops. Adv Agron 30:51–98. Stølen, O. and S. Andersen. 1978. Inheritance of tolerance to low soil pH in barley. Hereditas 88:101–105. Stone, J. M., J. P. Palta, J. B. Bamberg, L. S. Weiss, and J. F. Harbage. 1993. Inheritance of freezing resistance in tuber-bearing Solanum species: Evidence for independent genetic control of nonacclimated freezing tolerance and cold acclimation capacity. Proc Natl Acad Sci USA 90:7869–7873.

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Improvement of Cold 33 Genetic Hardiness in Bermudagrass Yanqi Wu and Jeffrey A. Anderson Contents 33.1 Introduction........................................................................................................................... 851 33.2 Importance and Economic Uses of Bermudagrass................................................................ 852 33.2.1 Forage Grass.............................................................................................................. 852 33.2.2 Turfgrass.................................................................................................................... 852 33.3 Germplasm Distribution: Endemic and Cosmopolitan Species............................................ 853 33.4 Development of Cold Hardy Forage and Turf Cultivars........................................................ 855 33.4.1 Forage Bermudagrass Cultivars with Improved Cold Hardiness.............................. 855 33.4.2 Turf Bermudagrass Cultivars with Improved Cold Hardiness.................................. 857 33.5 Evaluation of Cold Hardiness: Field-Based and Laboratory-Based Methods....................... 859 33.6 Inheritance, Physiological, and Molecular Mechanisms....................................................... 861 33.7 Future Prospects in Bermudagrass Cold Hardiness Improvement........................................ 862 References....................................................................................................................................... 863

33.1  Introduction The revised taxonomy of the genus Cynodon L. C. Rich. consisting of eight species (Harlan et al.1970a) has been widely adopted (Clayton and Renvoize 1989). “Bermudagrass” is a common name, widely used for several species of plants in the genus Cynodon, except for three large, robust, non-rhizomatous species originated in East Africa. Harlan (1970) noted that “star grass” is a proper name for the large East African species including C. aethiopicus Clayton et Harlan, C. nlemfuensis Vanderyst, and C. plectostachyus Pilger. Cynodon dactylon (L.) Pers. var. dactylon is widely known as “common bermudagrass” and C. transvaalensis Burtt-Davy as “African bermudagrass.” From the viewpoints of agriculture, economics, and environmental protection, bermudagrass is extremely important due to its extensive worldwide use for forage, turf, and soil and water protection. More recently, bermudagrass has been proposed as a potential cellulosic biofuel crop (Anderson et al. 2008). Bermudagrass is a warm-season, sod-forming, perennial grass that utilizes the C4 photosynthetic carbon fixation pathway (Krans et al. 1979). The optimum growth of bermudagrass plants occurs between 27°C and 35°C (Turgeon 2008). Cold hardiness is a major limiting factor determining the adaptation range of bermudagrass as its use extends beyond tropical and subtropical regions, the primary adaptation area for bermudagrass. Use at higher latitudes results in a greater risk of winterkill (Taliaferro et al. 2004a). For example, in the United States, bermudagrass has been used in the transition zone between the primary cool-season turfgrass belt in the northern states and primary warm-season turfgrass belt in the southern states. Although not well defined, the climatically variable transition zone is within the range of the U.S. Department of Agriculture (USDA) plant hardiness zones 5 through 7. Some harsh winters in the transition zone are so severe that bermudagrass stands can be extensively damaged or killed. The costs associated with extended loss of use and investments in reestablishment of bermudagrass are substantial (Anderson et al. 2005). Fortunately, bermudagrass freeze tolerance is a heritable trait, cold hardy bermudagrass germplasm exists in 851

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nature, and plant breeding is able to genetically incorporate this desirable trait into improved cultivars. Development and release of cultivars improved in freeze tolerance reduces the winterkill risk when bermudagrass is used in the transition zone and extends its use to higher latitudes. This chapter addresses the topics of economic significance, germplasm variability, and distribution related to cold hardiness, development of cultivars with improved cold hardiness for forage and turf use, evaluation and determination of freeze tolerance, related physiological and molecular mechanisms, and future potential to continue the improvement of bermudagrass freeze tolerance.

33.2  Importance and Economic Uses of Bermudagrass Bermudagrass has probably been grazed by herbivorous animals, used for turf, soil protection, or similar purposes for many hundred years or even longer in the geographic and climatic regions where environment factors are conducive for its growth. These past uses were probably sporadic and small in scale, with no documented records indicating any economic significance until recently. Bermudagrass, becoming a major grass cultivated in monoculture on millions of hectares for forage and turf is a phenomenon developed in the United States over the last two centuries, especially after improved cultivars were bred and released from organized breeding programs in the last six decades (Taliaferro et al. 2004b). Subsequently, improved forage and turf bermudagrass cultivars have received larger applications in many other countries in the world.

33.2.1  Forage Grass Hanna (2007) noted bermudagrass is one of the most widespread and heavily used grasses. Many inherent attributes make bermudagrass an important and popular forage grass. Desirable characteristics include high yield capability, perennial growth habit, drought tolerance, and tolerance to close defoliation from animal grazing (Taliaferro 2005). Bermudagrass has few devastating insect or disease problems. The aggressive spreading capability of bermudagrass reduces weed encroachment into its stands. Propagation of bermudagrass is easily realized by sowing seed or planting vegetative propagules. Bermudagrass responds well to management practices such as sufficient water availability, frequent cutting, and balanced fertilization of nitrogen, phosphorus, and potassium. Bermudagrass is well adapted to grazing because of its extensive stolons and rhizomes. New leaf growth following repeated defoliation is nutritious and digestible, although mature bermudagrass is low in forage quality. Bermudagrass forage cultivars improved in one or more desirable traits, such as forage yield, digestibility, cold hardiness, and drought tolerance, have contributed substantially to the rise in prominence and establishment of the species as a major warm-season forage grass in the southern United States. Bermudagrass, along with other warm-season perennial grasses, forms improved pastures and forage systems, which provide a feed base for an immense animal husbandry industry in the southern U.S. states and many other warm climate regions in the world. For example, Bouton (2007) estimated the economic value of the forage systems for beef cattle and calf production in 14 southeastern U.S. states (Oklahoma, Texas, Louisiana, Arkansas, Missouri, Tennessee, Mississippi, Alabama, Georgia, Florida, South Carolina, North Carolina, Kentucky, and Virginia) is approximately US $11.6 billion annually. Bermudagrass is a major component of the forage systems in the southeastern United States with an estimated 10–12 million hectares devoted to animal herbage (Taliaferro et al. 2004b).

33.2.2  Turfgrass Bermudagrass is the most widely used warm-season turfgrass in the world (Shearman 2006). In addition to the many desirable traits of bermudagrass discussed above, its dense sod-forming ability, tolerance to close mowing, long-lived perennial nature, and wide adaptation make bermudagrass an attractive turfgrass (Taliaferro et al. 2004a). Beard (1973) noted that bermudagrass is a good choice

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for many turf areas if the environment is suitable for its growth. He indicated the heat and drought tolerance of bermudagrass is excellent, but cold hardiness is poor in general. In warmer temperate, subtropical, and tropical regions, improved turf bermudagrass forms a very dense and uniform turf of high quality, making it suitable for establishment of a myriad of turf forms. Bermudagrass has excellent wear tolerance and recuperative capacity, which are important for athletic turf fields. Turf bermudagrass is extensively used on residential lawns, parks, institutional grounds, athletic fields, and roadside areas. It is also widely deployed to establish vegetations on golf courses, including fairways, tee boxes, putting greens and rough zones. In the United States and several other countries, the sod industry produces a large amount of turf bermudagrass sod, which can immediately cover the areas to be established. Establishing turf using bermudagrass seed is another widely used propagation means. Bermudagrass turf area in the United States is huge, though it is difficult to pinpoint the size. There is a total of 23 million hectares of managed turfgrass in the United States (Shearman 2006), with bermudagrass being the predominant warm-season turfgrass.

33.3  Germplasm Distribution: Endemic and Cosmopolitan Species Cynodon species vary widely in germplasm distribution, cold hardiness, and agronomic value for breeding forage and turf cultivars. Cynodon dactylon is the only ubiquitous and cosmopolitan taxon, and consequently it contains the greatest genetic diversity, the widest geographic distribution, and the greatest economic value for breeding forage and turf cultivars within the genus (Wu 2010). Cynodon transvaalensis is an endemic species in South Africa that has been very important for the development of clonal hybrid turf cultivars in hybridizations with C. dactylon (Burton 1965, 1991). Cynodon nlemfuensis is endemic in East Africa and the most promising and valuable stargrass species used in forage bermudagrass breeding (Burton 1972, Burton et  al. 1993). Selected C. nlemfuensis plants have been used to cross with C. dactylon genotypes in the development of interspecific forage hybrid cultivars improved in dry matter digestibility and/or biomass yield since C. nlemfuensis is genetically compatible with C. dactylon. However, germplasm distribution patterns and cold hardiness of the three species are distinct. It appears that natural distribution and cold hardiness in Cynodon species are closely related. Harlan et al. (1970a) noted there is little cold hardiness in C. nlemfuensis, C. aethiopicus, C. arcuatus, C. barberi, and C. plectostachyus. Those five species all have tropical and subtropical origins although their respective distribution patterns are different (Harlan et al. 1970b). Cynodon incompletus Nees, a species endemic to South Africa, is cold hardy but has little value as a forage grass (Harlan 1970). Germplasm of the latter species has not been used in turf bermudagrass breeding programs as well. Therefore, germplasm within C. dactylon appears to be the sole or major source to provide cold resistance genes for the development of cold hardy forage bermudagrass cultivars, while development of cold hardy turf bermudagrass cultivars can use germplasm from both C. dactylon and C. transvaalensis. The species C. dactylon consists of six botanical varieties: dactylon, afghanicus Harlan et de Wet, aridus Harlan et de Wet, coursii Harlan et de Wet, elegans Rendle, and polevansii (Stent) Harlan et de Wet (Harlan et al. 1970a). Variety dactylon is the only one which has a truly cosmopolitan geographic distribution in the world, being one of the most widely distributed of all plants (Harlan et al. 1970a). Its winter hardiness varies from none to very hardy. The taxon contains plants varying enormously in size from small statured varieties which have been used as turf grasses and in the development of modern turf cultivars, to large plants used as pasture and forage grasses and in breeding improved forage cultivars. Variety dactylon plants are distributed to about 45° south latitude in South America and 53° north latitude in northern Europe (Harlan and de Wet 1969). Harlan and de Wet (1969) indicated that var. dactylon is found in every country in Africa, in North, Central, and South America, Australia, New Zealand, and every country in Asia except Mongolia. In Europe, var. dactylon is ubiquitous and found in all countries except the most northern regions. In Nepal and southwest China, var. dactylon is found growing about 3000 m above sea level. Variety dactylon was introduced in the United States in or before 1751 (Taliaferro et al. 2004b).

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Currently, it is found in all southern and central states, and some northern states, such as New Hampshire, New York, Michigan, Montana, Idaho, and Washington in the United States, and British Columbia in Canada (USDA NRCS 2009). Taliaferro et al. (2004b) indicated var. dactylon occurs in greatest abundance in tropical and subtropical environments. Variety dactylon is less abundant at higher latitudes. Natural distribution to higher latitudes is indicative of cold hardy germplasm in var. dactylon. Harlan and de Wet (1969) proposed three races within var. dactylon on the basis of adaptation, distribution, and morphology. Tropical race plants are widely distributed in pantropical areas and 20 cm or less in stature. Plants in the tropical race have little winter hardiness. The temperate race resembles the tropical race in morphology, but is winter hardy. Plants of the temperate race are distributed in northern Europe, northern United States, and northern China. Seleucidus race plants are very coarse in leaf texture and distributed in Afghanistan, Turkey, Iran, Turkmenistan, Uzbekistan, Greece, Bulgaria, and Yugoslavia. Moving north to Europe, seleucidus plants become smaller and merge with the temperate race (Harlan and de Wet 1969). The seleucidus race gets its name because most typical plants of the race are distributed in the region from Turkey to Afghanistan once occupied by the Seleucid empire (Harlan and de Wet 1969). Harlan and de Wet (1969) have shown that plants typical of the seleucidus race are very cold hardy. The relative cold hardiness assessment of the above-mentioned three races was made in field observations by Harlan and colleagues in 1960s. No quantitative data are available to reveal cold hardiness variability of the three races. Variety aridus is naturally distributed in arid regions in South Africa, East African countries, Egypt, Israel, Arabia, India, Sri Lanka, and Myanmar. Plant size ranges from small in India to large in South Africa. Introduced var. aridus plants called “Giant bermudagrass” have been grown in Arizona. Harlan and de Wet (1969) indicated var. aridus plants in their collection from South Africa, India, Israel, and the Near East had no winter hardiness in field plots tested in Stillwater, Oklahoma. However, they can survive winters by producing deep rhizomes that escape freezing temperatures. Cooper and Burton (1965) reported that giant bermudagrass produces no more forage than common bermudagrass, which produces much less forage than the released cultivar “Coastal” bermudagrass in Georgia. Giant bermudagrass is inferior to common bermudagrass as a turf grass. However, from the evolutionary point of view, var. aridus may play an extremely important role as the progenitor of var. dactylon, which likely is an autotetraploid (Harlan and de Wet 1969). Variety afghanicus is so named because plants of this variety are only found in Afghanistan (Harlan and de Wet 1969). The endemic variety plants are tall and have long stolons. Plants of this variety normally are found growing in lowland steppes and along irrigation ditches. Although var. afghanicus has no rhizomes in diploid plants and has short rhizome-like structures in tetraploid plants, they have good winter hardiness since field-grown plants can survive winters at Stillwater, Oklahoma (Harlan and de Wet 1969, Harlan et al. 1970a). Harlan and de Wet (1969) believed the var. dactylon seleucidus race is derived from “the infusion of var. afghanicus germplasm into the temperate race of var. dactylon.” Cynodon transvaalensis is endemic to South Africa (de Wet and Harlan 1970, 1971, Harlan et al. 1970a). Plants of C. transvaalensis are unique and distinct from all other plant species including other Cynodon species. African bermudagrass plants are small in size, turf forming, and have purple, fine wirelike stolons. Other morphological characteristics of African bermudagrass include its slender leaf blade (4 4

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