Plant Cold Hardiness (Cabi)

Plant Cold Hardiness

From the Laboratory to the Field

FRIENDS IN SUNNY SASKATCHEWAN 8th International Plant Cold Hardiness Seminar, Saskatoon, Saskatchewan, Canada, August 3–9, 2007

Plant Cold Hardiness From the Laboratory to the Field

Edited by

Lawrence V. Gusta Department of Plant Sciences, University of Saskatchewan, Canada

Michael E. Wisniewski USDA-ARS, Appalachian Fruit Research Station, USA and

Karen K. Tanino Department of Plant Sciences, University of Saskatchewan, Canada

CABI is a trading name of CAB International CABI Head Office Nosworthy Way Wallingford Oxfordshire OX10 8DE UK Tel: +44 (0)1491 832111 Fax: +44 (0)1491 833508 E-mail: [email protected] Website: www.cabi.org

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© CAB International 2009. All rights reserved. No part of this publication may be reproduced in any form or by any means, electronically, mechanically, by photocopying, recording or otherwise, without the prior permission of the copyright owners. A catalogue record for this book is available from the British Library, London, UK. Library of Congress Cataloging-in-Publication Data Plant cold hardiness : from the laboratory to the field / edited by Lawrence V. Gusta, Karen K. Tanino, and Michael E. Wisniewski. p. cm. Includes bibliographical references and index. ISBN 978-1-84593-513-9 (alk. paper) 1. Plants–Effect of cold on. 2. Plant physiology. I. Gusta, Lawrence V. II. Tanino, Karen K. III. Wisniewski, Michael E. IV. Title. QK756.P536 2009 632'.11–dc22 2008046606 ISBN-13: 978 1 84593 513 9 Typeset by SPi, Pondicherry, India. Printed and bound in the UK by the MPG Books Group. The paper used for the text pages in this book is FSC certified. The FSC (Forest Stewardship Council) is an international network to promote responsible management of the world’s forests.

Contents

Contributors

ix

Preface

xv

PART 1: THE FREEZING PROCESS 1

Ice Nucleation, Propagation and Deep Supercooling: the Lost Tribes of Freezing Studies M.E. Wisniewski, L.V. Gusta, M.P. Fuller and D. Karlson

2

Low-temperature Damage to Wheat in Head – Matching Perceptions with Reality M.P. Fuller, J. Christopher and T. Fredericks

12

3

Freezing Behaviours in Plant Tissues: Visualization using NMR Micro-imaging and Biochemical Regulatory Factors Involved M. Ishikawa, H. Ide, W.S. Price, Y. Arata, T. Nakamura and T. Kishimoto

19

4

Factors Related to Change of Deep Supercooling Capability in Xylem Parenchyma Cells of Trees S. Fujikawa, J. Kasuga, N. Takata and K. Arakawa

29

PART 2: MOLECULAR BASIS FOR FREEZING TOLERANCE 5

THE

ACQUISITION

1

OF

Plant Cold-shock Domain Proteins: on the Tip of an Iceberg D. Karlson, K. Nakaminami, K. Thompson, Y. Yang, V. Chaikam and P. Mulinti

43

v

vi

Contents

6

Expressional and Functional Characterization of Arabidopsis Cold-shock Domain Proteins K. Sasaki, M.-H. Kim and R. Imai

55

7

Plasma Membrane and Plant Freezing Tolerance: Possible Involvement of Plasma Membrane Microdomains in Cold Acclimation A. Minami, Y. Kawamura, T. Yamazaki, A. Furuto and M. Uemura

62

8

Global Expression of Cold-responsive Genes in Fruit Trees C.L. Bassett and M.E. Wisniewski

72

9

Could Ethanolic Fermentation During Cold Shock Be a Novel Plant Cold Stress Coping Strategy? F. Kaplan, D.Y. Sung, D. Haskell, G.S. Riad, M. Popp, M. Amaya, A. LaBoon, Y. Kawamura, Y. Tominaga, J. Kopka, M. Uemura, K.-J. Lee, J.K. Brecht and C.L. Guy

80

PART 3:

LINKAGE BETWEEN DEVELOPMENTAL ARREST AND COLD HARDINESS

10

Bud Set – A Landmark of the Seasonal Growth Cycle in Poplar A. Rohde

91

11

An Epigenetic Memory from Time of Embryo Development Affects Climatic Adaptation in Norway Spruce Ø. Johnsen, H. Kvaalen, I. Yakovlev, O.G. Dæhlen, C.G. Fossdal and T. Skrøppa

99

12

The Influence of Temperature on Dormancy Induction and Plant Survival in Woody Plants L. Kalcsits, S. Silim and K. Tanino

108

PART 4: GENETIC BASIS

OF

SUPERIOR COLD TOLERANCE

13

Winter Hardiness and the CBF Genes in the Triticeae E.J. Stockinger

119

14

Regulation of Stress-responsive Signalling Pathways by Eudicot CBF/DREB1 Genes A. Nassuth and M. Siddiqua

131

PART 5: IMPACT 15

OF

GLOBAL CLIMATE CHANGE

ON

PLANTS

Evolution of Plant Cold Hardiness and its Manifestation along the Latitudinal Gradient in the Canadian Arctic J. Svoboda

140

Contents

vii

16

Ice Encasement Damage on Grass Crops and Alpine Plants in Iceland – Impact of Climate Change B.E. Gudleifsson

163

17

Impact of Simulated Acid Snow Stress on Leaves of Cold-acclimated Winter Wheat K. Arakawa, H. Inada and S. Fujikawa

173

18

Elevated Atmospheric CO2 Concentrations Enhance Vulnerability to Frost Damage in a Warming World M.C. Ball and M.J. Hill

183

19

The Occurrence of Winter-freeze Events in Fruit Crops Grown in 190 the Okanagan Valley and the Potential Impact of Climate Change H.A. Quamme, A.J. Cannon, D. Neilsen, J.M. Caprio and W.G. Taylor

20

Cold Hardiness in Antarctic Vascular Plants L.A. Bravo, L. Bascuñán-Godoy, E. Pérez-Torres and L.J. Corcuera

PART 6:

FROM THE LABORATORY BRIDGING THE GAP

TO

198

THE FIELD:

21

Patterns of Freezing in Plants: the Influence of Species, Environment and Experiential Procedures L.V. Gusta, M.E. Wisniewski and R.G. Trischuk

214

22

Going to Extremes: Low-temperature Tolerance and Acclimation in Temperate and Boreal Conifers G.R. Strimbeck and P.G. Schaberg

226

23

The Rapid Cold-hardening Response in Insects: Ecological Significance and Physiological Mechanisms M.A. Elnitsky and R.E. Lee, Jr

240

PART 7: PHOTOSYNTHESIS

AND

SIGNALLING

24

Conifer Cold Hardiness, Climate Change and the Likely Effects of Warmer Temperatures on Photosynthesis I. Ensminger, N.P.A. Hüner and F. Busch

249

25

Chemical Genetics Identifies New Chilling Stress Determinants in Arabidopsis J. Einset

262

26

Analysis of the Ascorbate Antioxidant Pathway in Overwintering Populations of Lucerne (Medicago sativa L.) of Contrasting Freezing Tolerance A. Bertrand, Y. Castonguay, S. Laberge, J. Cloutier and R. Michaud

271

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Contents

PART 8: SYSTEMS BIOLOGY 27

Identification of Proteins from Potato Leaves Submitted to Chilling Temperature J. Renaut, S. Planchon, M. Oufir, J.-F. Hausman, L. Hoffmann and D. Evers

279

28

Genomics of Cold Hardiness in Forest Trees J. Holliday

293

Index

The colour plate section can be found following p. 160

305

Contributors

Maria Amaya, School of Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA (previous address: Plant Molecular Cellular Biology Program, Environmental Horticulture, University of Florida, Box 110675, Gainesville, FL 32611, USA) Keita Arakawa, Research Faculty and Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. E-mail: [email protected] Yoji Arata, Water Research Institute, Sengen, Tsukuba, Ibaraki 305-0047, Japan Marilyn C. Ball, Functional Ecology Group, Building 46, Research School of Biological Sciences, Australian National University, Canberra, ACT 0200, Australia. E-mail: marilyn.ball@anu. edu.au Luisa Bascuñán-Godoy, Laboratorio de Fisiología Vegetal, Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Barrio Universitario, Casilla 160 C, Concepción, Chile Carole L. Bassett, US Department of Agriculture Agricultural Research Service (USDA-ARS), Appalachian Fruit Research Station, 2217 Wiltshire Road, Kearneysville, WV 25430, USA. E-mail: [email protected] Annick Bertrand, Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd, Quebec City, Quebec, Canada, G1V 2J3. E-mail: [email protected] León A. Bravo, Laboratorio de Fisiología Vegetal, Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Barrio Universitario, Casilla 160 C, Concepción, Chile. E-mail: [email protected] Jeffrey K. Brecht, Horticultural Sciences Department, University of Florida, Box 110690, Gainesville, FL 32611, USA Florian Busch, Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, Ontario, Canada, M5S 3B2 (previous address: Department of Biology and Biotron, University of Western Ontario, London, Ontario, Canada, N6A 5B7) Alex J. Cannon, Environment Canada, Pacific and Yukon Region, Vancouver, British Columbia, Canada, V6C 3S5. E-mail: [email protected] Joe M. Caprio, 1801 Willow Way Drive, Boseman, MT 59715, USA. E-mail: mjcaprio@juno. com Yves Castonguay, Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd, Quebec City, Quebec, Canada, G1V 2J3

ix

x

Contributors

Vijay Chaikam, Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506-6108, USA Jack Christopher, The Leslie Research Centre, Department for Primary Industries and Fisheries, PO Box 2282, Toowoomba, Queensland 4350, Australia. E-mail: Jack.Christopher@dpi. qld.gov.au Jean Cloutier, Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd, Quebec City, Quebec, Canada, G1V 2J3 Luis J. Corcuera, Laboratorio de Fisiología Vegetal, Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Barrio Universitario, Casilla 160 C, Concepción, Chile Ola Gram Dæhlen, Oppland Forest Society, Biri Nursery and Seed Improvement Centre, N-2836 Biri, Norway John Einset, Norwegian University of Life Sciences, N-1430 Ås, Norway. E-mail: john.einset@ umb.no Michael A. Elnitsky, Department of Biology, Mercyhurst College, Erie, PA 16546, USA (previous address: Department of Zoology, Miami University, Oxford, OH 45056, USA). E-mail: [email protected] Ingo Ensminger, Department of Forest Ecology, Forest Research Institute of Baden-Württemberg, D-79100 Freiburg, Germany; Department of Biology and Biotron, University of Western Ontario, London, Ontario, Canada, N6A 5B7. E-mail: [email protected] Daniele Evers, Centre De Recherche Public – Gabriel Lippmann, Department of Environment and Agrobiotechnologies, 41 rue du Brill, L-4422 Belvaux, Luxembourg Carl Gunnar Fossdal, Norwegian Forest and Landscape Institute, PO Box 115, N-1431 Ås, Norway Troy Fredericks, The Leslie Research Centre, Department for Primary Industries and Fisheries, PO Box 2282, Toowoomba, Queensland 4350, Australia. E-mail: [email protected]. gov.au Seizo Fujikawa, Research Faculty and Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. E-mail: [email protected] Michael P. Fuller, University of Plymouth, Plymouth PL4 8AA, UK. E-mail: mfuller@plymouth. ac.uk Akari Furuto, Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan Bjarni E. Gudleifsson, Agricultural University of Iceland, Modruvellir, 601 Akureyri, Iceland. E-mail: [email protected] Lawrence V. Gusta, Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan, Canada, S7N 5A8. E-mail: [email protected] Charles L. Guy, Plant Molecular Cellular Biology Program, Environmental Horticulture, University of Florida, Box 110675, Gainesville, FL 32611, USA. E-mail: [email protected] Dale Haskell, Plant Molecular Cellular Biology Program, Environmental Horticulture, University of Florida, Box 110675, Gainesville, FL 32611, USA Jean-François Hausman, Centre De Recherche Public – Gabriel Lippmann, Department of Environment and Agrobiotechnologies, 41 rue du Brill, L-4422 Belvaux, Luxembourg Michael J. Hill, Earth System Science and Policy, University of North Dakota, Clifford Hall, Stop 9011, 4149 Campus Drive, Grand Forks, ND 58202, USA Lucien Hoffmann, Centre De Recherche Public – Gabriel Lippmann, Department of Environment and Agrobiotechnologies, 41 rue du Brill, L-4422 Belvaux, Luxembourg Jason Holliday, Department of Forest Sciences, University of British Columbia, 3041-2424 Main Mall, Vancouver, British Columbia, Canada, V6T 1Z4. E-mail: [email protected] Norman P.A. Hüner, Department of Biology and Biotron, University of Western Ontario, London, Ontario, Canada, N6A 5B7 Hiroyuki Ide, Water Research Institute, Sengen, Tsukuba, Ibaraki 305-0047, Japan

Contributors

xi

Ryozo Imai, Crop Cold Tolerance Research Team, National Agricultural Research Center for Hokkaido Region, Hitsujigaoka 1, Toyohira-ku, Sapporo 062-8555, Japan. E-mail: rzi@ affrc.go.jp Hidetoshi Inada, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan Masaya Ishikawa, Environmental Stress Research Unit, National Institute of Agrobiological Sciences, Kan’nondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. E-mail: isikawam@affrc. go.jp Øystein Johnsen, Norwegian Forest and Landscape Institute, PO Box 115, N-1431 Ås, Norway. E-mail: [email protected] Lee Kalcsits, PFRA Shelterbelt Centre, Box 940, Agriculture and Agri-Food Canada, Indian Head, Saskatchewan, Canada, S0G 2K0; Department of Plant Sciences, College of Agriculture and Bioresources, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. E-mail: [email protected] Fatma Kaplan, Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610, USA (previous address: Plant Molecular Cellular Biology Program, Environmental Horticulture, University of Florida, Box 110675, Gainesville, FL 32611, USA) Dale Karlson, Monsanto Company, Research Triangle Park, NC 27709, USA (previous address: Division of Plant and Soil Sciences, West Virginia University, PO Box 6108, Morgantown, WV 26506-6108, USA). E-mail: [email protected] Jun Kasuga, Research Faculty and Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan Yukio Kawamura, The 21st Century Center of Excellence Program, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan Myung-Hee Kim, Crop Cold Tolerance Research Team, National Agricultural Research Center for Hokkaido Region, Hitsujigaoka 1, Toyohira-ku, Sapporo 062-8555, Japan Tadashi Kishimoto, Environmental Stress Research Unit, National Institute of Agrobiological Sciences, Kan’nondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan Joachim Kopka, Max Planck Institute of Molecular Plant Physiology, Am Muhlenberg 1, D-14476 Golm, Germany Harald Kvaalen, Norwegian Forest and Landscape Institute, PO Box 115, N-1431 Ås, Norway Serge Laberge, Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd, Quebec City, Quebec, Canada, G1V 2J3 Allison LaBoon, University of Miami Miller School of Medicine, 1600 NW 10th Avenue Suite 2099 Miami, FL 33136, USA (previous address: Plant Molecular Cellular Biology Program, Environmental Horticulture, University of Florida, Box 110675, Gainesville, FL 32611, USA) Kil-Jae Lee, Department of Biology, Korea National University of Education, Chung-buk 363791, Korea (previous address: Plant Molecular Cellular Biology Program, Environmental Horticulture, University of Florida, Box 110675, Gainesville, FL 32611, USA) Richard E. Lee, Department of Zoology, Miami University, Oxford, OH 45056, USA. E-mail: [email protected] Réal Michaud, Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd, Quebec City, Quebec, Canada, G1V 2J3 Anzu Minami, The 21st Century Center of Excellence Program, Iwate University, Morioka, Iwate 020-8550, Japan Prashanthi Mulinti, Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506-6108, USA Kentaro Nakaminami, Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506-6108, USA Toshihide Nakamura, Environmental Stress Research Unit, National Institute of Agrobiological Sciences, Kan’nondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan

xii

Contributors

Annette Nassuth, Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada, N1G 2W.1E-mail: [email protected] Denise Neilsen, Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Summerland, British Columbia, Canada, V0H 1Z0. E-mail: [email protected] Mouhssin Oufir, Centre De Recherche Public – Gabriel Lippmann, Department of Environment and Agrobiotechnologies, 41 rue du Brill, L-4422 Belvaux, Luxembourg Eduardo Pérez-Torres, Laboratorio de Biotecnología, INIA Quilamapu, Av. Vicente Méndez 515, Chillán, Chile Sébastien Planchon, Centre De Recherche Public – Gabriel Lippmann, Department of Environment and Agrobiotechnologies, 41 rue du Brill, L-4422 Belvaux, Luxembourg Michael Popp, Interdisciplinary Center for Biotechnology Research, Box 100156, Gainesville, FL 32610, USA William S. Price, Water Research Institute, Sengen, Tsukuba, Ibaraki 305-0047, Japan Harvey A. Quamme, Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Summerland, British Columbia, Canada, V0H 1Z0. E-mail: [email protected] Jenny Renaut, Centre de Recherche Public – Gabriel Lippmann, Department of Environment and Agrobiotechnologies, 41 rue du Brill, L-4422 Belvaux, Luxembourg. E-mail: renaut@ lippmann.lu Gamal S. Riad, Vegetable Research Department, National Research Center, Cairo 12622, Egypt (previous address: Horticultural Sciences Department, University of Florida, Box 110690, Gainesville, FL 32611, USA) Antje Rohde, Department Plant Growth and Development, Institute for Agriculture and Fisheries Research, Caritasstraat 21, B-9090 Melle, Belgium. E-mail: [email protected] Kentaro Sasaki, Crop Cold Tolerance Research Team, National Agricultural Research Center for Hokkaido Region, Hitsujigaoka 1, Toyohira-ku, Sapporo 062-8555, Japan Paul G. Schaberg, US Department of Agriculture Forest Service, Northern Research Station, South Burlington, VT 05403, USA Mahbuba Siddiqua, Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada, N1G 2W1 Salim Silim, PFRA Shelterbelt Centre, Box 940, Agriculture and Agri-Food Canada, Indian Head, Saskatchewan, Canada, S0G 2K0 Tore Skrøppa, Norwegian Forest and Landscape Institute, PO Box 115, N-1431 Ås, Norway Eric J. Stockinger, Department of Horticulture and Crop Science, The Ohio State University/ Ohio Agricultural Research and Development Center (OARDC), 1680 Madison Ave, Wooster, OH 44691, USA. E-mail: [email protected] G. Richard Strimbeck, Department of Biology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway. E-mail: [email protected] Dong Yul Sung, Cell and Developmental Biology Section, Division of Biological Sciences and Center for Molecular Genetics, University of California, San Diego, CA 92093, USA (previous address: Plant Molecular Cellular Biology Program, Environmental Horticulture, University of Florida, Box 110675, Gainesville, FL 32611, USA) Josef Svoboda, Department of Biology, University of Toronto at Mississauga, Mississauga, Ontario, Canada, L5L 1C6. E-mail: [email protected] Naoki Takata, Research Faculty and Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan Karen Tanino, Department of Plant Sciences, College of Agriculture and Bioresources, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, S7N 5A8. E-mail: karen.tanino@ usask.ca Bill G. Taylor, Environment Canada, Pacific and Yukon Region, Vancouver, British Columbia, Canada, V6C 3S5. E-mail: [email protected] Kari Thompson, Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506-6108, USA

Contributors

xiii

Yoko Tominaga, Département des Sciences Biologiques, Université du Québec à Montréal, Montréal, Québec, Canada, H3C 3P8 (previous address: Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan) Russell G. Trischuk, Dow Agrosciences Canada Inc., Saskatoon, Saskatchewan, Canada, S7N 4L8. E-mail: [email protected] Matsuo Uemura, The 21st Century Center of Excellence Program, Iwate University, Morioka, Iwate 020-8550, Japan; Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan. E-mail: [email protected] Michael Wisniewski, US Department of Agriculture, Agricultural Research Service (USDAARS), Appalachian Fruit Research Station, 2217 Wiltshire Road, Kearneysville, WV 25430, USA. E-mail: [email protected] Igor Yakovlev, Norwegian Forest and Landscape Institute, PO Box 115, N-1431 Ås, Norway Tomokazu Yamazaki, The 21st Century Center of Excellence Program, Iwate University, Morioka, Iwate 020-8550, Japan Yongil Yang, Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506-6108, USA

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Preface

This book is a collection of invited and selected papers on plant cold hardiness that were presented at the 8th International Plant Cold Hardiness Seminar (8IPCHS) hosted by the University of Saskatchewan (U of S) in August 3–9, 2007. It began at the U of S in time for our campus’ 100th anniversary. On the third day, the entire conference moved northward to the edge of the boreal forest at Elk Ridge Resort, Waskesiu, Saskatchewan. There were over 105 attendees representing 22 countries. The theme of the conference was: ‘From the Laboratory to the Field’. The collection of chapters in this book represent many of the topics that were presented during the conference. The conference and the book sought to integrate the most up to date basic and applied research on plant cold hardiness. Attendees at the conference included molecular biologists, plant physiologists, plant breeders, plant ecologists, microbiologists, agronomists, administrators, policy makers, and representatives from multinational companies. Due to the structure of the conference, scientists and students had ample time to personally discuss their research with colleagues and in many cases formulate collaborative research plans. The conference provided a better understanding of the stresses plants experience under field conditions compared to analyzing plants in a greenhouse, laboratory, or growth chamber. It also made applied researchers aware of new technologies to study freezing in plants. We sincerely thank the chapter authors for their important contributions. This conference and book would not have been possible without the generous support of: the University of Saskatchewan, the College of Agriculture and Bioresources and the Dept. of Plant Sciences; the Alberta Government; Alberta Agricultural Research Institute; Genome Prairie; Saskatchewan Agriculture and Food; Pioneer; Monsanto; BASF; Cargill; Performance Plants; Flir Systems; National Resources Canada; Conviron; Sasktel; and the National Research Council. We appreciate the work of our International Core Organizing Committee: Rajeev Arora, Yves Castonguay,

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Tapio Palva and Mat Uemura. Our International Advisory Committee: Leon Bravo, Geoffrey Fincher, Olavi Junttila, Alina Kacperska, Jenny Renaut, Fedora Sutton, Victor Voinikov. Many local organizing committee members devoted much time to making this conference a success: Kyla Shea, Markel Chernenkoff, Brian Fowler, Gloria Gingera, Gord Gray, Elaine Gusta, Lee Kalcsits, Wilf Keller, Ron Mantyka, M.P.M. Nair, Jie Qiu, Kerry Sproule, Russ Trischuk and Ruojing Wang. Our Local Advisory Committee consisted of: Yuguang Bai, Kirstin Bett, Peta Bonham-Smith, Ravi Chibbar, Degi Chuluunbaatar, Natalie Coetzee, Michelle Gallucci, Edward Kendall, Pramod Kumar, Rob Norris, Elaine Qualtiere, Martin Reaney, Steve Robinson, Isobel Parkin, Nirmala Sharma, Brian Sim, Perumal Vijayan, Susan Varughese, Grant Wood and Scott Wright. Special thanks to Randy Whitter of Elk Ridge. Our conference logo was the Inuksuk (‘In-ook-shook’, meaning ‘likeness of a person’). Built by the Inuit of the Canadian Arctic, it is a stone figure which was used for various purposes: to act as a guide for a safe journey, to warn of imminent danger, to mark a place of respect, to show the path for caribou hunting. The Inuksuk has grown to symbolize leadership, friendship and the spirit of sharing knowledge and wisdom. L.V. Gusta, M.E. Wisniewski and K.K. Tanino

1

Ice Nucleation, Propagation and Deep Supercooling: the Lost Tribes of Freezing Studies M.E. Wisniewski, L.V. Gusta, M.P. Fuller and D. Karlson

Introduction

Ice nucleation and propagation

Prior to the emphasis on the molecular biology of cold acclimation, considerable research was conducted on the processes of ice nucleation and deep supercooling. In many species, these two processes are critical to winter hardiness or surviving episodes of frost. Over the past two decades, however, research on these topics has diminished drastically. Research on these topics in major journals in some ways is much like the lost tribes of Israel, which were sent into exile in 722 BC. Where did the tribes go? So it may be asked of the topics of ice formation and deep supercooling in plants. The objectives of the current report are to review these topics and identify critical questions that still need answers, and to indicate how a greater understanding of these topics may lead to new strategies for improving cold hardiness in plants or new technologies for improving frost protection. The ideas and concepts presented have been developed over the past two decades through experimentation and conversations with a host of plant physiologists, horticulturists and microbiologists who have devoted significant time and thought to the process of ice formation in plants.

Beginning in the late 1970s, a considerable amount of research focused on ice-nucleating agents and their role in inducing plants to freeze at warm, subzero temperatures. Research focused on the identification of extrinsic, especially ice-nucleation-active (INA) bacteria, and intrinsic nucleation agents and their role in the freezing process. While published research in this area diminished greatly in the 1990s, new insights were gained when high-resolution IR thermography was employed to study the freezing process. Additionally, published reports on antifreeze proteins (exhibit hysteresis, bind to ice crystals and affect their morphology, and have the ability to inhibit nucleating compounds) and anti-nucleators (compounds that inhibit the activity of nucleating agents but do not exhibit hysteresis) have added to the complexity of our understanding of what induces a plant to freeze. A review of these findings is presented.

Deep supercooling Of the many aspects of biological ice nucleation and cold hardiness of plants, deep supercooling is perhaps the most problematic and

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

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difficult to study. The ability of some plants to maintain symplastic water in an unfrozen condition and without movement of water into the apoplast is a remarkable adaptation that has impressed both biophysicists and plant physiologists. Although the ability of woody plant tissues to avoid freezing by deep supercooling was first documented in the 1960s, the mechanism that allows small domains of water to avoid freezing, despite the presence of extracellular ice, remains little understood. While a great amount of attention has been placed on identifying genes responsible for cold acclimation and understanding their regulation, a similar effort on deep supercooling has been absent. While deep supercooling is considered largely a biophysical trait related to the composition of cell walls, evidence suggests this contention needs further evaluation and that even though cell wall composition and tissue structure play a critical role in deep supercooling, there are many aspects that must be genetically regulated either during development or even on an annual basis.

Ice Nucleation and Propagation In April 2007, the midwest, central and southern plains and south-east portions of the USA experienced a record-breaking freezing event that caused unprecedented damage to many economically important crops (Gu et al., 2008; Warmund, 2008). While heroic efforts were made to provide frost protection, most efforts were futile. Partially to blame for this failure is our continuing lack of knowledge about what makes plants freeze at a particular temperature and what frost protection methods should be used for different freezing weather conditions (Poling, 2008; Wisniewski et al., 2008). The need for a better understanding of ice nucleation and propagation in plants has been noted (Ball et al., 2002; Wisniewski et al., 2002a; Hacker and Neuner, 2007, 2008; Wisniewski et al., 2008). Podcasts of an overview of the subject of ice nucleation in plants and other topics associated with the 2007 US freeze are available at http://ashs.org/db/horttalks/detail. lasso?id=103. The temperature at which ice melts (0°C) is well defined but the temperature at which

water will freeze is not predetermined. Pure water has the ability to supercool to temperatures as low as −40°C (homogeneous nucleation temperature) and perhaps even to temperatures as low as −100°C (Franks, 1985; Chen et al., 2006) but freezes at much warmer, subzero temperatures due to the presence of heterogeneous nucleators that are very effective at inducing ice crystal formation (Franks, 1985). Stated in a simple manner, heterogeneous nucleators act as a template that make it easier for water molecules to begin to take on a crystalline arrangement. Once a core of water molecules has assumed this crystalline arrangement (ice nucleus), the ice nucleus acts as a catalyst to induce the freezing of the surrounding water molecules. Heterogeneous nucleators related to plant freezing can be of two sources, extrinsic and intrinsic, the former representing a foreign substance while the latter representing a natural component of the plant. Understanding the role and source of heterogeneous nucleators in ice nucleation of plants is extremely important because if methods can be developed for regulating their activity, significant advances could be made in limiting frost injury to freezing-sensitive plants (Wisniewski and Fuller, 1999). Part of the problem in trying to resolve how plants freeze has been the difficulty in determining where freezing is initiated in a plant and how the freezing process is propagated throughout the plant. Questions such as: can plants supercool in the absence of extrinsic nucleators, how many nucleation events are needed for a whole plant to freeze, do barriers exist within plants that influence the rates or avenues of ice propagation, how do cold acclimation, antifreeze proteins, anti-nucleators and elevated CO2 affect ice nucleation, and what determines the natural patterns of frost injury present in a field after a freezing event, remain unanswered. Part of the problem in trying to address these questions has been due to the available technology. Until recently, the main approach to monitoring freezing in plants has been through the use of thermocouples. Thermocouples provide localized information on the temperature at which a freezing event occurs, however they do not provide information on the initial site of nucleation or the rate of ice propagation. Additionally, there is

Ice Nucleation, Propagation and Deep Supercooling

evidence that the electrical field associated with thermcouples and/or the insertion of thermocouples into a plant can itself promote nucleation events that would otherwise not occur (Wisniewski et al., 1997; Weissbuch et al., 2003; Lahav and Leiserowitz, 2007). Wisniewski et al. (1997) demonstrated the advantages of using high-resolution IR thermography and this technology is now being used more routinely to study the freezing process in plants (Ball et al., 2002; Lutze et al., 1998; Sekozawa et al., 2004; Hacker and Neuner, 2007, 2008). NMR micro-imaging has also been used to better understand the freezing process in plants (Ishikawa et al., 1997; Ishikawa et al., Chapter 3, this volume). Using infrared technology, it has been possible to determine the temperature at which ice is initiated in a wide variety of plants, how plant structure affects ice nucleation and propagation, how specific patterns of freezing relate to patterns of freezing injury and the role of extrinsic nucleators in the freezing process (see review by Wisniewski et al., 2008). The sensitivity and versatility of this technology is shown in Fig. 1.1 in which droplets of water containing the INA bacterium, Pseudomonas syringae, are seen to have frozen on the petals of

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an apple flower. More recently, Hacker and Neuner (2007, 2008) have increased the ease of visualizing the freezing process by using the infrared camera in a differential thermal imaging mode.

Factors Affecting Ice Nucleation Moisture and extrinsic ice-nucleating agents Two critical elements that greatly contribute to determining the temperature at which plants will freeze are the presence of moisture and extrinsic nucleating agents (Lindow, 1983; 1995; Ashworth, 1992; Wisniewski and Fuller, 1999; Wisniewski et al., 2002a). Dry plants will supercool to a lower temperature than wet plants, although it is not clear whether the ice nucleation activity at warmer temperatures is due to the presence of moisture itself or the result of the moisture activating extrinsic icenucleating agents present on the plant surface. Leaves having drops of water on their surface containing INA bacteria will freeze at a warmer, subzero temperature than leaves with drops plain water (Wisniewski et al., 1997).

2 min 22 sec

Fig. 1.1. Frozen droplets of water containing ice-nucleation-active bacteria (Pseudomonas syringae) on the petals of an apple flower as seen with high-resolution IR thermography.

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The uniform response of plants sprayed with water freezing at warm, subzero temperatures compared with dry plants would suggest that moisture itself is sufficient to trigger freezing events at temperatures just below 0°C. However, data also indicate that if leaves can be kept dry a significant level of freezing avoidance, and hence frost protection, can be provided. During the major freeze of 2007 in the USA, it was reported (B. Poling, Raleigh, North Carolina, 2008, personal communication) that strawberries that were under row covers survived an episodic frost of −6.1°C even though flower tissues had a hardiness value of approximately −2.0°C. A plausible explanation is that the plants remained dry under the row covers and hence were able to supercool rather than freeze at warmer temperatures due to the activity of extrinsic ice-nucleating agents. The row covers themselves did not afford enough thermal protection to prevent freezing. Inherent in the argument that dry plants supercool more than wet plants is the premise that the plant in question does not contain intrinsic ice-nucleating agents that are active at warm temperatures (Ashworth and Kieft, 1995; Wisniewski and Fuller, 1999). This has been a controversial point of discussion in the literature (see review by Wisniewski et al., 2002a; Ishikawa et al., Chapter 3, this volume). While it appears that many herbaceous species are free of intrinsic ice-nucleating agents that are active in the same temperature range as INA bacteria, woody plants appear to be rich in active ice-nucleating agents that appear to play a role in establishing preferred sites of ice formation (see discussion below).

Hydrophobic barriers to ice propagation While the dynamics of intrinsic ice nucleation events has been examined by several researchers using high-resolution IR thermography (Ball et al., 2002; Gusta et al., 2004; Hacker and Neuner, 2007, 2008; Fuller et al., Chapter 2, this volume), the process by which ice on the surface of a plant induces the plant to freeze has been less studied. Using bean (Phaseolus vulgaris) plants, Wisniewski and Fuller (1999) demonstrated that ice crystals must physically grow through a crack in the cuticle, a broken epider-

mal hair or a stoma to induce ice nucleation within the plant. Furthermore, corroborating earlier work by Wisniewski et al. (1997) on rhododendron, they also demonstrated that the thick cuticle present on azalea leaves (Rhododendron spp.) was sufficient to block external ice from inducing an internal nucleation event. Workmaster et al. (1999) also reported on the ability of a thick cuticle to block ice nucleation in cranberry (Vaccinium macrocarpon). These results suggested that hydrophobic barriers could prevent external freezing events from inducing a plant to freeze. Tomato (Lycopersicon esculentum) plants coated with a hydrophobic particle film supercooled to as low as −6°C, despite having been sprayed with water containing INA bacteria (Wisniewski et al., 2002b). In contrast, control plants (uncoated and sprayed) froze at −2.5°C. Similar results were obtained by Fuller et al. (2003), who demonstrated potato (Solanum tuberosum), grape (Vitis vinifera) and citrus (Citrus limon) coated with a hydrophobic particle film supercooled more than control plants.

Plant structure and its role in ice formation and propagation It has commonly been observed that the formation of ice within a plant does not occur in a uniform manner but rather at select sites where ice preferentially accumulates (reviewed by Ashworth, 1992; Pearce, 2001; McCully et al., 2004; Ishikawa et al., Chapter 3, this volume). The factors associated with determining where ice forms and accumulates are not clearly understood but clearly must involve aspects of plant structure that have evolved to deal with ice formation within tissues and the activity of intrinsic nucleating agents, presumably of plant origin. The mid-rib of leaves, the base of petioles, areas near vascular bundles and phloem fibres have all been identified using infrared technology as common sites for initial nucleation events (Ball et al., 2002, 2004; Hacker and Neuner, 2008). McCully et al. (2004) conducted a detailed analysis of ice formation in the petioles of two frost-tolerant herbaceous plants (Trifolium repens and Eschscholzia californica). It was concluded that these plants have evolved a complicated arrangement of

Ice Nucleation, Propagation and Deep Supercooling

structural strengths and weaknesses within petiole tissues which enables them to accommodate large volumes of intercellular ice during freezing events. Upon thawing, the previously frozen tissue returned to its original structural organization. These findings raised several questions regarding the composition and quality of the cell walls associated with the points of strengths and weaknesses, and the mechanism of water movement to and from sites of ice for-

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mation. An example of selective locations of ice formation is presented in Fig. 1.2, where voids created by the formation of ice can be seen in bud scale tissues and in pith tissues subtending the floral bud of peach (Prunus persica). Wisniewski et al. (1997), Workmaster et al. (1999), Carter et al. (2001), Hacker and Neuner (2007) and Fuller et al. (see Chapter 2, this volume) have all noted the existence of barriers within a plant that influence the direction

Fig. 1.2. Longitudinal section through a peach flower and its subtending axis after being exposed to a freezing event. Voids in the pith tissue subtending the flower and within the surrounding bud scales are evident and result from the formation of ice during the freezing event.

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and rate of ice propagation. In some cases, this may directly affect the resulting pattern of injury observed after a potentially lethal freezing event has occurred. It appears that where ice forms, how it propagates and how it is accommodated are all important factors that affect the ability of a plant to survive freezing and may be as important as the ability to withstand the dehydrative stresses associated with ice formation. Unfortunately, while the latter topic has received much attention in the literature, the former topic has largely been neglected (Gusta et al., Chapter 21, this volume).

Ice nucleators, antifreeze proteins, sugars and anti-nucleators The ice nucleation activity of bacteria has been well characterized (Lindow, 1995) and the protein, and corresponding gene, have been isolated and identified (Lindow et al., 1989). In contrast, while ice nucleation activity of plant origin has been commonly observed, especially in woody plants (Ashworth and Kieft, 1995), the compounds responsible for the ice nucleation activity have not been identified. Reports on the composition of plant INA compounds have ranged from a soluble (Krog et al., 1979; Embuscado et al. 1996) or structural (cell wall) polysaccharide (Gross et al., 1988) to either a protein (Constantinidou and Menkissoglu, 1992) or a complex molecule such as a phospholipid (Brush et al., 1994). So, while there is strong evidence for the existence of plant INA compounds, their identity remains ambiguous, as does an understanding of their origin, development, distribution, turnover and role in adaptation to freezing temperatures. The temperature at which extrinsic nucleation occurs in plants appears to be an adaptive process requiring de novo synthesis, as it is influenced by cold acclimation which in turn is affected by ambient levels of CO2 (Lutze et al., 1998; Beerling et al., 2001; Ball and Hill, Chapter 18, this volume; Ishikawa et al., Chapter 3, this volume). Antifreeze proteins (AFPs), also known as hysteresis proteins (THPs), inhibit ice crystal growth in a non-colligative mechanism, lowering the freezing point of water below the melting point, thereby producing a thermal hysteresis

(DeVries, 1971; Duman and Olsen, 1993). First described in fish, AFPs have also been reported in insects (Duman, 2001) and plants (Griffith and Yaish, 2004). Thermal hysteresis activity of plant AFPs is low (0.2–0.5°C) compared with fish (0.7–1.5°C) and insects (3–6°C). Because of the low activity of plant AFPs, questions have been raised regarding their role in the survival of plants exposed to freezing temperatures. Griffith et al. (2005) demonstrated that rye (Secale cereale) AFPs did not exhibit any cryoprotective activity but rather interacted directly with ice in planta and reduced freezing injury by slowing the growth and recrystallization of ice. Gusta et al. (2004) reported that sugars had a much greater effect than proteins on determining rates of ice propagation in strips of filter paper. Wisniewski et al. (1999) reported that a dehydrin (PCA60) obtained from peach bark tissues exhibited both cryoprotective and antifreeze activity. The antifreeze activity was surprising since PCA60 is an intracellular protein and AFPs are generally secreted proteins. Postulating on the role of PCA60, they suggested that perhaps the main function of PCA60 was to act as an anti-nucleator, inhibiting the activity of ice-nucleating agents as had been described for other AFPs (Parody-Morreale et al., 1988; Zamecnik and Janacek, 1992). Interestingly, when Huang et al. (2002) expressed an insect AFP gene from the beetle, Dendroides canadensis, in Arabidopsis, supercooling of dry plants was enhanced. The increase in supercooling was much greater than the hysteresis activity, suggesting that the insect AFP blocked the activity of intrinsic ice-nucleating agents. Duman (2002) also reported that the inhibition of ice nucleators by insect AFPs could be enhanced by glycerol and citrate. Anti-nucleators (compounds that inhibit ice nucleation activity but do not exhibit hysteresis) have been identified from a variety of sources including microorganisms, insects, plants and synthetic polymers (see review by Holt, 2003; Fujikawa et al., Chapter 4, this volume). While they have been shown to inhibit ice nucleation activity in vitro, their role in defining the temperature at which a plant will freeze has not been definitely demonstrated. The practical application of these compounds in promoting supercooling has yet to be explored.

Ice Nucleation, Propagation and Deep Supercooling

Deep Supercooling in Buds and Xylem Tissues of Woody Plants Deep supercooling of bud (reviewed by Quamme, 1995) and xylem parenchyma tissues (reviewed by Wisniewski, 1995; Fujikawa et al., Chapter 4, this volume) of woody plants is one of the most enigmatic aspects of biological ice nucleation and cold hardiness. Water in these tissues exists in the liquid phase to temperatures as low as −50°C by being isolated from internal, heterogeneous ice nucleators including extracellular ice (Burke, 1979). Upon nucleation freezing occurs intracellularly, which is a lethal event. There is a strong correlation between the temperature range of deep supercooling and tissue injury (see review by Wisniewski, 1995). Deep supercooling in plant tissues can be monitored using differential thermal analysis (DTA) as outlined by Quamme et al. (1982), which relies on the use of thermocouples to detect the latent heat released by water in tissues as it undergoes a liquid to solid phase change. Despite deep supercooling being so integral to the cold hardiness of many woody plants, especially economically important tree fruit crops, it has received only minor research attention in the last 20 years. The majority of the research effort to understand deep supercooling in woody plants has been conducted by Fujikawa and colleagues (Fujikawa et al., Chapter 4, this volume). Early work indicated that deep supercooling of xylem tissues predominated in northern hardwood species, especially those having ring porous xylem structure (George et al., 1974; Becwar et al., 1981). Due to the homogeneous nucleation point of water (−38°C), it was suggested that species exhibiting deep supercooling would be limited to below the −40°C isotherm. Gusta et al. (1983) reported several exceptions to the −40°C isotherm limit and suggested that under prolonged exposure to freezing temperature (several weeks), xylem parenchyma cells could slowly dehydrate which resulted in depression of the freezing point. Additionally, Kuroda et al. (1997, 2003) have suggested that tropical, subtropical and boreal tree species also supercool (see review by Fujikawa et al., Chapter 4, this volume), although this has not yet been corroborated by

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other researchers. Karlson et al. (2004) indicated that within the genus Cornus, deep supercooling is an ancestral trait that was present prior to the development of freeze tolerance mechanisms that rely on the accumulation of cryoprotective proteins such as dehydrins. As stated by George and Burke (1977), in order for tissues to supercool, the cells within the tissue must: (i) be free of heterogeneous nucleating agents that are active at warm temperatures; (ii) have a barrier the excludes the growth of ice crystals into the supercooled cells from the surrounding apoplast; (iii) have a barrier that prevents the rapid loss of cellular water to sites of extracellular ice despite the presence of a large vapour pressure gradient; and (iv) have cell walls with sufficient tensile strength to counteract the negative hydrostatic pressures that result from a large vapour pressure gradient. Current theory suggests that the ability to deep supercool is largely a biophysical trait and defined by the physical properties of the apoplast rather than the symplast in which the capillary structure (porosity) of the cell wall plays an essential role (Wisniewski, 1995). While this is an attractive hypothesis it does not explain how the ability to deep supercool changes on a seasonal basis or how different species of temperate tree species that exhibit similar wood properties are either deep supercooling or not. Figure 1.3 is an electron micrograph of a xylem ray parenchyma cell in midwinter when supercooling is at its maximum. As reviewed by Wisniewski (1995), rather than the entire cell wall, the properties of the pit membrane and underlying protective layer may define whether or not a xylem cell would supercool. Removal of pectin from these portions of the cell wall dramatically decreased the ability of the cells to supercool, which suggests that seasonal changes in pit membrane structure could account for seasonal changes in deep supercooling. Wisniewski et al. (1999) also suggested that AFPs within the xylem parenchyma cells of peach xylem ray parenchyma may block the activity of intrinsic nucleators and hence allow the cells to supercool. Kasuga et al. (2006) have identified anti-ice nucleation activity in xylem extracts in woody plants. The anti-ice nucleators are mainly

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Fig. 1.3. Electron micrograph of a cross-section through xylem tissues of peach (Prunus persica L. Batsch) showing a xylem ray parenchyma cell that is located next to a vessel element. Note the complexity of the wall structure of the pit membrane, which is composed of a primary wall from adjacent cells, a middle lamella and a tertiary wall (protective layer) that is formed after completion and lignification of the secondary cell wall. Pit membranes are often also covered with a ‘black cap’ of carbohydrate material. Wisniewski (1995) proposed that it is the structure of the pit membrane that determines the degree of deep supercooling exhibited by the living cells in the xylem tissues of woody plants.

secondary metabolites and may play a key role in defining the supercooling ability of xylem parenchyma cells (Fujikawa et al., Chapter 4, this volume). These findings provide a new mechanism of regulating deep supercooling in woody plants.

Conclusions The past 20 years have seen an explosion of research in trying to identify genes involved in cold acclimation. Hundreds of genes are affected by exposure to low temperature but studies have mainly focused on genes that provide cryoprotection or tolerance to dehydrative stress. Some of the genes identified,

however, may also be involved in other aspects of adaption to low temperature. As Gusta et al. (see Chapter 21, this volume) have indicated, the freezing process, as well as a plant’s response to the presence of ice within its tissues, is complex and quite diverse. Factors such as ice nucleation activity, antifreeze proteins, anti-nucleators and plant structure should be considered to develop a holistic understanding of cold hardiness within any given species. Ice nucleation, ice propagation and deep supercooling are integral aspects of plant adaptation to freezing temperatures. A deeper understanding of the genetic regulation and inheritance of these traits will lead to new strategies and technologies for improving plant cold hardiness.

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Ball, M.C., Woldfe, J., Canny, M., Hofmann, M., Nicotra, A.B. and Hughes, D. (2002) Space and time dependence of temperature and freezing in evergreen leaves. Functional Plant Biology 29, 1259–1272. Ball, M.C., Canny, M.J., Huang, C.X. and Heady, R. (2004) Structural changes in acclimated and unacclimated leaves during freezing and thawing. Functional Plant Biology 31, 29–40. Becwar, M.R., Rajashekar, C., Hansen-Bristow, K.J. and Burke, M.J. (1981) Deep supercooling of tissue water and winter hardiness limitations in timberline flora. Plant Physiology 68, 111–114. Beerling, D., Terry, A., Mitchell, P., Callaghan, T., Gwynn-Jones, D. and Lee, J. (2001) Time to chill: effects of simulated global change on leaf ice nucleation temperatures of subarctic vegetation. American Journal of Botany 4, 628–633. Brush, R.A., Griffith, M. and Mlynarz, A. (1994) Characterization and quantification of intrinsic ice nucleators in winter rye (Secale cereale) leaves. Plant Physiology 104, 725–735. Burke, M.J. (1979) Discussion. Water in plants: the phenomenon of frost survival. In: Underwood, L.S., Tieszen, L.L., Callahan, A.B. and Folk, G.E. (eds) Comparative Mechanisms of Cold Adaptations. Academic Press, New York, New York, pp. 259–281. Carter, J., Brennan, R. and Wisniewski, M. (2001) Patterns of ice formation and movement in blackcurrant. HortScience 36, 1027–1032. Chen, S.-H., Mallamace, F., Mou, C.-Y., Brocdo, M., Corsavo, C., Faraone, A. and Liu, L. (2006) The violation of the Stokes–Einstein relation in supercooled water. Proceedings of the National Academy of Sciences USA 103, 12974–12978. Constantinidou, H.A. and Menkissoglu, O. (1992) Characteristics and importance of heterogeneous ice nuclei associated with Citrus fruits. Journal of Experimental Botany 43, 585–591. DeVries, A.L. (1971) Glycoproteins as biological antifreeze agents in Antarctic fishes. Science 172, 1152–1155. Duman, J.G. (2001) Antifreeze and ice nucleator proteins in terrestrial arthropods. Annual Review of Physiology 63, 327–357. Duman, J.G. (2002) The inhibition of ice nucleators by insect antifreeze proteins is enhanced by glycerol and citrate. Journal of Comparative Physiology 172, 163–168. Duman, J.G. and Olsen, T.M. (1993) Thermal hysteresis protein activity in bacteria, fungi, and phylogenetically diverse plants. Cryobiology 30, 322–328. Embuscado, M.E., BeMiller, J.N. and Knox, E.B. (1996) A survey and partial characterization of ice-nucleating fluids secreted by giant-rosette (Lobelia and Dendrosenecio) plants of the mountains of eastern Africa. Carbohydrate Polymers 31, 1–9. Fuller, M.P., Hamed, M., Wisniewski, M. and Glenn, D.M. (2003) Protection of plants from frost using hydrophobic particle film and acrylic polymer. Annals of Applied Biology 143, 93–97. Franks, F. (1985) Biophysics and Biochemistry at Low Temperatures. Cambridge University Press, Cambridge, UK. George, M.F. and Burke, M.J. (1977) Cold hardiness and deep supercooling in xylem of shagbark hickory. Plant Physiology 59, 319–325. George, M.F., Burke, M.J., Pellet, H.M. and Johnson, A.G. (1974) Low temperature exotherms and woody plant distribution. HortScience 9, 519–522. Griffith, M. and Yaish, M.W.F. (2004) Antifreeze proteins in overwintering plants. Trends in Plant Science 9, 399–405. Griffith, M., Lumb, C., Wiseman, S.B., Wisniewski, M., Johnson, R.W. and Marangoni, A.G. (2005) Antifreeze proteins modify the freezing process in planta. Plant Physiology 138, 330–340. Gross, D.C., Proebsting, E.L. and MacCrindle-Zimmerman, H. (1988) Development, distribution, and characteristics of intrinsic, nonbacterial ice nuclei in Prunus wood. Plant Physiology 88, 915–922. Gu, L., Hanson, P.J., Post, W.M., Kaiser, D.P., Yang, B., Nemani, R., Pallardy, S.G. and Meyers, T. (2008) The 2007 eastern US spring freeze: increased cold damage in a warming world? Bioscience 58, 253–262. Gusta, L.V., Tyler, M.J. and Chen, T.H. (1983) Deep undercooling in woody taxa growing north of the −40°C isotherm. Plant Physiology 72, 122–128. Gusta, L.V., Wisniewski, M., Nestbitt, N.T. and Gusta, M.L. (2004) The effect of water, sugars, and proteins on the pattern of ice nucleation and propagation in acclimated and nonacclimated canola leaves. Plant Physiology 135, 1641–1653. Hacker, J. and Neuner, G. (2007) Ice propagation in plants visualized at the tissue level by infrared differential thermal analysis (IDTA). Tree Physiology 27, 1661–1670. Hacker, J. and Neuner, G. (2008) Ice propagation in dehardened alpine plant species studied by infrared differential thermal analysis (IDTA). Arctic Antarctic and Alpine Research 40, 660–670.

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Holt, C.B. (2003) substances which inhibit ice nucleation: a review. CryoLetters 24, 269–274. Huang, T., Nicodemus, J., Zarka, D.G., Thomashow, M.F., Wisniewski, M. and Duman, J.G. (2002) Expression of an insect (Dendroides canadensis) antifreeze protein in Arabidopsis thaliana results in a decrease in plant freezing temperature. Plant Molecular Biology 50, 333–344. Ishikawa, M., Price, W.S., Ide, H. and Arata, Y. (1997) Visualization of freezing behaviors in leaf and flower buds of full-moon maple by nuclear magnetic resonance microscopy. Plant Physiology 115, 1515–1524. Karlson, D.T., Xiang, Q.-Y., Stirm, V.E., Shirazi, A.M. and Ashworth, E.N. (2004) Phylogenetic analyses in Cornus substantiate ancestry of xylem supercooling freezing behavior and reveal lineage of desiccation related proteins. Plant Physiology 135, 1654–1665. Kasuga, J., Mizuno, K., Miyaji, N., Arakawa, K. and Fujisawa, S. (2006) Role of intracellular contents to facilitate supercooling capability in beech (Fagus crenata) xylem parenchyma cells. CryoLetters 27, 305–310. Krog, J.O., Zachariassen, K.E., Larson, B. and Smidsrod, O. (1979) Thermal buffering in afro-alpine plants due to nucleating agent-induced water freezing. Nature 282, 300–301. Kuroda, K., Ohatani, J. and Fujikawa, S. (1997) Supercooling of xylem parenchyma cells in tropical and subtropical hardwood species. Trees 12, 97–106. Kuroda, K., Kasuga, J., Arakawa, K, and Fujikawa, S. (2003) Xylem ray parenchyma cells in boreal hardwood species respond to subfreezing temperatures by deep supercooling that is accompanied by incomplete desiccation. Plant Physiology 131, 736–744. Lahav, M. and Leiserowitz, L. (2007) Ice freezing induced by amphiphilic alcohols and local electric fields of polar crystals. In: Proceedings of the 2nd Annual Conference on the Physics, Chemistry, and Biology of Water, 18–21 October 2007, Brattleboro, Vermont; available at http://vermontphotonics.net/water_2007/ abstracts.html Lindow, S.E. (1983) The role of bacterial ice nucleation in frost injury to plants. Annual Review of Phytopathology 21, 363–384. Lindow, S.E. (1995) Control of epiphytic ice-nucleation-active bacteria for management of plant frost injury. In: Lee, R.E. Jr, Warren, G.J. and Gusta, L.V. (eds) Biological Ice Nucleation and Its Applications. APS Press, St Paul, Minnesota, pp. 239–256. Lindow, S.W., Lahue, E., Govindarajan, A.G., Panopoulos, N.J. and Gies, D. (1989) Localization of ice nucleation activity and the iceC gene product in Pseudomonas syringae and Escherichia coli. Molecular Plant– Microbe Interactions 2, 262–272. Lutze, J.L., Roden, J.S., Holly, C., Wolfe, J., Egerton, J.J.G. and Ball, M.C. (1998) Elevated atmospheric CO2 promotes frost damage in evergreen tree seedlings. Plant, Cell and Environment 21, 631–635. McCully, M.E., Canny, M.J. and Huang, C.X. (2004) The management of extracellular ice by petioles of frostresistant herbaceous plants. Annals of Botany 94, 665–674. Parody-Morreale, A., Murphy, K.P., DiCera, E., Fall, R., DeVrie, A.L. and Gill, S.J. (1988) Inhibition of bacterial ice nucleators by fish antifreeze glycoproteins. Nature 333, 782–783. Pearce, R.S. (2001) Plant freezing and damage. Annals of Botany 87, 417–424. Poling, B. (2008) Spring cold injury to winegrapes and protection strategies and methods. HortScience 43, 1652–1662. Quamme, H.A. (1995) Deep supercooling in buds of woody plants. In: Lee, R.E. Jr, Warren, G.J. and Gusta, L.V. (eds) Biological Ice Nucleation and Its Applications. APS Press, St Paul, Minnesota, pp. 183–199. Quamme, H.A., Chen, P.M. and Gusta, L.V. (1982). Relationship of deep supercooling and dehydrative resistance to freezing injury in dormant stems of ‘Starkcrimson Delicious’ apple and ‘Siberian C’ peach. Journal of the American Society of Horticultural Science 107, 299–304. Sekozawa, Y., Sugaya, S. and Gemma, H. (2004) Observations of ice nucleation and propagation in flowers of Japanes pear (Pyrus pyrifolia Nakai) using infrared video thermography. Journal of the Japanese Society of Horticultural Science 73, 1–6. Warmund, M. (2008) Temperatures and cold damage to small fruit crops across the eastern US associated with the April 2007 freeze. HortScience 43, 1643–1647. Weissbuch, I., Lahav, M. and Leiserowitz, L. (2003) Toward stereochemical control monitoring, and understanding of crystal nucleation. Crystal Growth and Design 3, 125–150. Wisniewski, M. (1995) Deep supercooling in woody plants and the role of plant structure. In: Lee, R.E. Jr, Warren, G.J. and Gusta, L.V. (eds) Biological Ice Nucleation and Its Applications. APS Press, St Paul, Minnesota, pp. 163–181.

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Wisniewski, M., and Fuller, M.P. (1999) Ice nucleation and deep supercooling in plants: new insights using infrared thermography. In: Maregesin, R. and Schinner, F. (eds) Cold-Adapted Organisms: Ecology, Physiology, Enzymology and Molecular Biology. Springer Verlag, Berlin, pp. 105–118. Wisniewski, M., Lindow, S. and Ashworth, E. (1997) Observations of ice nucleation and propagation in plants using infrared thermography, Plant Physiology 113, 327–334. Wisniewski, M., Webb, R., Balsamo, R., Close, T.J., Yu, X.-M. and Griffith, M. (1999) Purification, immunolocalization, cryoprotective, and antifreeze activity of PCA60: a dehydrin from peach (Prunus persica). Physiologia Plantarum 105, 600–608. Wisniewski, M., Fuller, M., Glenn, D.M., Gusta, L., Duman, J., and Griffith, M. (2002a) Extrinsic ice nucleation in plants: What are the factors involved and can they be manipulated. In: Li, P.H. and Palva, E.T. (eds) Plant Cold Hardiness: Gene Regulation and Genetic Engineering. Kluwer Academic/Plenum Publishers, New York, New York, pp. 211–221. Wisniewski, M., Glenn, D.M. and Fuller, M.P. (2002b) Use of a hydrophobic particle film as a barrier to extrinsic ice nucleation in tomato plants. Journal of the American Society of Horticultural Science 127, 358–364. Wisniewski, M., Glenn, D.M., Gusta, L. and Fuller, M.P. (2008) Using infrared thermography to study freezing in plants. HortScience 43, 1648–1651. Workmaster, B.A., Palta, J.P. and Wisniewski, M. (1999) Ice nucleation and propagation in cranberry uprights and fruit using infrared thermography. Journal of the American Society of Horticultural Science 124, 619–625. Zamecnik, J. and Janacek, J. (1992) Interaction of antifreeze proteins from cold hardened cereals with ice nucleation active bacteria. Cryobiology 29, 718–719.

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Low-temperature Damage to Wheat in Head – Matching Perceptions with Reality M.P. Fuller, J. Christopher and T. Fredericks

Abstract Wheat grown in eastern Australia can suffer severe frost damage during radiation frosts at ear emergence. Few studies have attempted to understand the characteristics of freezing and frost damage to wheat during late developmental stages. While the cultivars used in this region have adequate frost tolerance during their vegetative development, this is not maintained during ear emergence. It is perceived that this lack of head resistance is genetically controlled and that germplasm may be available for specific radiation frost resistance. Recent work has shown, however, that cold tolerance cannot be up-regulated at these developmental stages and that conventional screening of germplasm has little chance of producing field resistant material. However, wheat in head can tolerate a limited amount of freezing without damage and furthermore can supercool substantially in both controlled environments and in the field. The present chapter demonstrates the evidence for these findings and suggests new approaches for screening for resistance in an attempt to make headway in this recalcitrant phenomenon.

Introduction In subtropical areas and some Mediterranean regions, spring wheat is sown in the late summer and autumn to allow flowering in midwinter to avoid water stress during the summer months (Woodruff and Tonks, 1983). Such cropping strategies expose the flowering crop to episodic frost risk. Significant crop losses in eastern Australia due to frost damage have resulted in economic losses in excess of $AU100 million per annum. Occasional late frosts also cause frost damage to wheat in head in several other regions including the Mediterranean, South America and in continental Canada, Russia and the USA (Shroyer et al., 1995). In Queensland and northern New South Wales (NSW), Australia, yield losses associated with frost damage are significant and gross yield reductions of 10% as a direct result of frost injury are common. Record losses occurred in the 1965 season when 50% of

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the NSW wheat crop was lost due to heavy frosts (Boer et al., 1993). Individual regions can suffer losses in excess of 85% (Paulsen and Heyne, 1983) and individual growers risk total crop loss. Growers frequently minimize frost risk by delayed planting and using longer-season varieties which delays flowering beyond the most significant frost risk periods (Gomezmacpherson and Richards, 1995; Shackley and Anderson, 1995). These measures, however, result in delayed maturity and can in themselves lead to even greater yield and quality losses if grain-filling is pushed into a significant drought period or the beginning of the rainy season (Woodruff and Tonks, 1983; Woodruff, 1992). Thus frost damage limits yield not only by causing actual damage but also by restricting use of the most effective flowering period. A compromise between the effects of frost and drought has been sought using computer-aided decision-making systems such as ‘WHEATMAN’ (Woodruff and Tonks, 1983).

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

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Tolerance to freezing damage during the vegetative stage does not appear to confer tolerance in the reproductive stages in wheat (Fuller et al., 2007). Many elite winter wheats are tolerant to temperatures of −20°C in the vegetative stages (LT50) but suffer severe damage at much more moderate temperatures (−5 to −7°C) during the reproductive stages. Despite the apparent high levels of freezing tolerance in winter wheats, there appears to be little or no additional protection from frost injury in the reproductive stages compared with spring varieties (Paulsen and Heyne, 1983; Marcellos and Single, 1984; Cromey et al., 1998; Fuller et al., 2007). Vegetative freezing tolerance requires a period of 7 to 14 days’ exposure to temperatures less than 8°C before there is significant increase in frost tolerance in winter wheat. Unfortunately, the temperatures to confer acclimation in winter wheat are rarely experienced for prolonged periods during the flowering stages of either winter or spring wheats in eastern Australia; therefore it is possible that the frost damage during flowering in wheat is a result of a lack of expression of acclimation. The physical processes occurring during freezing temperatures in wheat plants during the reproductive stages are yet to be fully characterized and this work is complicated by the difficulty of working with natural frost events. Screening using natural frosts in the field can be problematic due to the unpredictable nature of frost events in terms of both timing and severity. The effect of natural frosts on winter cereals post ear emergence is difficult to simulate, but it may not be necessary to faithfully reproduce the conditions of a natural frost to successfully identify plants with increased resistance. Unfortunately, there are no conclusive data to indicate that this can be achieved with any of the frost cabinets tested to date (Fredericks et al., 2004). Despite more than a century of interest in improving in-head frost resistance in Australian wheats (Farrer, 1900 cited in Single, 1985), very little genetic gain has been made. In the current chapter we present some initial findings on freezing of in-head wheat aimed at examining the effect of simulated freezing in an attempt to characterize ice

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nucleation and frost resistance and to help develop sensible research strategies.

Materials and Methods A number of experiments were undertaken to investigate the freezing behaviour of wheat in ear. Results presented are observations taken from a range of these experiments. All plant material was spring habit wheat including the cultivars Hartog, SeriM82 and a number of experimental genotypes. Hartog is a cultivar widely grown in the Australian subtropical grain belt.

Frost testing Three freezing cabinets were used. Convective freezing was conducted in a Sanyo M533 incubator where the set temperatures were preprogrammed according to the desired regime. Radiative freezing was conducted in a custombuilt radiation freezing chamber (Fuller and Le Grice, 1998) and in a custom-built radiation chamber at the Australian Genome Research Facility (AGRF), Adelaide (www.agrf.org.au) (Long et al., 2005). The AGRF radiation frost chamber is a recently built chamber which provides a unique facility to address the Australian cereal frost problem. It utilizes three ceilingmounted freezing batteries cooled to approximately −20°C in a walk-in chamber with no air movement. It is being used to routinely screen genetic material in the Australian legume breeding programmes and is being tested for use with wheat and barley. Typically, plants were allowed to equilibrate to a pre-set chamber temperature of 2 to 4°C for 1 to 2 h prior to being subjected to subzero temperatures. Following exposure to subzero temperatures, the plants were held at 2 to 4°C for several hours to defrost. Monitoring of plants in the field at Kingsthorpe, Queensland was also undertaken during natural radiative freezing conditions. An IR camera (Inframetrics model 760) was used to observe the plants’ exposure to subzero temperatures in order to determine the temperature and location of ice nucleation

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and the freezing pattern of plants and plant parts, as described previously by Wisniewski et al. (1997), Fuller and Wisniewski (1998) and Pearce and Fuller (2001).

Results In many of the experiments undertaken it was apparent that plants or isolated ears of wheat demonstrated supercooling, i.e. ice did not occur in their tissues. Although plants were misted with water and droplets of water on the leaves froze, freezing was not detected in up to 90% of the culms (Fig. 2.1). In the experiment illustrated in Fig. 2.1, only one culm showed damage and this was the culm identified to have frozen and visualized by IR thermography. When analysed using Thermotechnix™ software, the culm that froze in this experiment showed a characteristic two-phase pattern of freezing (Fig. 2.2) with an initial quick freeze, interpreted as the freezing of the apoplastic water, followed by a second slower freeze interpreted as freeze dehydration. Symptoms of frost damage included bleaching of the head, which only occurred if

freezing in the tissues also occurred (Fig. 2.3). It was not possible to reproduce partial floret damage to the heads in controlled frost tests although partial floret damage is readily observable in the field (Fig. 2.3). Following two nights of field observation when the air temperature fell to −7°C, not a single ear of wheat was observed to freeze and detected by IR thermography; neighbouring vegetative wheat plants however were observed to freeze (Fig. 2.4). A standard freezing programme utilized for screening the Australian wheat and barley germplasm (2°C for 2 h, slow freeze to −4.5°C for 8 h, slow defrost to 2°C) was conducted on four pots of wheat at ear emergence. Analysis of this experiment by IR thermography revealed that only 15% of the plant material and only one ear froze while the remaining material supercooled (Fig. 2.5). It was possible to discern frozen tissue from unfrozen tissue during rewarming the chamber to −0.5°C (Fig. 2.5). Plants that had frozen are depicted as hatching in Fig. 2.5 in contrast to those which supercooled, which are circled (see Plate 1 where green represents plants that had frozen and red those

Fig. 2.1. Greyscale IR image of wheat plants at ear emergence during radiative freezing. Structures which appear white are undergoing freezing (exhibiting exotherms) and include water droplets and one culm (far left of picture).

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0.35 0.30

Temperature (d, °C)

0.25 0.20 0.15 0.10 0.05 0.00

-0.05 -0.10 -0.15 0.00

10.00

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Fig. 2.2. Temperature (d, °C) trace taken from a freezing wheat ear showing two phases of freezing.

Fig. 2.3. Observed frost damage to heads of wheat following freezing. Left: following frost testing, the head on the left froze and the three heads on the right supercooled; right, typical head damage observed in the field during head development. NB: see Plate 1 for colour representation.

which supercooled). When these plants were thawed, only the ear that was observed to freeze showed frost damage as illustrated in Fig. 2.3. Vegetative material that was observed to freeze showed no frost damage. Follow-up observations on the plants at grainfill showed that both control (unfrozen but with a moderate baseline levels of sterility) and treated (supercooled) plants had floret infertility and this was significantly higher (P3000 m); and (iii) African (Atlas Mountains, Kilimanjaro). Löve and Löve (1974) maintain that the oldest alpine flora can be traced through the Tibetan range to a mountain chain north of Tethys Sea, before separation of the continents. These cosmopolitan plants can also be found in the Southern Rocky Mountains, and might even be related to the Antarctic elements which escaped from glaciation. This suggests that alpine flora, although diverse, yet having

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common features, has evolved in the high altitudes. Some of its elements are older than the mountains which are presently harbouring them. They have evolved from the ordinary populations of lowland floras due to gradual changes in altitude owing to orogeny, and/or latitude owing to continental drift, over a long period of time. They are therefore a typical product of microevolution. True cold-hardened arctic and alpine species are extremophiles belonging to a specific subgroup of cryophytes. The cryophytes replaced the lush Nemoral vegetation even in the low-lying areas when the continents entered higher latitudes and the global climate deteriorated. The altitudinal treeline, and the permanent snow line above it, delineate the viable zone for alpine plants. In the tropics, the treeline is at ~4000 m level. With increasing latitude it descends, and in the highest Arctic/ Antarctic it drops to sea level. At these latitudes, the alpine and arctic tundra practically merge. As we find later, the descending alpine zone represents a corridor through which the alpine flora could reach the bare arctic landscape after the continental ice retreat.

Glaciations It goes against common sense to argue that the Ice Ages were not only unsupportive for the evolution of cold-hardened plants, but that they also strongly assisted in weakening and eradication of their populations around the world. This for the simple reason that the expanding continental ice sheet and surging mountain glaciers suffocated all higher structured life underneath the fallen snow which would not melt in summer and thus became perennial by changing to ice. During its geological history, planet Earth experienced several deep cooling periods known as Ice Ages. The oldest known was the Huronian Glaciation, 1.8 By ago, followed by the Eo-Cambrian (650 My), African (450 My), Permo-Carboniferous (280 My) and finally Quaternary, as recent as 2 My ago. Less prominent glacial advances may have occurred at other times as well. Although there are plausible theories interpreting climate and ice fluctuations within the glaciation period, the causes

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of the widely separated great Ice Ages are still unknown. The Quaternary Glaciation was heralded by a progressive temperature decrease in the late Tertiary, during the ‘rainy’ epoch, the Pliocene. Thick sand and gravel deposits preceding the younger glacial drift deposits, found in North Athabasca River beds, suggest heavy precipitation at the end of Tertiary. During the subsequent Pleistocene epoch, rapid fluctuations of temperature resulted in repeated glaciations and deglaciations of both hemispheres, changes in sea level and migration of flora and fauna. As many as ten glacial advances and retreats have been identified over the last 1.8 My. The major glacial periods got their names after the present North American states, indicating how far south their ice reached and where they deposited terminal moraines. From the oldest known to the most recent ones, these were: Pre-Nebraskan, Nebraskan, Kansan, Illinoian and Wisconsinan glacials (~100,000 years each). Interglacials, the in-between warm periods, with their particular names, lasted longer (~200,000 years). We are still in the midst of the Ice Age but live in the early phase of an interglacial called the Holocene. The epoch started about 11,000 years ago, after the Wisconsinan ice sheet began to rapidly melt away. The present global temperature is some 5°C higher than that during the full-blown glacial, but also by about the same margin lower than was the ‘global normal’ before and up to the mid-Tertiary. Prior to the present Ice Age, cryophytes, which had evolved over many millions of years on high plateaus, on top of the mountains and their north-facing slopes, were prolific in their particular ranges and specialized habitats. When the climate began cooling down, mountains developed ice caps, even ice fields, which covered and killed the extant cold-hardened and often also all the vegetation of the particular range. As the ice expanded, the physical realm of the cryophytes shrunk, forming only a girdle around the mountains. This alpine zone became sandwiched between the permanent snowline above and the altitudinal tree line below. None the less, alpine environments with their extreme conditions, cold-adapted plants and other biota must have always

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existed. They have been dispersed over all latitudes otherwise there would be no coldhardened plants at present. The Pleistocene glaciations spread over a significant portion of the affected continents. At the peak of expansion, 18,000 years ago, the ice covered almost a third of the global land surface compared with ~10% at present (Lange, 2005). The Wisconsinan ice sheet occupied 40.3 million km2, while the rapidly melting ice of the present day still covers 14.9 million km2, most of it being confined in the Antarctic.

Survival in (Marginal) Cold-region Environments Arctic plants are derivatives of their alpine cousins, and these, in turn, evolved from a ‘general’ non-specialized flora of a particular geographical region and era. In spite of their multisource origin, the cryophytes share common requirements for survival in their non-necessarily supportive, if not hostile environment. While the inter- and intraspecific competition for space and resources is the main struggle for plants in the mesic temperate and tropical ecosystems, the cryophytes simply strive to survive in their physically taxing niches, often each plant on its own.

Climate and microclimate Cold regions denote a cold climate. The climate (or macroclimate), in short, is defined by long-term weather patterns prevailing in a large region or a zone. Weather is described by short-term atmospheric conditions such as solar radiation, temperature, precipitation, humidity, wind and other physical parameters. However, plants, and organisms in general, do not recognize the climate. They respond to a climate near the ground, the microclimate, which, with the microtopography, soil moisture, nutrient availability, presence of other organisms, canopy shading, etc., determines the habitat’s microenvironment. The thickness of the microclimate layer varies. Generally, it diminishes with latitude (or altitude) and so does the height of the vegeta-

tion canopy. This, in turn, determines the nature, structure and composition of the plant community in a particular site or zone. Remarkably, during the July sunny noon, the nearground temperatures of the tropical forest and tundra vegetation are a surprisingly similar ~30°C. This suggests that even tundra plants require warm spells, to experience the ‘tropical’ milieu from which they have evolved, to initiate flowering and seed ripening. In polar regions the few centimetres of warm air near the ground make a difference between the vegetated and bare sites. Often such sheltered conditions exist only in a tiny cavity between rocks (Fig. 15.1). Curiously, while cold-hardened plants are not limited by light for photosynthesis, they are very much temperature (heat) limited. At the Alexandra Fiord upland, Ellesmere Island, the mean July temperature is ~5°C. Yet, when we tried to grow some of the local plants at 5°C in the growth chamber, they hardly produced new leaves and never flowered. In the tropics, the warm microclimate prevails for most of the year, in the temperate zone during the warmer part of the growing season and in the Arctic only for several days during the short summer.

Pre-selection and pre-adaptation The basic characteristic of evolution is its proverbial slowness. Sudden changes destroy the species, slow changes promote speciation. Pre-adaptation has become advantageous in the process of cold hardening. Only plants able to miniaturize, to squeeze within the shallow layer of the favourable microclimate, could occupy the cold-environment niche. In other words, as terrains have been slowly rising due to orogeny, only species producing smaller and smaller forms were predisposed to be preserved by natural selection. In time, they became cold-hardened and UV-tolerant alpine species. Only a few tree species could do it. Certain conifers changed slowly into prostrate shrubs and krummholz (Pinus mugo, Pinus banksiana). Plants dependent on the presence or protection of other species in a complex community (cf. ‘dominant’ versus ‘rare’ species) have died out.

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Fig. 15.1. The tiny herb Epilobium arcticum in a shelter niche among the rocks.

Although the cryophytes belong to a number of families and genera, there is a preponderance of monocots, mainly grasses and sedges. These graminoids are short even in warmer regions and are therefore pre-adapted (predisposed) for harsher environment. According to Raunkier’s classification, cold-hardened dicots are cryptophytes with perennating buds under the ground surface or hemi-cryptophytes, protected by a litter layer. Less often they are chamaephytes, mostly dwarf shrubs with overwintering buds protected by snow against exposure, desiccation and abrasion by fast-moving snow crystals. Other pre-adaptations favoured by natural selection include wind- and selfpollination even in species with showy flowers (apparent relict of their pre-alpine origin with the abundance of insects), agamospermy (asexual production of seed without fertilization by

pollen; e.g. genus Antennaria), apomixis (vegetative propagation by bulbils, stolons, rhizomes and roots; e.g. Polygonum viviparum, Saxifraga cernua) and polyploidy (duplication of chromosome numbers, which increases species survival in extreme high-latitude conditions), all a result of natural selection.

Adaptation We mentioned that a speciation across the environmental spectrum symbolizes a microevolution, a lateral genetic shift in a degree. Alpine and arctic semi-deserts and deserts represent extreme environments where the main task of any living creature is survival (Svoboda, 1978). No major evolutionary

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advancements are likely in such marginal conditions, especially since the genetic pool allowing for the generation of mutants is small due to smaller metapopulation sizes and the very long time (ranging in decades) required from seed germination to reproductive stage of the plant. Nevertheless, the necessity to survive has led to a development of adaptations, and to amazingly diverse survival strategies in order to grow, endure the hardships and to reproduce. Many authors became preoccupied with the way alpine and arctic plants have established in the cold-challenging environment, and with their long-term survival (Löve, 1963; Saville, 1972; Billings, 1974; Bell and Bliss, 1977; Körner and Larcher, 1988; Crawford, 2005; and others). Measured by human standards, the ten months frozen and two months cold Arctic was deemed extreme for man as well as for ‘beast’ (cf. Robert Frost’s poems and Jack London’s stories). The actual reality with respect to arctic biota, vascular plants included, is not far from this deep-rooted perception. Adaptations or not, marginal arctic and alpine zones, such as wind-swept ridges and plateaus, represent living conditions at the

very edge of survival almost for every creature (Svoboda and Henry, 1987).

Survival strategies Billings (1974) describes the adaptations of arctic and alpine plants in terms of their general morphology and their physiology. The more severe the milieu, the more likely the plants become dwarfed to fit within the thin layer of warmer air – the more favourable microclimate. Shrubs, if any, are found pressed to rock faces, in winter seeking protection in snow drifts and snow beds. Semi-erect and prostrate willows (Salix arctica) are often severely grazed right after the spring thaw and frost-heaved from the ground during their lifespan. Yet they show the most remarkable resilience to physical stresses and other privation (Fig. 15.2). Herbs, almost all perennials, develop extensive root systems (root/shoot ratio 5:1 and higher) to draw nutrients and store carbohydrates, yet there are exceptions. On frozen grounds with the shallowest active layer, some herbs produce spider web-like roots under ‘warm’ thin rocks, having almost no weighable biomass. At the

Fig. 15.2. Grad student Glenda Jones in the willow ‘forest’ (Salix arctica) in Sverdrup Pass, Ellesmere Island. Pressed to the ground, hundreds of years old plants are beaten by the elements and continuously grazed.

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end of the seasonal life cycle, shoots of some species die back to ground level where their perennating buds are protected over the winter. Annual herbs are rare and even these often exist as perennial ecotypes. There are three main types of tundra perennials: graminoids, leafy dicots and cushion dicots. Grasses, sedges and rushes are omnipresent, owing their prevalence to their natural ability to miniaturize and adapt from aquatic (Carex aquatilis) to the driest habitats (Carex nardina). They flower late, ~20 days after the snowmelt, yet produce viable seed. The dicots’ common denominator is that during the growing season they produce a pre-formed shoot and flower primordia and overwintering buds. Flowering occurs early next spring and ripening of seeds takes place shortly after, so that the entire process is completed within 40–50 days, usually in early August. Thus these plants complete their life cycle in two, rather than in a single season. Longevity is a winning ticket (Svoboda, 1977; Fig. 15.3). The purple saxifrage (Saxifraga oppositifolia) blooms usually 3–5 days after its release from snow. Its pre-formed flower buds enlarge while still snow-covered because they absorb

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solar radiation and form a tiny bubble greenhouse around each bud. The flowers open their petals just at the plant surfaces to keep the ovaries warm, yet during the seed ripening, the flower peduncle starts to elongate high enough for the wind to shake the fruit and disperse the seeds. Arctic avens (Dryas integrifolia) and other arctic/alpine species utilize the same reproductive and dispersal strategy. These species also display phenomenal phenotypic plasticity. In favourable (mesic and sheltered) habitats the purple saxifrage and arctic avens produce loose, creeping patches; in a less protected site they form compact clumps or cushions; and on an exposed ridge they grow as thin prostrate mats. Dryas cushions as semi-closed micro-ecosystems In extreme conditions, plants develop structures and defence mechanisms to be almost self-sustaining and self-contained in the hostile environment (cacti in deserts, tree islands in the Subarctic). In polar semi-deserts, Dryas cushions serve as an example of such an ingenious and efficient arrangement of features by

Fig. 15.3. An 800-year-old individual (>100 cm diameter) of arctic avens (Dryas integrifolia) lives only on its periphery. The core of the clump has died out and weathered away. A large stand of similar old clumps which escaped the Little Ice Age neoglaciation was found on a gravel terrace of Coats Island.

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combining slow incremental growth with a prolific seed production. Arctic avens and the purple saxifrage are the most representative species of polar semidesert communities. They maintain a large above-ground biomass compacted into a shape of a cushion. At its surface, Dryas produces a layer of tiny green leaves which are photosynthetically active for two years. When they die, they remain attached and form a distinctly coloured layer similar to a tree ring. By counting these ‘rings’, the age of the clump can be established. This standing dead mass remains useful in several ways. It holds moisture and decays slowly. Its nutrients are released gradually within the cushion to be resorbed and recycled by the secondary roots developed within the cushion. The plant also catches fine dust particles which fill the cushion core providing minerals for the secondary roots. Mycorrhizal associations with Dryas roots (and other arctic dicots) infer their N-fixing function (Bledsoe et al., 1990; Kohn and Stasovski, 1990). Nematodes and tiny invertebrates cohabit in the cushions’ protected environment to a mutual benefit. Physiology Phenotypic and morphological difference among various ecotypes, ecoforms and perhaps even acclimated populations of cold-hardened plants result in physiological dissimilarities as well. These might be related to timing their life cycle, favouring self-pollination, apomixis over sexual reproduction, germination, dormancy, frost resistance, and many other physiological and eco-physiological characteristics, studied by a great number of researchers. As an example, at Alexandra Fiord (79°N), during the summer continuous daylight, we measured a 24 h transpiration activity of S. cernua. In spite of a full midnight sun, albeit with a lower light intensity, there was a 2 h ‘midnight’– a pause in transpiration due to stomata closure. We believe that was a residual property carried over from the species’ low-latitude alpine origin with a regular circadian rhythmicity still active. McNulty and Cummins (1987) found that dark respiration rate in S. cernua collected at the same locality and grown in chambers at a constant 10°C was higher than in plants

grown at 20°C. Crawford (2008b) reported distinct yet opposing metabolic strategies between the extreme forms of S. oppositifolia. As there are many environmental gradients, so there are gradients of evolutionary progression from induced acclimation, to temporary ecoforms, to more stable ecotypes and finally to a new species. Over a long timespan and several generations, some of the acquired physiological responses may ultimately serve the particular populations to cope better in their accustomed habitat. ‘Although the ecoforms are merely temporary, their production is continuous and may be one of the ways that the ancient autochthonous flora of the Arctic has survived’ (R.M. Crawford, St Andrews, Scotland, 2008, personal communication). However, unless such shifts become genetically fixed, they will remain a mere temporary phenomenon, prone to be reversed when the living conditions change.

Postglacial Plant Reinvasion During the Pleistocene, polar regions were subjected to long- and short-term climate oscillations resulting in vegetation and soil burials under the ice, circum-continental and longterm exposure of continental shelves due to sea level drop, damping of glacial drifts over the formerly vegetated lands, subsequent terrain erosion, sea and periglacial flooding, isostatic rebound, re-colonization, etc. All of these forces and factors had a considerable impact on the pre-Ice Age and interglacial flora: preserving it in some areas, moving it around as the situation changed, and, indeed, mostly decimating it during the great glaciations but also during minor neo-glaciations, as was the recent ‘Little Ice Age’ (Bergsma et al., 1984; Lévesque and Svoboda, 1999). The Pleistocene climatic upheavals which started the elimination, mixing and sorting of the biota have continued also in Holocene, in reality until the present. One must wonder that patterns of any floristic elements can be still recognized and that indigenous plant populations can be traced to their sources where they carried on in exile during the glaciations. The reconstruction of the dramatic saga has been possible largely due to the advancement of modern analytical techniques.

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After the Pleistocene’s repeated ice advances and retreats, a smaller number of the cryophytes and proportionately even fewer animal species survived to return to the deglaciated terrain. Ice-free Alaska and Siberia together with fringe tundra zones adjacent to the glacial fronts, and for millennia exposed continental shelves during glaciations, had been the most plausible sources of the cryophytes’ reinvasion. Hultén (1972) described in detail radiation pathways of arctic plants from various continental, coastal, amphi-Atlantic, arctic-montane and boreal centres and refuges into their deglaciated realms. Some species may have survived within the glaciated landscape on nunataks, i.e. on rocky mountain tops protruding the ice field, giving a foundation for the ‘nunatak refugia hypothesis’ (Ives, 1974). However, at least at high latitudes, the potential pool of cryophytic species on these rock islands has been very limited. The presentday new-world nunataks still harbour remnants of the depaupered ice age flora. Only a small number of species at negligible frequency has persevered on them, although the present climate is kinder than was that during the fullstrength glacial. Of the world’s 235,000 species of flowering plants (Tudge, 2000), only about 1700 taxa populate the Nearctic realm of mostly Beringia, Alaska and Yukon Territory at present (Hultén, 1974). A mere 0.4% of the known vascular plant species are established in the Arctic (Billings, 1992). For the continental Northwest Territories of Canada, Porsild and Cody (1980) list 1100 species, mostly overlapping with those of Hultén (1972). However, both lists include many taxa of the species-rich tundra– taiga ecotone, of which about 500 comprise the flora of the Alaskan Arctic slope (Murray, 1995) and 350 occur in the Canadian Arctic Archipelago (Porsild, 1964). With increasing latitude, the number of vascular plants diminishes rapidly, dropping to less than 20 species on the vast regions of polar uplands at a dismally low total ground cover of 0.3% (Bliss et al., 1984). These uplands are a true polar desert. Most of the high arctic plant and animal life is concentrated in sheltered bays and lowlands, called thermal oases. At the Alexandra Fiord lowland, 79°N, Ellesmere Island, 92 vascular plants were identified (Ball and Hill, 1994).

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By contrast, the largest northernmost oasis at Lake Hazen, 82°N, Ellesmere Island, sustains 117 vascular species (Soper and Powell, 1985). The non-vascular elements are represented by 750 bryophyte and 1200 lichen species (Murray, 1992).

The tardy journey to the promised land The Wisconsin glaciation ended some 11,000 years ago, when the continental ice ceased spreading and, in turn, started melting vigorously, first at its margins. The Holocene, the recent ameliorated era and likely a new interglacial, began. It took 8000 years for the continental ice masses to incompletely melt away (Greenland and eastern islands of the Arctic archipelago are still glaciated, and polar regions had been subjected to temporary neoglaciations during minor cold climate anomalies). At the south, huge periglacial lakes developed in front of the end moraines. The largest of them, Lake Agassiz (1100 km×400 km), was draining its waters for thousands of years. The forest–tundra plant communities adjacent to the glacial front entered the spatial void left by the shrinking continental ice. However, much of the vegetation, which followed the glacial retreat, drowned in these newly formed lakes. Nevertheless, the vegetation had steadily advanced. A plethora of temperate and boreal species invaded the icefree terrain; not all of them, however, succeeded in entering it. Walker (1995) describes the extant arctic plant diversity as a community of species which have passed through a series of ‘filters’. The ‘pores’ of the first filter have been so fine that only a tiny fraction of the existing vascular flora could pass through it. The second and third sets of filters are represented by the climatic, geological and habitat gradients, and biological interactions. In this imaginative scenario, these ‘filters’ further screened, and thus eliminated, many taxa which passed through the first filter. The melting of the continental ice sheet was triggered by a relatively sudden climate warming and also the climate in the melting zones became much more favourable for plant growth. A great number of species began marching north and would do well. However, a fierce interspecific competition made many species only temporarily successful. As the ice

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receded into higher latitudes, a climatic gradient began emerging. Only those better adapted to the increasingly harsh conditions (shorter and cooler growing season, shallow active layer, etc.) would prevail migrating to higher latitudes. The less resilient species would remain behind, holding on their achieved positions. The Ice Age was a continental phenomenon and so was the phenomenon of plant recolonization of the immense ice-free landscape. The terrain to be reclaimed stretched over 40° of latitude from the present Wisconsin State to the top of Ellesmere Island, and acted as a large sheet of chromatographic paper. Cohorts of vascular species had travelled along it as far north as they could tolerate the increasingly hostile conditions and positioned themselves in the most plausible area or zone, out of reach of their less resilient competitors. For the sake of completeness, it has to be added that the deglaciated realm has been invaded also from the western and north-western side in a rather semicircular fashion; nevertheless, the pattern of re-colonization was very similar. Walker (1995) pointed to a remarkably high correlation between the regional summer climate and the number of species of the regional high arctic floras, ranging from more than 200 species at 9°C to less than 50 species at 3°C of the July mean temperature. At present, the North American tundra complexes represent a huge biome composed of various ecosystems characterized by similar vigour, competitive strength and tolerance of the environmental conditions (Bliss, 1997). In this biome distinct vegetation zones have reached a dynamic equilibrium with the present climate. However, the zones have shifted in the past and are bound to shift even more, as the climate gets warmer (Crawford, 2008a).

Competition versus stress tolerance Svoboda and Henry (1987) described the postglacial plant advancement and classified the process as a primary succession of continental dimensions. Three ongoing successional phases can be recognized. 1. Phase I: directional replacement succession, typical in mesic temperate environments and also manifested in the low-arctic contigu-

ous tundra. The invading plant cohorts meet little resistance here; however, due to fierce competition, only the winners have survived and established more or less permanent plant communities. 2. Phase II: directional non-replacement succession in near-marginal environments, where populations of invading species keep progressing slowly without much competitive interference. 3. Phase III: non-directional non-replacement succession, a virtually stagnant succession in marginal, extremely unfavourable environments. Plant propagules reach the area, of which some may germinate but most of the seedlings will sooner or later die. A handful of diminutive individuals may very slowly reach maturity, some even produce viable seed. Eventually, these few cold-hardened pioneers establish their presence in areas marginal for survival of any higher-structured life. Out of several thousands of the initially invading species, these final cold-hardened pioneers proved to be best pre-adapted. Their journey was the longest and they now grow far beyond the reach of their closest potential competitors. In other words, as the conditions along the latitudinal gradient worsened, the emphasis became less on species replacement and more on the establishment and survival of an individual. One more point: all the pioneer finalists of the long journey were the underdogs at the start line. They were the ‘rare species’ in the original plant community of the ‘big and tall’ dominants, composed of trees, shrubs, heath and graminoids. Presently, in the huge expanse of the Nearctic tundra, phase I predominates in the climatically least unfavourable zone, known as the Low Arctic. Phase II prevails in the Mid-Arctic, climatically more severe. Phase III is typical in the High Arctic polar desert with the most severe climate, being the extreme end of the latitudinal severity gradient. It should to be emphasized that the tundra plant communities, as we find them spread from the lowest to the highest latitudes (and altitudes in their alpine version), are still in the phase of colonization, ready to move forward when the conditions improve. In fact, this occurred during the warm hypsithermal period 6000 to 4000 years ago, at which time the treeline extended up to 350 km north of the

Plant Cold Hardiness in the Canadian Arctic

present forest–tundra boundary. This has also been happening after the termination of the more recent cold climatic episode, the Little Ice Age, and is gaining momentum during the present global warming (Svoboda et al., 1995; Crawford, 2008b). Time-lapse photography would show a busy activity of individual tundra plants and their entire cohorts, gaining ground and losing it again, as the year-to-year weather and decade-to-decade climate differ in completion of snowmelt, in precipitation patterns, in total degree-days, and other relevant parameters. As the Red Queen explained to Alice: ‘it takes all the running, to keep in the same place’.

Distribution of closely related species The arctic tundra vegetation is made of a relatively small number of species, yet these belong to an even disproportionately smaller number of genera. Moreover, certain genera are widely out of the expected range in terms of species they contain. Thus Porsild and Cody (1980) list 105 species of sedges (Carex spp.), 57 willows (Salix spp.), 29 buttercups (Ranunculus spp.), 27 blue grass (Poa spp.), 25 saxifrages (Saxifraga spp.), 23 cinquefoils (Potentilla spp.), 22 dandelions (Taraxacum spp.), 21 drabas (Draba spp.) and 20 oxytropis (Oxytropis spp.). Only a few of these species fully overlap in their ranges of distribution. For instance, Taraxacum lacerum, Taraxacum hyparcticum and Taraxacum phymatocarpum, and another set of closely related species, Salix planifolia, Salix arctophylla and Salix arctica, occupy separate zones in the Low, Mid and High Arctic (Svoboda and Henry, 1987). The topographical distribution of all these species reflects the source of their origin or, in the case of the above-named dandelions and willows, a fine-tuning of their cold hardiness along the latitudinal severity gradient. While, at the generic level, the dandelions and willows can be easily recognized by a non-taxonomist, each of the many saxifrage species looks different, as they arrived to the mountain tops and lastly to the arctic tundra domain via quite heterogeneous phylogenetic paths. Aside from their essential cladistic similarities (easily overlooked by a non-expert), they developed organs and use-

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ful adaptations particular to each species. The spider plant (Saxifraga flagellaris) sends out long naked stolons, terminated by rooting rosettes; the most common purple saxifrage (S. oppositifolia) exists in dry habitats as a densely tufted (cushion-like) form and in wet snowbanks as a loosely trailing mat; the tiny nodding saxifrage (S. cernua) reproduces prolifically by clusters of white bulblets at its base and by clusters of small purple bulbils along the stem; the brook saxifrage (Saxifraga rivularis) specializes in wet places and reproduces frequently by thread-like rooting stolons; in contrast the prickly saxifrage (Saxifraga tricuspidata) is very successful in dry rocky places. Its succulent-like leaves are arranged on densely crowded shoots. Long flowering stems produce plenty of wind-dispersed seeds. In contrast to the homogeneous dandelions or willows which tend to occupy separate climatic zones, many of the heterogeneous saxifrages coexist in various habitats of the same, curiously, the most severe environment; both types being an amazing example of the various convergent cold-hardening pathways and, indeed, of the natural selection.

Small but prolific, or big yet rare? Why this dilemma? Plant vigour and species ground cover along the increasing severity and decreasing competition gradient reveal an opposite trend. In mesic habitats, at their southern rim of distribution, the wide-range species produce robust individuals but they are usually only a minor component of the plant community. Plant vigour continually diminishes because of increasing gradient severity. In contrast, due to decreasing competition, the species’ ground cover builds up, peaking at a certain midpoint of the distribution range, and then starts to decrease up to the point of extinction, as the physical environment becomes too hostile. In other words, as the less-adapted species keep dropping out of the picture and the competitive drive diminishes, the more stress-hardened species are able to cover more ground. The trade-off is a reduction of physical vigour for a higher presence (Svoboda and Henry, 1987).

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Arctic avens (D. integrifolia) may serve as a classic example and model of arctic plants’ strategy of survival. This official flower of the Canadian Northwest Territories belongs to the most common species in the Nearctic tundra. As described above, at its southern margins, Dryas forms semi-erect shrubs, yet its ground cover is very low; at mid-range it becomes

prolific and produces substantial cushions; while at its northern fringes it becomes sporadic again forming thin, scattered mats. At the lower margin the strong competition with other shrubs and heath limit the species presence while at its northern margin, the intolerable severity of the physical environment becomes the ultimate limiting factor (Fig. 15.4).

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Low Latitude/severity gradient Fig. 15.4. Effects of interspecific competition and environmental stress on (a) vigour and distribution of a single species, (b) relative vigour of a series of species and (c) relative abundance/standing crop of a series of species along the latitudinal stress gradient, as demonstrated on a Dryas integrifolia model. (Used with permission by Arctic and Alpine Research Journal.)

Plant Cold Hardiness in the Canadian Arctic

The Non-vascular Components The most ‘dwarfed’ cryophytes are mosses, lichens, algae, fungi and bacteria. These opportunistic organisms take advantage of the momentary changes of temperature and moisture, able to turn their metabolism on and off in minutes. They flourish far beyond the range of vascular plant distribution and some function even in subfreezing temperatures. They can also remain dormant for a very long time. Lichens grow at elevations ≥6000 m and their plaques and discs are known for their extreme longevity (>5000 years), allowing use for dating purposes (lichenometry). Various microbiota were found in deep ice cores (Miteva and Brenchley, 2005; Mosier et al., 2006) and in the permafrost (Steven et al., 2006). Algae flourish in the snow and glacial ice. As in the primeval times of algal invasion on to bare land, they still play a crucial role in re-colonization of the modern open terrain. In high latitudes, the resulting exposed landscape, the polar desert, is a climatic remnant of the past ice age. Here, cyanobacteria (blue-green algae) are first to operate. They fix N on the surface of the pre-washed, N-starving regosols, accompanied in tandem by the green algae. Together they produce the first organic matter and accumulate biomass, storing nutrients which, after their slow decomposition, are made available to fungi, mosses and soon vascular plants (Elster and Benson, 2004). Thus these photosynthetically active, cold-hardened microbes are essential in the process of initial primary succession following landscape deglaciation. Algal fertilization and ground preconditioning are highly efficient in the barren landscape revitalization and tundra ecosystem restoration.

Field studies In Sverdrup Pass (79°N), Ellesmere Island, we have conducted research on algal diversity, abundance and productivity in a glacial stream running from the melting front of the large Teardrop Glacier and merging about 500 m down with the fast rivulet in the pass’s valley (Elster and Svoboda, 1995, 1996). At the glacial front, the meltwater was only 0.5°C,

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increasing gradually to a maximum of 10°C at the merger with the rivulet. We made the following observations. 1. The dissolved nutrient loading (nitrate- and ammonia-N, ortho- and total phosphate, Ca and Mg) was highest in the glacial front zone and diminished along the stream. 2. This wet and coldest zone was inhabited mainly by Cyanoprocaryota (blue-greens) clearly responsible for the highest reading in available N, mostly fixed by them. Further from the glacier, the blue-greens were largely replaced by the green algae. 3. As the water temperature increased with distance from the glacier, distinct taxonomical groups showed a proclivity for separation along the stream, proving that even algae are coldhardened to different degrees and thus temperature-sensitive. 4. The algal visible biomass (mostly dense filamentous curtains) peaked about 100 m from the glacial front and diminished virtually to zero before the stream merged with the rivulet about 400 m further on. The algae consumed practically all of the dissolved nutrients, thus impeding their own growth in the lower section. Here, the stream bottom showed the original substrate (clean sand and gravel) with no visible coating by algae. In contrast, the elevated stream banks consisted of a thick peat layer, rich with mosses and vascular plants. This was a classical demonstration of a gradual build-up of organic matter as a function of time and distance from the receding glacial front (Fig. 15.5). At the end of the growing season, the glaciers stop melting and the stream beds dry out and freeze. The algal biomass breaks and is blown into the valley, fertilizing the already established vegetation and facilitating further growth. A significant amount (kilograms) of dry algal biomass is being produced by a single stream every season, and thousands of such streams and seepages run down a great number of outlet glaciers descending into the 80 km long Sverdrup Pass from the surrounding ice fields at Ellesmere Island alone. Evidently, the quantity of organic matter produced and contributed by algae annually and summed up over years, even centuries, has been staggering. Presently, the ice margins of the 2000 km2 glaciated area in the circumpolar Arctic (1975 estimate) are melting, nourishing algal growth

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Fig. 15.5. One of many glacial streams at Sverdrup Pass, Ellesmere Island. At the glacial front, the stream is loaded with algae. Their floating biomass diminishes along the stream, and the water and the gravelly stream bottom are free of visible algae before the stream merges with the valley rivulet.

in zones adjacent to their glacial fronts. As a consequence, also the neighbouring landscapes abound with life. The revitalized periglacial environment represents a net carbon sink, scrubbing CO2 from the atmosphere and depositing it as organic carbon upon the land. The cold-hardened meltwater algae are the primary facilitators of the initial primary succession after deglaciation.

Cold origin of life? A prolific presence of prokaryotes in ice cores and the permafrost points to their ability to metabolize in subfreezing temperatures, condi-

tions existing as well on cold astral bodies. These are the true extremophiles! Microbes which fit into micro-veins of liquid water within the ice can utilize nutrients and energy the chemo-autotrophic way. In a well-corroborated review paper, Price (2007) explains that: On the early Earth, and on icy planets, prebiotic molecules in veins in ice may have polymerized to RNA and polypeptides by virtue of the low water activity and high rate of encounter with each other in nearly one dimensional trajectories in the veins.

This is certainly a fascinating idea, contrasting with the more well-known theory of the hot underground and oceanic hydrothermal vents origin.

Plant Cold Hardiness in the Canadian Arctic

Horticultural Experiment Near 80°N In the early 1980s our research group was involved in a 7-year-long ecological study of the polar oasis at Alexandra Fiord lowland (79°N), Ellesmere Island (Svoboda and Freedman, 1994). The lowland’s mesoclimate seemed to be favourable enough to grow some lettuce and radishes in the local soil, under plastic mulch, for the camp consumption. The results were promising and the next year we arrived equipped with light, specially designed umbrella-shaped structures covered with Du Pont Fabrene® tear-resistant plastic. Twelve of them were reach-in (3 m diameter) and two were walk-in (6 m diameter) structures. We set them at a sunny site and for their circular shape, reflecting the 24 h path of the sun, we named them ‘igloos’. Two outlet glaciers nearby created a contrasting background. We called the experiment the Green Igloos Farm (Fig. 15.6). A parallel experiment, the Keewatin Gardens, a set-up of 40 A-frame rectangular greenhouses, was assembled at Rankin Inlet, (63°N), Northwest Territories, with the objective to grow native arctic plants as potential food crops. What was intended as a side project to our polar oasis study has developed into a seri-

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ous research endeavour (four Masters theses) at two arctic localities in collaboration with Guelph University and supported by a significant grant (Romer et al., 1981; Cummins et al., 1988). At Alexandra Fiord, the local soil was worked out, fertilized, and used to fill black plastic grow-bags to avoid root contact with the permafrost. The bags were arranged in circles in the sun-heated greenhouses. A large variety of vegetables, potatoes and ornamental plants were grown with unanticipated success. Many were grown from seeds (radishes, beets, carrots, beans, etc.), others from seedlings (cabbages, lettuces, broccoli, tomatoes) or tubers (seven varieties of potatoes) (Fig. 15.7). In addition to the southern cultivars, two native herbs, arctic poppy (Papaver radicatum) and bulblet saxifrage (S. cernua), were extracted from the nearby tundra soon after the snowmelt and transplanted to one empty greenhouse. The very first season the transplants responded to the warm greenhouse environment by growing five times taller than the tundra controls and the next season they produced clusters from seeds and bulblets dropped the previous autumn (Figs 15.8 and 15.9).

Fig. 15.6. Green Igloos Farm at Alexandra Fiord, Ellesmere Island, with two outlet glaciers in the background.

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Fig. 15.7. Assortment of vegetables, grown in black plastic grow-bags, in the walk-in ‘igloo’ greenhouse.

Fig. 15.8. Gigantic clumps of arctic poppy (Papaver radicatum – yellow flowers) and bulblet saxifrage (Saxifraga cernua – the reddish inflorescences), produced in an ‘igloo’ from seed dropped by single plants at the end of the previous growing season. NB: See Plate 3 for colour representation.

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Fig. 15.9. Control plant collected in the open tundra (left of the scale bar), a single plant and a clump of bulblet saxifrage (Saxifraga cernua) grown in the ‘igloo’ greenhouse (right of the bar).

The horticultural experiment demonstrated that the southern produce plants respond well to a favourable greenhouse microclimate in spite of the continuous daylight. The formation of sizeable tubers under prolifically flowering potato plants surprised the Guelph experts who predicted that since potatoes originate in the equatorial zone with 12 h/12 h photoperiod, no tubers would be formed. The tundra transplant experiment showed clearly that their native environment was much below a desired optimum. Their immediate and vigorous growth confirmed that these plants, although tolerant to the stressful polar conditions, were still genotypically and phenotypically ready to take advantage of the ameliorated conditions which resembled the environment of their southern alpine origin. There is a fine yet critical distinction between plant adaptation (to feel home at the site) and plant tolerance (to be able to cope with the taxing situation).

Conclusion Over their evolutionary history, organisms have been adapting to most diverse environments. Plants, from the green algae to the most advanced angiosperms, have diversified in their forms and survival strategies to fill all reachable niches over a great range of conditions. Those living in the most contrasting habitats are called extremophiles (‘lovers’ of extremes: hot/cold, wet/dry, alkaline/acid, etc.), although, as manifested, some of them may not necessarily ‘love’ but rather only tolerate their habitat conditions. The cold-hardened arctic and alpine species, the cryophytes, belong to this category. They evolved in regions subjected to orogeny by being slowly carried up with the rising mountains to high altitudes and, in some cases, had been rafted to the polar regions by the northward migration of the continents. During the Interglacials and after the last Ice Age, the clean slate of the deglaciated

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North American continental landmass acted as a large sheet of chromatographic paper along which the plant species travelled as far north as they were able to tolerate the increasingly hostile conditions. Some tundra species are more cold-hardened than others, although additional limiting factors are also involved. According to the degree of cold and stress tolerance, various species reached and established at different geographical positions and now form separate or overlapping ranges of their spatial distribution. All tundra species would prefer more favourable environments than the one they occupy most. In a less supporting environment, these plants are under stress. However, they recover fast if the conditions change for the better. At present, vegetation complexes of similar vigour, competitive strength and stress tolerance form distinct vegetation zones in the North American tundra biome. The vegetation of these zones is in a dynamic equilibrium with the extant climate. However, the zones have shifted in the past and are bound to shift again, as the climate ameliorates. The cold-hardened algae and bryophytes play a crucial role in the colonization of freshly deglaciated terrain by fixing N and

building the first biomass for the higher plants’ establishment. The horticultural experiment with southern cultivars at Alexandra Fiord confirmed that even warm-climate vegetables grow well in an artificially ameliorated space bubbles (‘igloos’) in a generally hostile climate. Similarly, the native tundra plants, transplanted into the same sun-heated greenhouse, grew much taller and produced several times more seeds and bulblets than plants in the nearby tundra.

Acknowledgements I am very grateful to Professor Karen Tanino for inviting me, an ageing arctic ecologist, to present an inaugural lecture to mostly the agrobiologists at the 8th International Plant Cold Hardiness Seminar, and for her involvement in editing of my manuscript. I am also thankful to Professor Robert M. Crawford, University of St Andrews, Scotland, for his constructive comments on the manuscript. Michael Svoboda provided vital assistance by resolving some defiant computer glitches and with preparing the figures for the publication.

References Ball, P. and Hill, N. (1994) Vascular plants at Alexandra Fiord. In: Svoboda, J. and Freedman, B. (eds) Ecology of a Polar Oasis Alexandra Fiord, Ellesmere Island, Canada. Captus University Publications, Toronto, Ontario, Canada, pp 255–256. Basinger, J.F., Greenwood, D.R. and Sweda, T. (1994) Early Tertiary vegetation of Arctic Canada and its relevance to paleoclimatic interpretation. In: Boulter, M.C. and Fisher, H.C. (eds) Cenozoic Plants and Climates of the Arctic. NATO ASI Series, Vol. 127. Springer Verlag, Berlin, pp. 175–198. Bell, K.L. and Bliss, L.C. (1977) Overwinter phenology of plants in a polar semi-desert. Arctic 30, 118–121. Bergsma, B.M., Svoboda, J. and Freedman, B. (1984) Entombed plant communities released by a retreating glacier at central Ellesmere Island, Canada. Arctic 37, 49–52. Billings, W.D. (1973) Arctic and alpine vegetations: similarities, differences, and susceptibility to disturbance. BioScience 23, 697–704. Billings, W.D. (1974) Arctic and alpine vegetation: plant adaptations to cold summer climates. In: Ives, J.D. and Barry, R.G. (eds) Arctic and Alpine Environments. Methuen, London, pp. 403–443. Billings, W.D. (1992) Phytogeographic and evolutionary potential of the arctic flora and vegetation in a changing climate. In: Chapin, F.S. III, Jefferies, R.L., Reynolds, J.F., Shaver, G.S. and Svoboda, J. (eds) Arctic Ecosystems in a Changing Climate: An Ecophysiological Perspective. Academic Press, San Diego, California, pp. 91–108. Bledsoe, C., Klein, P. and Bliss, L. (1990) A survey of mycorrhizal plants on Truelove Lowland, Devon Island, N.W.T., Canada. Canadian Journal of Botany 68, 1848–1856. Bliss, L.C. (1997) Arctic ecosystems of North America. In: Wielgolaski, F.E. (ed.) Polar and Alpine Tundra. Ecosystems of the World 3. Elsevier, Amsterdam, pp. 551–683. Bliss, L.C., Svoboda, J. and Bliss, D. (1984) Polar deserts, their plant cover and plant production in the Canadian High Arctic. Holarctic Ecology 7, 305–324.

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Plate 1. Observed frost damage to heads of wheat following freezing. Left, following frost testing, left head froze, right 3 heads supercooled. Right, typical head damage observed in the field during head development. Plate 2. Infrared images taken during freezing and thawing of wheat plants at ear emergence.

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Plate 3. Integrative molecular timetable of bud development in poplar. Autumnal bud development is a composite of bud formation (red), acclimation to dehydration and cold (blue), and dormancy (orange). Selected genes or processes that specifically belong to one of these aspects are highlighted accordingly. Simultaneous with bud development, elongation growth (green) gradually ceases in young derivatives that are displaced from the apex. Bud development is characterized by the sequential activation of light, ethylene, and ABA signal transduction pathways. The major transcriptional changes at the regulatory and target process levels are depicted at the time that the respective genes show their maximal change in expression. The two major phases of transcriptional and metabolic response are indicated by grey boxes. Below, tentative levels of cellular responses and/or the quantity of major metabolites are indicated with a graded scale. Arrows connect regulators and transcription factors to their putative downstream processes, without implying a genetic or direct molecular interaction. Because of its putative nature, the link between low sugar and ethylene signal transduction is shown with a dashed arrow. Genes shown in grey and within brackets are only found differentially expressed in ABI3-overexpressing poplars. Plate 4. Gigantic clumps of arctic poppy (Papaver radicatum - yellow flowers), and bulblet saxifrage (Saxifraga cernua - the reddish inflorescences), produced in an ʻIglooʼ from seed dropped by single plants at the end of the previous growing season.

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Ice Encasement Damage on Grass Crops and Alpine Plants in Iceland – Impact of Climate Change B.E. Gudleifsson

Introduction

Winter Damage in Iceland

Many kinds of stresses attack plants during winter (Griffith et al., 2001). The stresses cause strains in plants which subsequently can cause injury or death (Levitt, 1980). Frost stress is the most studied winter stress to plants and much knowledge on frost damage has been collected. Another type of winter stress, ice encasement, appears occasionally or frequently in some areas of the world. Ice encasement has especially damaged grasses and winter cereals or other perennial herbage plants in northern Scandinavia, Iceland, eastern Canada and northern Japan. The process of ice encasement damage in plants is not as thoroughly studied as the process of frost damage. Ice encasement damage is related to the absence of oxygen and the exact cause of death has mainly been studied on winter cereals in Canada (Andrews and Pomeroy, 1991). In the present chapter, information is presented on the impact of winter damage, especially ice encasement, on grass survival and agriculture in Iceland in the past, present and future. The impact of winter climate and the principal cause of plant death in ice encasement are explained and the impact of predicted climate change is discussed.

In Iceland, freezing damage is mainly harmful to trees because buds, the most sensitive organs to freezing, experience the ambient air temperature directly. The surviving organs of grasses and cereals, on the other hand, are located close to the soil surface where the temperature usually fluctuates around zero (Baadshaug, 1973) due to snow and old straw insulating the grass buds from extremely low air temperatures (Andrews and Pomeroy, 1977). The impact of different types of winter damage on hayfields and forest trees in Iceland has been evaluated by the author (Guðleifsson and Örvar, 2000). This survey confirms the dominance of frost damage of trees and ice damage of grasses (Table 16.1). Tolerance to these two stresses should be taken into account when species and cultivars are chosen for cultivation. This is particularly important in forestry as almost all plant species used in afforestation in Iceland are imported and therefore not adapted to the northern oceanic climate of the island. Also, the trees planted today will grow for 70–100 years and therefore will experience the climate of the future, which will surely be subject to climate change.

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

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Table 16.1. Relative impact of different types of winter damage in hayfields and forests in Iceland. Evaluation is based on information, studies and written records from 1950 to 2000. (From Guðleifsson and Örvar, 2000.) Stress Frost heaving Drought Frost Starvation

Strain

Drought Drought Freezing Energy shortage Flooding Suffocation Ice encasement Suffocation Snow moulds Rotting Ice-nucleating Freezing bacteria

Hayfields Forests (%) (%) 1 2 5 +

8 5 85 ?

+ 90 2 ?

? − ? 2

Impact of Ice Damage on Agriculture in Iceland The agriculture in Iceland is very single-tracked. Milk and meat production is based on cattle and sheep, which need to be fed indoors for 7–8 months a year. In earlier times, the hay production relied completely on uncultivated pastures and periodically years occurred when the yield was severely reduced because of climatic extremes. This had catastrophic consequences as the herd had to be decreased to match the food supply and, with fewer livestock, the human population was sometimes reduced as a consequence. Thus, from the 17th to the 19th century, annals describe between 25% and 37% of the years as ‘grassless years’ (Friðriksson, 1954). In the 20th century hay was mainly harvested from cultivated permanent hayfields. The figures on hay yield and hayfield area are rather unreliable regarding average hay yield. No figures are available on winter damage, but subjective evaluations could be found from descriptions of the agricultural situation each year. The years could only be classified as years with no damage, years with little damage and years with great damage. In the 20th century, there were seventeen years with great winter damage and twenty-one with little damage, i.e. 38% of the years. All these rather unreliable data have been used to produce Fig. 16.1, where the

annual yields for 1900–2006 for the whole country are presented. The years with great damage are marked (grey columns) and annual mean temperature in Stykkishólmur in western Iceland is inserted. In most cases, years with great damage have decreased mean yield and, during these years, the annual average temperature was fairly low.

Climate and Ice Damage The yield (Fig. 16.1) is not a very exact indication of the intensity of winter damage in hayfields in Iceland. This is partly because data were collected for purposes other than yield evaluation. There are many other factors besides winter climate and winter damage that influence hay yield, summer climate being one of these. The information on winter damage indicates that the distribution of such damage is highly localized, and mean yield for the whole country may therefore not be a very revealing figure. As an example, when damage is intense in northern Iceland it may be absent in the rest of the country and vice versa. Also it should be remembered that climatic factors other than temperature are involved in winter damage in the field. In spite of these weaknesses, the correlations between monthly temperature measurements and hay yield in 1900–2006 were calculated, indicating that March and April temperatures were the most closely related to hay yield (r=0.40, P