Advances in Botanical Research, Volume 29

Advances in BOTANICAL RESEARCH incorporating Advances in Plant Pathology VOLUME 29 Advances in BOTANICAL RESEARCH ...

Author: J. A. Callow

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Advances in

BOTANICAL RESEARCH incorporating Advances in Plant Pathology

VOLUME 29

Advances in

BOTANICAL RESEARCH incorporating Advances in Plant Pathology Editor-in-Chief J. A. CALLOW

School of Biological Sciences, University of Birmingham, UK

Editorial Board J. H. ANDREWS

University of Wisconsin-Madison, Madison, USA J. S. HESLOP-HARRISON John Innes Centre, Norwich, UK Universite' de Puris-Sud, Orsay, France M. KREIS R. M. LEECH University of York, York, UK R. A. LEIGH University of Cambridge, Cambridge, UK University of California, Riverside, USA E. LORD I. C . TOMMERUP CSIRO, Perth, Australia

Advances in

BOTANICAL RESEARCH incorporating Advances in Plant Pathology Series editor

J. A. CALLOW School of Biological Sciences, University of Birmingham, Birmingham, UK

VOLUME 29

1999

ACADEMIC PRESS San Diego London Boston Ncw York Sydney Tokyo Toronto

This book is printed on acid-free paper Academic Press 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http:/lwww.apnet.com Academic Press Limited 24-28 Oval Road, London NW1 7DX, UK http:llwww.hbuk.co.uWap/ Copyright 0 1999 by ACADEMIC PRESS All rights reserved No part of this publication may be reproduced or transmitted in any form or by any means electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. A catalogue record for this book is available from the British Library

ISBN 0-12-005929-0

Typeset by Keyset Composition, Colchester, Essex Printed in Great Britain by MPG Books Limited, Bodmin, Cornwall

99 00 01 02 03 04 MP 9 8 7 6 5 4 3 2 1

CONTENTS

CONTRIBUTORS TO VOLUME 29 CONTENTS OF VOLUMES 18-28 PREFACE

.............................................................

ix

...............................................................

xi

.........................................................................................................

xxi

The Calcicole-Calcifuge Problem Revisited J . A. LEE I.

............................................................................................

2

I1. Edaphic Factors ................................................................................................ A . Acid Soils ...... ..................................................................................... B . Calcareous Soils .......................................................................................

2 4 4

111.

Introduction .....

Controlled Environment Experimentation on Individual Edaphic Factors ........ A . Aluminium and Acidity .............................................................................. B . Iron and Manganese Toxicity ................................................................... C . Bicarbonate Toxicity and Iron Deficiency ............ ............................... D . Phosphate ........................................................................................... E. Calcium ........................................................... .... F. Nitrogen .......................................................... .... G . Microelements ..........................................................................................

IV. Conclusion ...................................................................................

4 4 10 11

13 14 17 20 22

Acknowledgements ...........................................................................................

25

References ........................................................................................................

25

Ozone Impacts on Agriculture: an Issue of Global Concern M . R . ASHMORE and F. M . MARSHALL I . Introduction

......................................................................................................

I1. Ozone Impacts on Agricultural Crops .............................................................. A . Exposure-Response Studies ..................................................................... 111.

Rural Ozone Levels in Developing Countries ..................................................

32 33 34 36

vi

CONTENTS

IV. Direct Evidence of Adverse Effects on Crops ................................................. A . Studies with Field Chambers .................................................................... B . Studies with Ozone Protectant Chemicals ................................................

39 39 41

V. Responses to Ozone of Tropical Crops and Cultivars ........................................ A. Experimental Studies .................................................................................. B . Factors Influencing Ozone Sensitivity in the Field ....................................

43 43 45

VI . Future Concentrations and Impacts of Ozone ....................................................

46

VII . Conclusions ........................................................................................................

46

Acknowledgements ............................................................................................

48

References ..........................................................................................................

49

Signal Transduction Networks and the Integration of Responses to Environmental Stimuli G . I . JENKINS I . Introduction ........................................................................................................ A . Networks Versus Pathways ......................................................................... B . Achieving an 'Appropriate' Response ........................................................ 11.

.

111

54 55 56

Interactions Within Signalling Networks ............................................................ A. Evidence of Negative Regulation ............................................................... B . Evidence for Synergism ..............................................................................

57 57 63

Approaches to Identify the Mechanisms Involved in Interactions Between Signalling Pathways ..........................................................................................

67

Conclusions ........................................................................................................

69

Acknowledgements ............................................................................................

70

References

..........................................................................................................

70

Mechanisms of Na' Uptake by Plants A. AMTMANN and D. SANDERS 1. Introduction ........................................................................................................ A. Salinity Toxicity and Salinity Tolerance ..................................................... B . Exclusion . Uptake and Sequestration of Na+ .............................................

76 76 77

I1. Electrochemical Potential Differences for Na+ Across the Plasma and Vacuolar Membranes ......................................................................................................... 78

CONTENTS

vii

111. Carrier-mediated Entry of Na’ ...........................................................................

80

IV. Channel-mediated Entry of NaC ......................................................................... A. Ionic Selectivity of Ion Channels B. Inward-Rectifying Channels .... C. Outward-Rectifying Channels . D. Voltage-Independent Channels E. Co-residency of Different Channel Types ..................................................

82

89

V. Contributions of Channel Types to Na+ Entry in Physiological Conditions ...... 91 9I A. Semiquantitative Dissection of Fluxes ....................................................... B. Relative Activity of Different Channel Types Determines Rate of Na+ Uptake ......................................................................................................... 95 VI. Regulation of Monovalent Cation Influx Across the Plasma Membrane ........... 96 ....................................................................... 97 A. Voltage ......................... B. External Ca” and pH .. C. Cytosolic Ca’+ and pH ........ D. External and Cytosolic Na+ E. ATP .............................. F. Other Regulators .......... VII. Comparison of Salt-Sensitive and Salt-Tolerant Genotypes or Cell Lines ....... 103 VIII. Future Work ......................................................................................................

103

Acknowledgements ...........................................................................................

104

References ........................................................................................................

104

The NaC1-induced Inhibition of Shoot Growth: The Case for Disturbed Nutrition with Special Consideration of Calcium Nutrition D. B. LAZOF and N. BERNSTEIN I.

11.

Ill.

IV.

Introduction to the Inhibition of Shoot Growth by Salinity ............................. A. Growth Inhibitions: General Considerations ............................................. B. NaCI-induced Inhibition of Shoot Growth: General Hypotheses ............. C. A Nutritional Effect of NaCl on Shoot Growth ........................................

115

115 116

119

Inhibition of Shoot Growth in Dicots and Monocots ...................................... A. The Timing of the Growth Inhibition .... B. Salinity Effects on Cell Extension ......... C. Salinity Effects on Primordium Formatio D. Salinity Effects on Cell Division in Leaves

12 I

NaCI-induced Disruptions of Nutrient Transport ............................................. A. Influence of Some Experimental Conditions ..... . B. Effects on Whole Shoot Nutrient Accumulation ......................................

I26 126 128

Nutrient Transport to Growing Shoot Tissue Under Salinity ...........................

132

...

CONTENTS

Vlll

A. B. C. D. E. F. G. H.

Protection of Growing Tissues ................................................................. Levels of Na and K in Young Tissues ...................................................... Disturbed Ca Status in Young Tissues ...................................................... Other Nutrient Disruptions in Young Shoot Tissues ................................ Effects in Young Tissues Compared to Effects in Mature Tissues ........... Genotypic Salinity Effects in Young Tissues ........................................... Lactucu surivu: a Model Dicot System .................................................... Summary: Salinized Nutrition of Growing Shoot Tissues .......................

V. The Shoot Meristems: Special Nutrient Transport Challenges ......................... A. The Nutrition of Rapidly Dividing Cells: Possible Effects of Salinity B . Transport to Zones Proximal to the Meristem in Poaceae .......................

....

146 148

149

VI . Phloem Transport and Ion Recirculation Under Salinity ................................. A. Remobilization of Nutrients from Ageing Shoot Tissues. ‘Long-term Recirculation’ ............................................................................................ B. XylemRhloem Transfer, ‘Short-term Recirculation’ ................................ C. Calcium Recirculation in the Shoot .......................................................... D. Summary of Salinization and Recirculation ............................................. VII .

133 133 137 138 139 141 141 143

Salinization and Shoot Nutrition: Specific Nutrients ........................................ A. Potassium ............... ........ B. Calcium .................. ........ C. Magnesium ............................................................................................... D. Phosphorus ................................................................................................ E . Nitrogen ............................... ............................................................. F. Micronutrients ...........................................................................................

150 151 154 155 156 157 157 157 158 158 160 162

VIII. The Study of Nutrient Status and Transport on the Microscale ........ A. Kinematic Growth Analysis and Elemental Deposition Rates ................ B . Microdissection ......................................................................................... C . Specimen Preparation Considerations ...................................................... D. Electron Probe X-ray Microanalysis ......................................................... E . Secondary Ion Mass Spectrometry ........................... ........ ... F. Some Other Microanalytical Techniques ..................................................

162 163 166 166 167 168 170

IIIX . Summary and Future Prospects ........................................................................ A . Reassessment of Current Status ................................................................ B . Model Systems .. C. In Situ Elemental ...........................................

171 171 173 174

Acknowledgements References

....

.......

..... 175

........................................................................................................

175

AUTHOR INDEX ............................................................................................

191

............................................................................................

203

SUBJECT INDEX

Plates are located between p p . 74-75 .

CONTRIBUTORS TO VOLUME 29

A. A M T M A " The Plant Laboratory, Biology Department, PO Box 373, University of York, York YO1 SYH? UK M. R. ASHMORE Department of Environmental Science, University of Bradford, West Yorkshire, BD7 IDP, U K N. BERNSTEIN Institute of Soil Water; The Volcani Center; PO Box 6, Bet Dagan 50250, Israel G. I. JENKINS Plant Molecular Science Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Bower Building, University of Glasgow, Glasgow G I 2 8QQ, UK D. B. LAZOF Department of Chemistry, CB 3290, University of North Carolina, Chapel Hill, North Carolina 27599, USA J. A. LEE Department of Animal and Plant Science, University of Shefield, Shefield SIO ZTN, UK F. M. MARSHALL Centre for Environmental Technology, Imperial College of Science, Technology and Medicine, 48 Princes Gardens. London SW7 2PE, UK D. SANDERS The Plant Luboratoq Biology Department, PO Box 373, University of York, York YO1 5YW UK

This Page Intentionally Left Blank

CONTENTS OF VOLUMES 18-28

Contents of Volume 18 Photosynthesis and Stornatal Responses to Polluted Air, and the Use of Physiological and Bacterial Responses for Early Detection and Diagnostic Tools

H. SAXE Transport and Metabolism of Carbon and Nitrogen in Legume Nodules J. G. STREETER Plants and Wind

P. VAN GARDINGEN and J. GRACE Fibre Optic Microprobes and Measurement of the Light Microenvironment within Plant Tissues T. C. VOGELMANN, G. MARTIN, G. CHEN and D. BUTTRY

Contents of Volume 19 Oligosaccharins

S. ALDINGTON and S. C. FRY Are Plant Hormones Involved in Root to Shoot Communication?

M. B. JACKSON Second-Hand Chloroplasts: Evolution of Cryptomonad Algae G. I. McFADDEN

The Gametophyte-Sporophyte Junction in Land Plants R. LIGRONE, J. G. DUCKETT and K. S. RENZAGLIA

xii


Contents of Volume 20 Global Photosynthesis and Stomatal Conductance: Modelling the Controls by Soil and Climate E I. WOODWARD and T. M. SMITH

In vivo NMR Studies of Higher Plants and Algae

R. G. RATCLIFFE Vegetative and Gametic Development in the Green Alga Chlamydomonas H. VAN DEN ENDE

Salicylic Acid and its Derivatives in Plants: Medicines, Metabolites and Messenger Molecules W. S. PIERPOINT

Contents of Volume 21 Defence Responses of Plants to Pathogens E. KOMBRINK and I. E. SOMSSICH

On the Nature and Genetic Basis for Resistance and Tolerance to Fungal Wilt Diseases of Plants C. H. BECKMAN and E. M. ROBERTS

Implication of Population Pressure on Agriculture and Ecosystems A. H. EHRLICH

Plant Virus Infection: Another Point of View G. A. DE ZOETEN

The Pathogens and Pests of Chestnuts S. L. ANAGNOSTAKIS


Fungal Avirulence Genes and Plant Resistance Genes: Unraveling the Molecular Basis of Gene-for-Gene Interactions P. J. G. M. DE WIT

Phytoplasmas: Can Phylogeny Provide the Means to Understand Pathogenicity? B. C. KIRKPATRICK and C. D. SMART

Use of Categorical Information and Correspondence Analysis in Plant Disease Epidemiology S. SAVARY, L. V. MADDEN, J. C. ZADOKS and H. W. KLEIN-GEBBINCK

Contents of Volume 22 Mutualism and Parasitism: Diversity in Function and Structure in the “Arbuscular” (VA) Mycorrhizal Symbiosis F. A. SMITH and S. E. SMITH

Calcium Ions as Intracellular Second Messengers in Higher Plant A. A. R. WEBB, M. R. McAINSH, J. E. TAYLOR and I. M. HETHERINGTON

The Effects of Ultraviolet-B Radiation on Plants: A Molecular Perspective B. R. JORDAN

Rapid, Long-Distance Signal Transmission in Higher Plants M. MALONE

Keeping in Touch: Responses of the Whole Plant to Deficits in Water and Nitrogen Supply A. J. S. McDONALD and W. J. DAVIES

...

Xlll

xiv


Contents of Volume 23 PATHOGEN INDEXING TECHNOLOGIES The Value of Indexing for Disease Control Strategies D. E. STEAD, D. L. EBBELS and A. W. PEMBERTON

Detecting Latent Bacterial Infections S. H. DE BOER, D. A. CUPPELS and R. GITAITIS

Sensitivity of Indexing Procedures for Viruses and Viroids H. HUTTINGA

Detecting Propagules of Plant Pathogenic Fungi S. A. MILLER

Assessing Plant-Nematode Infestations and Infections K. R. BARKER and E. L. DAVIS

Potential of Pathogen Detection Technology for Management of Diseases in Glasshouse Ornamental Crops

I. G. DINESEN and A. VAN ZAAYEN Indexing Seeds for Pathogens J. LANGERAK, R. W. VAN DEN BULK and A. A. J. M. FRANKEN A Role for Pathogen Indexing Procedures in Potato Certification S. H. DE BOER, S. A. SLACK, G. VAN DEN BOVENKAMP and I. MASTENBROEK

A Decision Modelling Approach for Quantifying Risk in Pathogen Indexing C. A. LEVESQUE and D. M. EAVES

Quality Control and Cost Effectiveness of Indexing Procedures C. SUTULAR


Contents of Volume 24 Contributions of Population Genetics to Plant Disease Epidemiology and Management M. G. MILGROOM and W. E. FRY

A Molecular View through the Looking Glass: the Pyrenopeziza brussicae-Brussica Interaction A. M. ASHBY The Balance and Interplay Between Asexual and Sexual Reproduction in Fungi M. CHAMBERLAIN and D. S. INGRAM The Role of Leucine-Rich Repeat Proteins in Plant Defences D. A. JONES and J. D. G. JONES Fungal Life-styles and Ecosystem Dynamics: Biological Aspects of Plant Pathogens, Plant Endophytes and Saprophytes R. J. RODRIGUEZ and R. S. REDMAN

Cellular Interactions between Plants and Biotrophic Fungal Parasites M. C. HEATH and D. SKALAMERA Symbiology of Mouse-Ear Cress (Arubidopsis thulianu) and Oomycetes E. B. HOLUB and J. L. BEYNON

Use of Monoclonal Antibodies to Detect, Quantify and Visualize Fungi in Soils F. M. DEWEY, C. R. THORNTON and C. A. GILLIGAN Function of Fungal Haustoria in Epiphytic and Endophytic Infections

P. T. N. SPENCER-PHILLIPS

xv

xvi


Towards an Understanding of the Population Genetics of Plant-Colonizing Bacteria B. HAUBOLD and P. B. RAINEY

Asexual Sporulation in the Oomycetes A. R. HARDHAM and G. J. HYDE

Horizontal Gene Transfer in the Rhizosphere: a Curiosity or a Driving Force in Evolution? J. WOSTEMEYER, A. WOSTEMEYER and K. VOIGT

The Origins of Phytophthoru Species Attacking Legumes in Australia J. A. G. IRWIN, A. R. CRAWFORD and A. DRENTH

Contents of Volume 25 THE PLANT VACUOLE The Biogenesis of Vacuoles: Insights from Microscopy F. MARTY

Molecular Aspects of Vacuole Biogenesis D. C. BASSHAM and N. V. RAIKHEL

The Vacuole: a Cost-Benefit Analysis J. A. RAVEN

The Vacuole and Cell Senescence P. MATILE

Protein Bodies: Storage Vacuoles in Seeds G. GALILI and E. M. HERMAN

Compartmentation of Secondary Metabolites and Xenobiotics in Plant Vacuoles M. WINK


xvii

Solute Composition of Vacuoles R. A. LEIGH

The Vacuole and Carbohydrate Metabolism C. J. POLLOCK and A. KINGSTON-SMITH

Vacuolar Ion Channels of Higher Plants G. J. ALLEN and D. SAUNDERS

The Physiology, Biochemistry and Molecular Biology of the Plant Vacuolar ATPase U. LUlTGE and R. RATAJCZAK

The Molecular and Biochemical Basis of Pyrophosphate-Energized Proton Translocation at the Vacuolar Membrane R.-G. ZHEN. E. J. KIM and P. A. REA

The Bioenergetics of Vacuolar H+ Pumps J. M. DAVIES Transport of Organic Molecules Across the Tonoplast E. MARTINOIA and R. RATAJCZAK

Secondary Inorganic Ion Transport at the Tonoplast E. BLUMWALD and A. GELLI

Aquaporins and Water Transport Across the Tonoplast M. J. CHRISPEELS. M. J. DANIELS and A. W E E

Contents of Volume 26 Developments in the Biological Control of Soil-borne Plant Pathogens J. M. WHIPPS

xviii


Plant Proteins that Confer Resistance to Pests and Pathogens P. R. SHEWRY and J. A. LUCAS

The Net Primary Productivity and Water Use of Forests in the Geological Past D. J. BEERLING

Molecular Control of Flower Development in Petunia hybrids L. COLOMBO, A. VAN TUNEN, H. J. M. DONS and G. C. ANGENENT

The Regulation of C4Photosynthesis R. C. LEEGOOD

Heterogeneity in Stomata1 Characteristics J. D. B. WEYERS and T. LAWSON

Contents of Volume 27 CLASSIC PAPERS The Structure and Biosynthesis of Legume Seed Storage Proteins: A Biological Solution to the Storage of Nitrogen in Seeds D. BOULTER and R. R. D. CROY

Inorganic Carbon Acquisition by Marine Autotrophs J. A. RAVEN

The Cyanotoxins W. W. CARMICHAEL

Molecular Aspects of Light-harvesting Processes in Algae T. LARKUM and C. J. HOWE

Plant Transposable Elements

R. KUNZE, H. SAEDLER and W.-E. LONNIG


xix

Contents of Volume 28 Protein Gradients and Plant Growth: Role of the Plasma Membrane H+-ATPase

M. G. PALMGREN The Plant Invertases: Physiology, Biochemistry and Molecular Biology Z. TYMOWSKA-LALANNE and M. JSREIS

Dynamic Pleomorphic Vacuole Systems: Are They Endosomes and Transport Compartments in Fungal Hyphae? A. E. ASHFORD

Signals in Leaf Development T. P. BRUTNELL and J. A. LANGDALE

Genetic and Molecular Analysis of Angiosperm Flower Development V. F. IRISH and E. M. KRAMER

Gametes, Fertilization and Early Embryogenesis in Flowering Plants C. DUMAS, F. BERGER, J. E.-FAURE and E. MATTHYS-ROCHON


PREFACE

One of the classical problems of experimental plant ecology for much of this century, has been the elucidation of mechanisms which control the extreme growth preferences many plants show for either acidic or calcareous soils. The chapter by Lee evaluates both the historical evidence on this ‘calcicole-calcifugeproblem’, and more recent information on plant ion transport processes and calcium signalling. The potential contribution of molecular technologies is considered. The article stresses the importance of studying the interactions between edaphic factors rather than single factors and emphasises the important, but rather neglected contribution of the rhizosphere microflora. The impact of ozone pollution on crop production in the developed world is well documented but its importance in the more vulnerable economies of the developing countries of Asia, Africa and Latin America is less clear. The article by Ashmore and Marshall considers the effects of ozone on the physiology and yield of crop plants and identifies thresholds for impacts on crops of less-developed countries. The article then shows that actual ozone concentrations in LDCs may indeed reach inhibitory concentrations for sensitive species, that yields of staple crops may be adversely affected, and that there is considerable potential for a worsening situation over the next two decades. Ozone impacts on agriculture must now be considered as a global issue and the authors identify a clear and urgent need for collaborative international experimental programmes. As sedentary organisms the survival of higher plants depends critically on their abilities to detect and respond appropriately to a range of environmental signals, including those which are potentially stressful such as UV light and molecules produced by pathogens. Such responses involve mechanisms which detect the primary signals and which transduce those signals into a form that can promote the coordinated expression of specific sub-sets of genes. The article by Jenkins shows that these signals are not perceived and transduced in isolation. Rather, there are mechanisms for interaction within signal transduction networks which allow the plant to exhibit a response which is coordinated and integrated with other signals and processes including those which regulate normal development. A strength of the article is an evaluation of the exciting information emerging from the combined use of newer cell technologies such as microinjection, and the measurement of ion channel activities and intracellular calcium transients, with the approaches of molecular genetics including transgenic manipulation of the signalling pathway. One of the major limitations to crop growth on marginal soils, and some agriculture-intensive, irrigated soils is salinity. Lack of understanding of the mechanisms of salt tolerance and salt-induced inhibition of growth have hampered attempts to develop new crop cultivars with improved salt tolerance, and two articles

xxii

PREFACE

consider these two complementary aspects of the problem. The article by Amtmann and Sanders explores salinity tolerance from the perspective of recent advances in our knowledge of the sodium transport pathways across cell membranes. Data on selectivity, conductance, abundance and regulation of major cation uptake channel types is considered and integrated into a simple model which can be used to explore physiological questions on how some plants cope with saline environments. The article by Lazof and Bernstein focuses on the inhibition of shoot growth by NaCl which is one of the earliest responses to salinity. The physiological basis of this is unclear and the main thesis of the authors is that physiologists have tended to concentrate on whole or mature tissues, or tissues of ill-defined developmental and physiological status and that insufficient attention has been paid to the events in dividing and rapidly expanding cells and tissues. They hypothesise that the primary cause of salt-induced growth inhibition is a disturbance of mineral nutrition in these minute zones. Recommendations are made for advanced analytical methods that might be used to quantify alterations in nutrient transport and status within areas of rapid growth. As usual the Editor would like to thank all the contributors to this volume, for their patience and cooperation in making his task easier. J. A. Callow

The Calcicole-Calcifuge Problem Revisited

J. A. LEE

Department of Animal and Plant Sciences, University of Shefield S10 2TN, UK

I.

Introduction ...........................................................................................................

2

...............................

2 4 4

11. Edaphic Factors

A. B.

Acidic Soils .................................................................................................... Calcareous Soils ............

111. Controlled Environment Experimentation on Individual Edaphic factors ............. 4 4 A. Aluminium and Acidity ....................... B. Iron and Manganese Toxicity ...................................................................... 10 C. Bicarbonate Toxicity and Iron Deficiency 11 D. Phosphate ................................................... 13 E. Calcium . .............................. 14 F. Nitrogen ............................. 17 G. Microelements .............................................................................................. 20

IV. Conclusion ........................................................................................................... Acknowledgements .............................................................................................. References .................................

22 25 25

The adaptations shown by plants to growth in acidic and calcareous soils have ,fascinated ecologists during much of the 20th Century, but have not so f a r been elucidated entirely. This paper describes the major edaphic factors operating in these soils, arid discusses recent advances in our understunding of the udaptutions of calcicole arid calcifuge species to them. It concentrates on an evaluation of the results from controlled environment experimentation on variations in individual edaphic juctors. Adaptations to the ,following edap1zic.factor.sare considered in detail: low p H , toxicities of aluminium, bicarbonate and manganese deficiencies and to-ricities qf calcium and iron, and nitrogen and phosphorus availability. Although most experiments have coricentruted on udaptutions to individual eduphic factors, the importance of studying interactions between these factors is stressed. The paper also eniphasizes the potential importance of rhizosphere microorganisms in modibing plant Advances In Botanical Research Vul 29 incorporating Advances In Plant Pathology ISBN 0- 12-005929-0

Copyright 0 199’) Academic Press 411right,

reproduction

any rOml reserved

2

J. A. LEE

responses to edaphic factors. Important recent advances in our understanding of plant adaptations to aluminium and calcium supply in particular highlight the need to harness molecular biological tools and modern methods of studying ion transport processes to improve our understanding of the major factors involved in determining the distribution of plants on acidic and calcareous soils.

I. INTRODUCTION The origin of the modern era of experimental plant ecology can be traced to Tansley (1917) and was based on attempts to understand the factors governing plant distribution at a small scale. Tansley observed that Galium saxutile L. was confined to deep brown Podzolic soils on the limestone peneplain of Derbyshire, UK whereas Gulium stemeri Ehrend was confined to shallow rendzinas on the limestone dales. The deep acidic soils of the peneplain were later demonstrated to result from the deposition of wind-blown loess at the time of the last glaciation (Pigott, 1962). Although these two soil types are frequently found only a few metres apart, their floras are always distinct. Tansley conducted an experiment in the Cambridge Botanic Garden sowing the seed of the two Galium species alone and in competition with one another on a range of soils. Gulium saxutile grew much more vigorously on acidic soils when grown in monocultures, but a few survived and flowered on the calcareous soil. Gulium stemeri grew well on the calcareous soil, but some seedlings survived even in competition with Galium saxatile on acid peat. However, when grown in competition with Galium stemeri, Gulium saxatile seedlings only survived on acidic soils. Tansley concluded that soil factors combined with competition resulted in the distribution of the two species in the field. It was not until the 1950s that field experiments were first established to test the validity of Tansley’s findings. Rorison (1960a), in an experiment on the chalk and greensand of southern England, demonstrated that competition from other species was not the primary factor affecting seedling survival of calcicoles and calcifuges in respectively acid and calcareous grassland. Later experiments by Grime and his co-workers (see e.g. Grime and Curtis, 1976) demonstrated, for example, that seeds of calcifuge species germinated on calcareous soils in Derbyshire, but seedling mortality was particularly associated with frost and drought. The field experiments of Grime and Rorison supported the earlier view of Hope-Simpson (1938) that the primary factor governing the distribution of calcicoles and calcifuges was soil chemistry.

11. EDAPHIC FACTORS The fact that large differences in soil chemistry can occur over differences of only a few metres in the limestone dales of Derbyshire is illustrated in Table. I. The extractable calcium is c.24 times, the magnesium c.6 and the ammonium c.2 times greater in the rendzina than in the brown podzolic soil. The latter has no detectable

TABLE I Some extractable element contents ( p g g-’ soil) from two soils over limestone at Coombesdale, Derbyshire, UK. Cations and phosphorus were extracted with 1M ammonium acetate (pH 7.0), and nitrate-N and ammonium-N with IM KCI. Figures are means of a minimum of 10 samples (cations and phosphorus) and 8 samples (N03-N and NH3-N)2 1 S.D. pH was determined in a 1:1 (wt) aqueous suspension. Data of J. R. L e a h

Rendzina Podzolic brown earth

Ca

Mg

Fe

K

P

NOyN

N€&-N

PH

6958 ? 472 291 5 2 9 1

172 2 13 2925

1?1 25 5 2

8529 95 ? 35

3.8 ? 1.6 6.0 5 4.6

1922 0

21 ? 3 921

7.0 5 0.1 4.65 0.2

4

J. A. LEE

nitrate and a 25 times greater extractable iron content than the former. In contrast the two soils have similar extractable potassium and phosphorus contents. However, much of our knowledge of the chemical composition of soil solutions and its effects on plant growth in acidic and calcareous soils comes from agriculture (see e.g. Hewitt, 1952). This information can be summarized as follows. A. ACIDIC SOILS

Soil acidity factors include deficiencies of calcium, magnesium, potassium and molybdenum, increased solubility and toxicity of aluminium, manganese and iron, reduced availability of phosphate, and an impaired nitrogen cycle. In agricultural soils (other than those on periodically waterlogged acid sulphate soils) there is no evidence that hydrogen ion concentration per se is directly damaging to plant growth. B. CALCAREOUS SOILS

Shallow soils over chalk or limestone are typically highly porous, freely draining and saturated with calcium carbonate. The predominant ions in the soil solution are Ca2* and HCO;, the concentration of the latter in particular being dependent on the partial pressure of carbon dioxide in the soil atmosphere. Associated with a pH of the soil solution close to neutrality (pH 7-8), low plant availability of iron, cobalt, boron and phosphate are potentially important factors. The predominance of Ca” in the soil solution may also pose problems for the uptake of, for example, K + . However, in calcareous soils it is assumed that there is no impairment of the nitrogen cycle, and that NO3- is the major form of available nitrogen for plants (but see Table I) whereas NH: predominates in acidic soils.

111. CONTROLLED ENVIRONMENT EXPERIMENTATION ON INDIVIDUAL EDAPHIC FACTORS The 1960s represented the most intensive period of experimentation on the calcicolecalcifuge problem. This reflected the new availability of improved controlled environment facilities and the great interest in autecological problems. Emphasis was very much placed on an understanding of the relative importance of individual soil factors in determining plant distribution. A strong feature of these investigations was a comparison of the responses of calcicole and calcifuge species to individual edaphic factors. A. ALUMINIUM AND ACIDITY

The potential importance of aluminium as a differential edaphic factor can be judged by the fact that it is the most abundant metallic element in the soil, it is toxic to many plants at low concentrations in solution, and is present mostly in insoluble

THE CALCICOLE-CALCIFUGE PROBLEM REVISITED

5

forms above pH 5.0. Rorison (1960b) demonstrated that the calcicole species Scahiosa columburia L. produced stunted roots when grown on an acidic sand which were similar to those produced when Scabiosu was grown on a solution containing 5 0 m g l - ' Al. He suggested that the principal cause of the failure of Scnbiosa on acidic soils was aluminium toxicity. Subsequently a series of investigations demonstrated that calcicole species showed marked growth inhibition by aluminium in solution, whereas calcifuge species were largely unaffected or even stimulated at low concentrations (see e.g. Clarkson, 1966). The importance of aluminium as a soil acidity factor can also be inferred from intraspecific studies. Thus Davies and Snaydon ( 1973) demonstrated that populations of Anthoxanthurn odoraturn L. in acidic and calcareous (limed) plots from the Park Grass Experiment at Rothamsted differed in their response to aluminium in solution. Acidic populations showed little or no inhibition of root growth by up to 54 mg I F ' Al whereas calcareous populations were inhibited throughout the range of aluminium concentrations used. This difference in response to aluminium evolved within 65 years of liming treatments being imposed, pointing to the importance of aluminium as a soil acidity factor. New information on the toxicity of aluminium species in solution has led to an awareness of the toxicity of A17+,the extreme toxicity of the Al13polymer and the non-toxicity of AI(0H)'- and various aluminium chelates (see e.g. Kinraide and Parker, 1990; Kinraide, 1991; Kinraide and Ryan, 1991). The major factors affecting the rate of dissociation of minerals in soils appear to be pH and the availability of ions to react with the dissolving surface. The release of aluminium from kaolinite, for example, is a function of pH in the range where H+ ions are adsorbed by the clay, and reaches a maximum at pH 0.05

30 01'

Succinate

+ malate Oxalate

Citrate

lsocitrate

tionsof rhe Royul Sociey of London, B 341, 67-74. Ruiz, L. P. and Mansfield, T. A. (1994). A postulated role of calcium oxalate in the regulation of calcium ions in the vicinity of stomata1 guard cells. New Phytofogist 127. 473481. Runge, M. ( 1974). Die Stickstoff-Mineralisation im Boden eines Sauerhumus-Buchenwaldes. 11. Die Nitratproduktion. Oecologiu Plunrrrrum 9, 219-230.

30

J. A. LEE

Ryan, P. R., Delhaize, E. and Randall, P. J. (1 995). Characterisation of Al-stimulated efflux of malate from the apices of Al-tolerant wheat roots. Planta 196, 103-110. Scheible, W-R., Gonzalez-Fontes, A., Morcuende, R., Lauer, M., Geiger, M., Glaab, J., Gajon, A., Schulze, E-D. and Stitt, M. (1997). Tobacco mutants with a decreased number of functional nia genes compensate by modifying the diurnal regulation of transcription, post-translational modification and turnover of nitrate reductase. Planta 203, 304-319. Snowden, R. E. D. and Wheeler, B. D. (1995). Chemical changes in selected wetland plant species with increasing iron supply, with specific reference to root precipitates and iron tolerance. New Phytologist 131, 503-520. Takagi, S., Nomoto, K. and Takemoro, T. (1984). Physiological aspects of mugeneic acid a possible phytosiderophore of graminaceous plants. Journal of Plant Nutrition 7 , 469471. Tansley, A. G.(1917). On competition between Galiurn saxatile L. (G. hercynicurn Weig) and G. sylvestre Poll (G. asperurn schreb) on different types of soil. Journal of Ecology 5, 173-179. Taylor, G. (1995). Overcoming barriers to understanding the cellular basis of aluminium resistance. In “Plant Soil Interactions at Low pH” (R. A. Date, N. J. Grundon, G. E. Rayment and M. E. Probert, eds) pp. 255-269. Kluwer Academic Publishers, Dordrecht . Tyler, G. (1992). Inability to solubilise phosphate in limestone soils - key factor controlling calcifuge habit of plants. Plant and Soil 145, 65-70. Tyler, G. (1994). A new approach to understanding the calcifuge habit of plants. Annals of Botany 73, 321-330. Tyler, G. and Olsson (1993). The calcifuge behaviour of Viscaria vulgaris. Journal of Vegeration Science 4, 29-36. Tyler, G.and Strom, L. (1995). Differing organic acid exudation patterns explain calcifuge and acidifuge behaviour of plants. Annals of Botany 75, 75-78. Wallihan, E. F. (1961). Effects of sodium bicarbonate on iron absorption by orange seedlings. Plant Physiology 36, 52-53. Webb, A. A. R., McAinsh, M. R., Taylor, J. E. and Hetherington, A. M. (1996). Calcium ions as intracellular second messengers in higher plants. Advances in Botanical Research 22, 45-96. Wieland, E. and Stumm, W. (1992). Dissolution kinetics of kaolinite in acidic aqueous solutions at 25OC. Geochimica Cosmochimica Acta 56, 3339-3355. Woolhouse, H. W. (1966a). Comparative physiological studies on Descharnpsia fiexuosa, Holcus mollis, Arrhenatherum elatius and Koeleria gracilis in relation to growth on calcareous soils. New Phyrologist 65, 22-3 1. Woolhouse, H. W. (1966b). The effect of bicarbonate on the uptake of iron in four related grasses. New Phytologist 65, 372-375. Woolhouse, H. W. (1969). Differences in the properties of the acid phosphatases of plant roots and their significance in the evolution of edaphic ecotypes. In “Ecological Aspects of the Mineral Nutrition of Plants” (I. H. Rorison, ed.) pp. 357-380. Blackwell Scientific Publications, Oxford. Woolhouse, H. W. (1983). Toxicity and tolerance in the responses of plants to metals. In “Physiological Plant Ecology 111. Responses to the Chemical and Biological Environment”. Encyclopedia of Plant Physiology New Series, 12C (0. L. Lange, P. S. Nobel, C. B. Osmond and H. Ziegler, eds) pp. 245-300. Springer-Verlag. Berlin. Yermiyahu, U., Brauer, D. K. and Kinraide, T. B. (1997). Sorption of aluminum to plasma membrane vesicles isolated from roots of Scout 66 and Atlas 66 cultivars of wheat. Plant Physiology 115, 1119-1125.

Ozone Impacts on Agriculture: An Issue of Global Concern

M. R. ASHMORE and F. M. MARSHALL

Centre for Environmental Technology, Imperial College of Science Technology and Medicine, Silwood Park, Ascot, Berks SL5 7PI: UK*

I.

Introduction .........................................................................................................

11. Ozone Impacts on Agricultural Crops ..... ....... ............................. A. Exposure-Response Studies .......................................................................

32 33 34

111. Rural Ozone Levels in Developing Countries ........

36

IV. Direct Evidence of Adverse Effects on Crops ._...... A. Studies with Field Chambers ...................................................................... B. Studies with Ozone Protectant Chemicals ..................................................

39 39 41

V.

Responses to Ozone of Tropical Crops and Cultivars ....................................... A. Experimental Studies ................................................................... ................ . B. Factors Influencing Ozone Sensitivity in the Field ....................................

V1. Future Concentrations and Impacts of Ozone .................................................... VII. Conclusions ............................................ Acknowledgements .. References ................

43 43 45

46 46 48 49

While ozone has been shown to be the most important air pollutant affecting national crop production in North America and western Europe, mainly because it is found at phytotaxic concentrations over large areas, its impact in the developing countries qf Asia, Africa and Latin America, where the economic and social consequences of loss of production may be much greatec is uncertain. This review assesses the current and future significance c$ ozone impacts on agriculture in these countries. Although the information available on rural ozone *Address for correspondence: Department of Environmental Science, University of Bradford, Bradford, W. Yorks, BD7 1DP Advances in Botanical Rexarch Vol. 29

incorporating Advance, in Plant Psthulogy ISBN 0-12-11059?Y-0

Copynghi 0 19YY Academic Press All rigtits o l reproduction in any lurm reserved

32

M. R. ASHMORE and F. M . MARSHALL

concentrations is very limited, it does show that these concentrations can be high enough to have adverse effects on sensitive species, while a limited number of experimental studies have shown decreases in yield of staple crops due to ambient ozone. Current projections oj' rising emissions of ozone precursors suggest that the impacts on agriculture may increase very rapidly over the next two decades. There is an urgent need for more rural studies to determine ozone concentrations, and their impact on major crop species. in order to assess the current scale of the problem, and to develop models to estimate the future impacts of increased emissions.

I. INTRODUCTION The impacts of air pollution have long been recognized as an issue for concern in agriculture both in North America and Europe. Research programmes in both continents have provided extensive information on the physiological effects of pollutants, the relative sensitivity of different crop species and cultivars, and the air pollutant concentrations at which adverse impacts are found (e.g. Heck et al., 1988; Jager et al., 1993). Attempts have also been made to quantify the impacts of different pollutants on crop yields at national or regional levels (Adams et a/., 1988; van der Eerden et al., 1988). These assessments consistently indicate that, although pollutants such as sulphur dioxide and particulates may have significant local impacts on crop production, the air pollutant which is clearly the most important in terms of regional or national economic impacts on agriculture is ozone. Ozone is not emitted directly into the atmosphere. Instead, high atmospheric concentrations are produced as a result of a complex series of reactions in the atmosphere, which involve emissions both of nitrogen oxides and of certain reactive hydrocarbons. These emissions are produced from a range of sources, but the most important of these in both North America and western Europe is undoubtedly motor vehicles. The reactions leading to ozone formation are favoured by high temperatures and light intensities, and thus it is characteristically a pollutant of hot summer days. A key feature of such ozone episodes is that high concentrations of the pollutant are not restricted to urban or industrial areas, where nitrogen oxides and hydrocarbons are emitted; instead, high concentrations of ozone may be found over large agricultural regions, and even in remote rural areas (UK PORG, 1993). Indeed, ozone concentrations are often lower in cities than in the surrounding rural areas. It is this wide distribution of high ozone concentrations in rural, as well as urban, areas which is the key reason for its importance as an air pollutant affecting agriculture. Although impacts of ozone on agriculture in North America and western Europe have received considerable attention, there has been little recognition of its potential impacts in the developing countries of Asia, Africa, and South and Central America. Significant impacts of ozone in such countries, where the need to increase food production to meet the requirements of growing populations, and earn foreign exchange, is often vital, could be of much greater economic or social importance than in those parts of the world currently experiencing agricultural

OZONE IMPACTS ON AGRICULTURE

33

surpluses. Emissions of major air pollutants are growing rapidly in many of these countries, with industrialization, urbanization and the growth of transport, while the high temperatures and high solar radiation typical of many of these countries are favourable for the production of high concentrations of ozone. This review aims to provide a critical evaluation of current knowledge of ozone impacts on agriculture in these regions of the world, and to assess the current and future significance of the problem. The first section briefly describes the impacts of ozone on crop physiology and yield, and identifies thresholds for significant impacts on sensitive crops derived from studies in North America and western Europe. These are then compared with the limited data on rural ozone concentrations in other parts of the world to assess the potential impacts of ozone. The available evidence of ozone impacts in the field in developing countries is then described, and the sensitivity of local crops considered. Finally, the significance of the predicted trends of increased emissions of ozone precursors is evaluated.

11.

OZONE IMPACTS ON AGRICULTURAL CROPS

Ozone may have impacts at different levels of organization, from the cellular to individual organs and plants, and to plant communities and ecosystems (Ashmore, 1991). After passing through the stomatal pore, ozone can react with organic molecules in the intercellular space, or with components of the extracellular fluid, leading to the formation of secondary oxidants which can react with the cell membrane. Such reactions can be prevented or reduced by antioxidants, such as ascorbate and polyamines. High concentrations of ozone can cause cells to collapse. leading to visible foliar injury, and effects on the plasma membrane can cause changes in membrane functions which can reduce photosynthetic processes in the chloroplasts. Reduction in COz fixation is typically found in leaves exposed to ozone over longer periods of time (Lenherr et al., 1988). Stimulated dark respiration often also occurs, probably due to increased respiration associated with maintenance and repair (Amthor and Cumming, 1988). The reduced C02 assimilation and increased respiratory C 0 2 loss leads to an overall reduction of assimilate production and export from the source leaves. In leaves of crop species exposed to ozone over long periods, the onset of senescence is typically accelerated (Grandjean and Fuhrer, 1989), and the period with positive net assimilation of CO? is diminished. The reduced overall production of assimilates and altered carbon allocation patterns result in reduced grain or seed yield. Ozone is rarely the only stress factor for crops, and its impact may be modified by a range of other factors. Soil water stress and atmospheric vapour pressure deficit can cause reductions in stomatal conductance and hence in ozone uptake, which may lead to reduced ozone impacts on yield (Fangmeier et nl., 1993). The chemical. physiological and morphological changes to leaves caused by ozone can also alter plant sensitivity to other stresses. There is evidence of such effects for tolerance of cold stress, attack by herbivorous insects and attack by fungal

34


pathogens. In the case of insect and fungal attack, these effects can be induced by relatively low ozone exposures; for example, ozone at ambient concentrations in south-east England has been shown to increase the performance of insect herbivores on field bean (Ashmore et af., 1987), and increased infestation of fungal pathogens on wheat was observed after one month’s exposure to a low concentration of ozone (von Tiedemann et al., 1991). It is well-established that there are differences between species in their sensitivity to ozone. However, many of the lists of sensitive species are based on visible injury induced by acute ozone exposures; although these are relevant to instances of visible injury in the field, they may not be related to relative sensitivity based on the effects on growth or physiology of longer-term exposures. It is not currently possible to provide comprehensive lists of relative sensitivity of species to these longer-term exposures. Furthermore, the cultivars of widely distributed crops, such as wheat, grown in tropical and subtropical regions may differ substantially in their growth pattern and physiology, and hence their response to air pollutants, from cultivars of more temperate regions.

A. EXPOSURE-RESPONSE STUDIES

The most important source of information on the impacts of ozone on crop yield comes from studies using field chambers in which crops have been exposed to a range of ozone concentrations, and exposure-response relationships for crop yield have been established. Qpically, open-top chambers are placed over field plots of soil-grown plants and supplied with filtered air, unfiltered air, or unfiltered air with ozone added. Such chambers provide climatic conditions that are similar, but not identical, to those outside (Colls et al., 1993). Some reservations about extrapolation to field conditions remain, and recent data (Pleijel et al., 1994) suggest that, due to forced turbulence, the ozone flux in such chambers is normally higher than that outside. This would lead to a tendency to overestimate the adverse effects of a given ozone concentration. Exposure-response data may also be derived from closed chamber studies in glasshouses or controlled environment facilities, although more care is needed in this case in interpreting the significance for field conditions. Complex chamber systems may not always be appropriate or practicable methods of assessing impacts of ozone on crops in developing countries. A useful alternative for ozone is the use of chemical ozone protectants, such as ethylenediurea (EDU), since this avoids the need for experimental enclosures or a power supply (Sanders et al., 1993). Plants treated with EDU can be used as a control and compared with untreated plants, thus providing an estimate of ozone effects. However, because the extent of plant protection from ozone provided is uncertain, these experiments may underestimate the impact of ozone on crops. The first major programme to determine exposure-response relationships for crops was the US National Crop Loss Assessment Network (NCLAN), which was


35

established in the late 1970s, with five experimental sites chosen to reflect the variation in climatic conditions and cropping systems across the country (Heck et al., 1988). Experimental studies involved the use of a standard design of open-top chamber, with a range of ozone concentrations being used in each experiment to generate dose-response relationships between ozone exposure (expressed as the seasonal mean ozone concentration for the seven hours between 9.00 and 16.00) and crop yield. This period was used as it is often the time of day at which the highest ozone concentrations are found. A total of 10 crops were examined (corn, soybean, wheat, hay, tobacco, sorghum, cotton, peanuts, barley and dry beans), representing altogether 85% of the US acreage. Soybean and cotton proved to be among the most sensitive crops to ozone, and barley the least. The programme was designed to allow an estimate to be made of the national impact of air pollution on crop yield. In addition, the dose-response data were integrated with economic models to estimate the value of the loss, and the financial benefits of measures to reduce pollution. The estimate of yield loss nationally requires two other sets of geographical information; data on ozone concentrations nationwide and data on the distribution of the key crops studied in the programme. The results of the study nationally were that a decrease in ozone concentrations of 40% would provide a net annual economic benefit of $3000 million, or about 2.8% of national production (Adams et al., 1988). However, the percentage losses were much higher in particular regions and for specific crops. For example, another study has estimated yield losses due to ozone in California to be 19% for cotton, 23% for dry beans and 24% for onions (Olszyk et ul., 1988). The exposure-response data derived in the NCLAN studies also allow yield losses for different crops to be estimated for a given ozone exposure. For a seasonal mean concentration of 50 ppb, estimated yield reductions for soybean, cotton and forage exceed lo%, whereas that for winter wheat is slightly under 10%; in contrast, at this ozone exposure, estimated yield losses for less-sensitive crops, such as sorghum and rice, were below 5% (Adams et ul., 1988). During the 1980s, an effort was made by the European Commission to develop a similar coordinated assessment of air pollutant impacts on agriculture (Jiiger et ul., 1993). This European Crop Loss Assessment Network had more sites than its US counterpart, but because it was collaborative it had less standardization of chamber design and experimental protocols. A smaller number of crops was studied, with work focusing on wheat and beans; in addition, some studies were conducted on oats and barley. The results showed significant effects of ambient air pollution on the yield of beans and wheat at several locations, although oats and barley appeared to be less sensitive. Unlike NCLAN, the European experimental programme was not specifically designed to estimate regional crop losses through integration with pollution and agricultural datasets. Nevertheless, the data have been employed in a form of agricultural risk assessment, albeit in a rather different way. This is through the establishment of new air quality standards, also termed ‘critical levels’, defined as the air pollutant concentration above which significant adverse effects can occur.

36

M. R. ASHMORE and E M. MARSHALL

The critical level for ozone derived from the European data uses a different method of expressing long-term ozone exposure - the accumulated exposure above a threshold concentration of 40 ppb (AOT40), during daylight hours (Ashmore, 1993; Fuhrer, 1994). Using such an index, linear relationships with crop yield are commonly found. For crop yield losses of 5% and lo%, the AOT40 value has been calculated, based on data for wheat, to be 3000 ppb.h or 6000 ppb.h. respectively (Fuhrer, 1996). The 7 h seasonal mean concentration of 50ppb over an 80 day growing season would correspond to an AOT40 value of 5600 ppb.h, sufficient to cause a 10% yield reduction in wheat. However, it is likely that other European crops, such as Phaseolus vulgaris, for which adequate data are not available to define a critical level, may be more sensitive to ozone (Fuhrer, 1994). No attempts have been made to estimate yield losses due to ozone across Europe. However, for the Netherlands, van der Eerden er czl. ( I 988) estimated that the total yield loss due to all air pollutants was 5%, with 3.4% being due to ozone alone. The impacts of ozone were estimated to be greatest on legumes, potatoes, fodder crops, vegetables and cut flowers. However, these estimates were based on earlier Canadian exposure-response data, rather than the results of the European programmes. Visible foliar injury is a common response to exposure to episodes of high pollutant concentrations, and may adversely affect the value of crop, as well as providing a route for secondary infection. Collaborative European studies of the impact of ozone on visible injury have been conducted using EDU, which provides some protection against ozone injury. The results of the European exercise show a clear north-south gradient, with visible injury being more likely in the warmer areas of southern and central Europe; they also show that there is a risk of visible injury to sensitive crops throughout Europe, except in northern Britain and Scandinavia (Benton et al., 1996).

111. RURAL OZONE LEVELS IN DEVELOPING COUNTRIES The results of the North American and western European studies provide indications of the concentrations of ozone over a crop growing season which may significantly reduce yield. These suggest that yield reductions of 10% or more might be found in sensitive crops when the seasonal mean concentration in the middle of the day exceeds 50 ppb. Extrapolation of these studies to field conditions in the tropics can only be made with caution, but comparison of measured rural concentrations in developing countries can indicate the potential for adverse effects on crop species. However, whereas urban air pollution in cities such as Mexico City, Delhi and Beijing has received considerable attention, and international urban monitoring networks have been established (WHOLJNEP, 1992), there has been very limited coordinated monitoring of air pollution in rural agricultural areas in Asia, Africa or South America. Thus, at the current time, it is only possible to draw on isolated studies in a small number of countries. Table I summarizes information from studies which provide seasonal mean ozone concentrations in the middle ot

37


TABLE I Ozone concentrations in rural areas in developing countries Concentration

Site ..

Brazil - two rural sites in the middle of the sugar cane area in the state of Sao Paolo Brazil - site in the savannah region of central Brazil Mexico - forest area 25 km southwest of Mexico City at an

-.

Reference -

.

7 h mean over 6 days during the dry season at two sites in the range 45-50 ppb

Kirchhoff et al. (1991)

7 h mean for the month of August 1990 about 50 ppb

Kirchhoff et al. ( 1992)

7 h mean concentration about 75 ppb for both summer and winter

Miller et al. (1994)

elevation of 2970 m Egypt - Western Desert 12 h daytime mean 49 ppb 12 h nighttime mean 44 ppb Summer 6 h mean 75-80 ppb Egypt - rural area SO km north of Cairo Seasonal mean midday South Africa - 25 krn concentration about SO ppb from centre of Johannesburg

Gusten et al. (1996) Farag et al. ( 1993)

Stevens ( I 987)

the day, based on continuous monitoring with techniques which measure ozone specifically. One country for which rural ozone data do exist is Egypt, where agricultural production is concentrated in a small area in the Nile valley and delta, which also has high population densities, considerable industrial activity and high traffic densities in some areas. One of the most detailed studies at a rural site in Egypt was carried out by Farag et al. (1993), who made continuous measurements of ozone over the course of one year. The site chosen was about 55 km north of Cairo in an agricultural area. Concentrations reached a maximum between 12.00 and 18.00, and in spring and autumn mean ozone concentrations in this time period were only 30-35 ppb. However, during the summer months, the average concentration was over 75 ppb in this period, with occasional values above 100 ppb. Although these concentrations were lower than those in the city centre and an industrial area of Cairo, they nevertheless indicate the potential for very significant adverse effects on local crops, since substantial impacts of ozone at seasonal mean concentrations of 80 ppb have been shown on a range of American and European crops. The authors suggested that the photolysis of locally applied pesticides could contribute to the high concentrations, but it seems more likely that emissions from Cairo are the major factor involved. These measurements are consistent with data recorded in the summer of 1991

38

M. R. ASHMORE and F. M . MARSHALL

within Cairo by Gusten et al. (1994). The diurnal variation was similar, and the mean afternoon concentrations in mid-summer were about 85 ppb; however, the urban concentrations on occasion reached 120 ppb. Concentrations have also been measured at a remote site in the Western Desert (Gusten et al., 1996). There, as expected, concentrations were lower, but mean daytime concentrations were still close to 50 ppb. In South Africa, Stevens (1987) reported ozone concentrations in the greater Johannesburg region. Although this was essentially an urban study, two of the stations were outside the city boundaries about 25 km from the centre. Concentrations at these sites were higher than in the city centre, and averaged about 50ppb over the summer months during the midday period. On occasional days, the concentration exceeded 120 ppb. Very large concentrations of most major air pollutants have been recorded in Mexico City, where ozone concentrations have reached 400 ppb, and frequently exceed 150 ppb. In Brazil, high concentrations of ozone are common in Sao Paolo and Cubatao, with values frequently exceeding 80 ppb, and reaching 200 ppb on occasions. In both countries there are few comparable data on ozone levels in the agricultural areas surrounding the large cities. However, the limited information which is available does indicate the potential for ozone impacts on vegetation. Ozone concentrations have been monitored in a mountain area close to Mexico City, where damage to forests has been reported (Miller et al., 1994), and compared to those in forests around Los Angeles where extensive ozone damage has been documented. The concentrations are lower at the Mexican site in summer, but higher in winter, and the Mexican site experiences 7 h mean concentrations in the middle of the day averaging about 75 ppb, throughout the year. A number of studies have been carried out in remote areas of South America as part of studies of global background tropospheric ozone. In general, these studies reveal ozone concentrations of 15-30 ppb, below those of any concern in terms of impacts on vegetation. However, there is some evidence of increased ozone concentrations during the dry season, when biomass burning is occurring in the region. Thus, Kirchoff et al. (1992) measured ground-level ozone at sites in an area of central Brazil dominated by cerrado vegetation during the dry season; the sites were chosen to avoid the influence of any urban sources of ozone precursors. Concentrations of ozone, expressed as monthly 7 h mean concentrations, reached 50 ppb during this period. Kirchoff et al. (1991) also demonstrated that burning of sugar cane fields could contribute to the formation of significant ozone concentrations; this source has increased considerably since the introduction of the use of alcohol as a substitute for gasoline in the early 1980s. Measurements made at two rural sites in the state of Sao Paolo, during the dry season of 1990, showed mean ozone concentrations in the middle of the day reaching about 50 ppb. These values were similar to those recorded simultaneously at an urban site, indicating that sugar cane burning can contribute to ozone formation at this time of year to a similar extent to urban transport and industry.


39

Data from the field studies in Pakistan described in Section IV demonstrate the presence of elevated ozone concentrations in the middle of the day at a site in the suburbs of Lahore, with mean midday concentrations reaching 60 ppb in some summer months (Maggs ef al., 1995). However, these measurements were made using a chemical method which may respond to other atmospheric oxidants, such as nitrogen dioxide, and thus they must be interpreted with some caution. Other data from urban sites in India, using a range of different analytical methods, have demonstrated 7 h mean ozone concentrations in summer months which exceed 40 ppb, and in some cases approach 60 ppb (Thimmiah, 1996). The evidence from the Indian subcontinent generally suggests the potential for generation of ozone in concentrations high enough to damage sensitive crops in and around large cities, but there is a lack of continuous monitoring using ozone-specific methods in rural areas. In summary, the limited data available demonstrate clearly that seasonal mean ozone concentrations at a number of sites fall in the range 50-80ppb, at which adverse effects on the yield of sensitive crops might be expected. At several of these sites, pollutant emissions from neighbouring cities are likely to be primarily responsible, but seasonal biomass burning may be a further additional source in certain areas and seasons. There is a clear need both to collate additional datasets which are not accessible through journal publications, and to increase the extent of ozone measurement in agricultural areas likely to be affected by urban or biomass emissions.

IV. DIRECT EVIDENCE OF ADVERSE EFFECTS ON CROPS The data summarized in Table I thus raise the possibility of adverse impacts of ozone on agricultural crops in developing countries. Direct experimental evidence to support this assertion comes from two major sources: field chamber studies in which air has been filtered to remove ambient pollutants, and field trials in which plants have had ozone protectant chemicals applied. Table I1 summarizes some of the key studies which have been carried out.

A.

STUDIES WITH FIELD CHAMBERS

The use of field chambers which are ventilated with unfiltered air, or with air filtered through activated charcoal to remove air pollutants, has proved an effective tool in identifying adverse effects of the ambient air pollutant mix in Europe. However, few such experiments have so far been carried out with field chambers in developing countries. The most important series of experiments of this nature is that using local cultivars of rice and wheat at a site on the outskirts of Lahore, in an area where these crops are being grown (Maggs et al., 1995a,b; Wahid et al., 1995a,b). Two cultivars of each species were grown in pots in the chambers under

TABLE I1 Field experiments on the effects of ozone on crops in developing countries Method

Reference

Response ~

Pakistan Punjab in the viciniw of Lahore

Or~m sativa cv Basmati-385 and IRRI-6

Pakistan Punjab in the vicinity of Lahore

Triticum aestivum cv Pal-81 and cv Chakwal-86

Pakistan Punjab; 3 locations in the vicinity of Lahore Indian Punjab

Glycine

cv NARC 1

) 7 2 ~

Open-top chambers with charcoal-filtered air to remove 0, and other pollutants Open-top chambers with charcoal-filtered air to remove O3 and other pollutants Application of EDU

Solanum tuberosurn cv. Kufri jyoti

Dusting with activated charcoal or addition of EDU

Abbis 35 km south of Alexandria, Egypt

Raphanus sativus and Brassica rapa

Application of EDU

Montecillos, Mexico; a rural site in the Valley of Mexico

Phaseolus vulgaris cv Canario 107 and Pinto 111

Application of EDU

42% yield loss in unfiltered air for Basmati-385; 37% loss for LRRI-6 46.7% yield loss in unfiltered air for Pak-81; 34.8% for Chakwal-86 49% reduction in seed wt. in untreated plants at the rural site Treated plants did not develop visible injury, whereas untreated plants did Root and shoot dry weight decreased by 30 and 17% in radish, and 17 and 11% in turnip, in untreated plants 4.5% yield reduction in untreated plants of Canario 107; 40.7% yield reduction in untreated plants of Pinto III

~

~~

~

Wahid et al. (1995a)

Wahid et al. (1995b)

Wahid, A. and Shamsi, S. R. A. (pers. comm.) Bambawale (1986)

Hassan et al. (1995)

Laguette Rey et al. (1986)


41

local cultivation conditions in two successive growing seasons (NovemberApril/May for wheat, and May/June-OctoberlNovember for rice). Plants were also grown outside the chambers, to demonstrate that there was relatively little effect of the chamber enclosure on crop growth and yield. All four experiments showed a large and significant effect of filtration on the yield of both species, with yield in unfiltered air being reduced by 34% and 45% for the two wheat cultivars (Pak-81 and Chaknwal-86), and by 37% and 46% for the two rice cultivars (Basmati-385 and IRRI-6), averaged over the two years. The most important yield component affected by filtration was the number of ears, or number of panicles, per plant, and in all cases, leaf sensescence was accelerated in unfiltered air. In contrast, effects of filtration on 1000 grain weight were relatively small. The concentrations of sulphur dioxide at this site were negligible, but there were significant concentrations of nitrogen dioxide and ozone. Mean nitrogen dioxide concentrations were typically about 25 ppb, except during the monsoon season, when they were much lower. Concentrations of ozone, determined using a chemical method which determines total oxidant levels, varied between 40 and 60-70 ppb, as a 6 h mean concentration, on individual days, except during a heavy monsoon period and cool winter periods. Concentrations of ozone were generally higher in the rice season than the wheat season. The recorded concentrations of nitrogen dioxide are lower than those normally found to significantly reduce crop growth and yield, and this was confirmed by laboratory studies of these particular cultivars (Maggs, 1996). Thus it is likely that the large yield reductions observed in this experiment were primarily due to ozone. The size of the yield reductions found is larger than has been reported at similar concentrations in controlled fumigation studies with wheat and rice (e.g. Kats er al., 1985; Kohut et ui., 1987; Pleijel et nl., 1991; Kobayashi, 1993). This may be due to differences in cultivar sensitivity or climate. The results of these experiments imply that ozone may be having a substantial impact on rice and wheat yields in the Pakistan Punjab, but until the work is repeated at other locations, it is impossible to be sure that the results do not represent the impact of an unusual air pollutant mix at the particular experimental site.

B. STUDIES WITH OZONE PROTECTANT CHEMICALS

The first experiment in a developing country to use the ozone protectant N-[2-(2-oxo- I -imidazolidinyl)ethyl]-N’-phenylurea(EDU) to assess the impacts of ambient ozone in a rural location was that of Bambawale (1986), who tested the hypothesis that leaf spot on Solanurn tuberosurn was caused by ozone. The work was carried out near Jalanghar, in northern India. and showed that application of both EDU and activated charcoal dust to screens above the plants reduced the prevalence of the symptom. Work reported in the same year by Laguette Rey et al.

42


( 1986) in Montecillos in Mexico demonstrated that application of EDU increased the yield of one cultivar of Phaseolus vulgaris by 41%, although the effect on a second variety was only 5%. This work was carried out in an area close to Mexico City where characteristic visible foliar symptoms of ozone damage have been found in a number of crops. Given the diagnostic value of this work, it is surprising that relatively few further studies have been carried out over the subsequent decade. The only recent published study with EDU in developing countries appears to be that of Hassan et al. (1 993, who grew Egyptian cultivars of radish and turnip at two sites: one in the suburbs of Alexandria, and one in a village (Abbis) in the Nile delta, 35 km south-east of Alexandria. The harvested dry weights of both species were significantly reduced at Abbis, by 30% for radish and by 17% for turnip, in plants not treated with EDU; at Alexandria, the dry weight of turnip was not significantly altered by EDU treatment, whereas that of radish was reduced by 24% in untreated plants. EDU also effectively reduced visible symptoms of ozone injury which appeared on radish at both sites, and on turnip only at the village site. The fact that the value of EDU protection was greater at the remote rural site than at the suburban site is consistent with measurements of ozone concentrations at the two sites, which showed higher values at the more rural site. This result is also consistent with studies in the US and in Europe, and can be explained by the removal of ozone by reaction with nitric oxide (NO) at more urban sites. The 6 h mean oxidant levels recorded during the experiment, in February and March, were 55 ppb in Alexandria and 67 ppb in the village site. It is probable that higher concentrations, with the potential for larger effects on yield, would be found in the summer months. Recently, A. Wahid and S . R. A. Shamsi (personal communication) have completed a similar experiment, with a Palustani cultivar of Glycine mar, in and around Lahore. A key element of the experiment was a comparison of the protective effect of EDU at the site on the outskirts of Lahore used in the chamber experiments, and at a rural site about 35 km east of the city. The seed weight per plant was 32% lower in the untreated plants, compared with the EDU-treated plants, at the suburban site, and 49% lower at the rural site. As in the Egyptian experiment, the larger difference in yield between the EDU-treated and control plants at the more rural site was associated with higher atmospheric oxidant levels. The studies with EDU in Mexico, Pakistan and Egypt clearly demonstrate the potential for ozone to cause large impacts on yield at rural sites close to major cities. Without further research it is impossible to be certain to what extent this effect would extend into more remote rural areas. However, given the large size of the effects observed in these experiments, and the fact that EDU may not prevent all adverse effects of ozone on crop yield (Hassan er al., 1995). this limited body of evidence clearly indicates that the impacts of ozone may be a very significant problem in the field.


43

V. RESPONSES TO OZONE OF TROPICAL CROPS AND CULTIVARS A. EXPERIMENTAL STUDIES

Studies using chambers in glasshouses or controlled environment rooms cannot replicate field conditions, but they may indicate the sensitivity of crops to ozone, as well as determining the mechanisms leading to the observed effects of ozone. Again, limited experimental data are available on the response of tropical crops or cultivars to ozone, and many of the experiments which have been carried out have used seasonal daylight mean concentrations well above the maximum of 80 ppb so far recorded at a rural site in a developing country (Table I). More experiments exist on the responses of crops such as wheat and rice, but these have used North American, European or Japanese cultivars, the responses of which may be quite different from the local cultivars. One important series of laboratory fumigation experiments which has been recently completed is that of Maggs (1996), who studied the response to ozone of the Pakistani cultivars of wheat and rice which were used in the filtration studies with open-top chambers on the outskirts of Lahore (Wahid et al., 1995a,b). The results are summarized in Table 111. The experiment with wheat involved exposing both of the cultivars of wheat used in the field chamber experiments to ozone for 8 h per day, on 5 days per week, over a period of 11 weeks. The 8 h mean concentration on the fumigation days was 65 ppb, although when expressed as a mean over the entire 1 I weeks it was 46 ppb. The plants were also exposed to 30 ppb nitrogen dioxide, alone or in combination with ozone; however, there was no significant effect of this concentration of nitrogen dioxide on plant growth or yield. In contrast, ozone reduced the yield per plant by 48% for Pak-81, and by 31% for Chak-86. Both cultivars showed a similar reduction, of about 30%, in 1000 grain weight, and the reason for the larger yield effect in Pak-81 was additional significant ozone effects on grain number per plant and percentage sterility. This experiment strongly suggests that the impacts of ambient air at the study site in Lahore were due to ozone, rather than nitrogen dioxide, and also demonstrates the sensitivity of the cultivars to ozone. However, the nature of the yield components most affected by ozone in these laboratory experiments differed from those in the field study. A similar experiment to examine the effects of ozone and nitrogen dioxide, alone or in combination, was carried out by Maggs (1996), with two cultivars of rice. However, this experiment was only of 40 days duration, and used lower concentrations - the 8 h mean ozone concentration on fumigation days was 47 ppb, whereas the 24 h mean nitrogen dioxide concentration was 17 ppb. As for wheat, there was no significant effect of nitrogen dioxide, alone or in combination with ozone, but ozone alone did significantly reduce vegetative biomass, by 19% in the case of Basmati-385 and by 18% in the case of IRRI-6. In this experiment, as in the

TABLE 111 Fumigation studies with ozone on crops or cultivars grown in developing countries Crop

Method

_ _ _ _ _ _ _ ~ ~ . _ _ _ _ _ ~

O3 exposure ~

Cicer arietinum

Fumigation in closed polythene chambers

80 ppb O3 for 2 hlday for 20 days

vicia faba

Fumigation in closed polythene chambers

80 ppb O3 for 1.5 hlday for 60 days

Triticum aestivum

Fumigation in closed glasshouse chambers

65 ppb O3 for 8 h/day for 5 days/week for 11 wks

Oryza sativa

Fumigation in closed glasshouse chambers

67 ppb O3 for 8 hlday for 40 days

Oryza sativa

Closed glasshouse chambers Fumigation in greenhouse domes

54 ppb O3 for 8 Wday for 133 days 70 ppb O3 for 7 Wday for 6 weeks

13 leguminous species and I fibre crop

~

_

Response _

_

Grain wt. reduced by 77% Reduced nodule size and number Chlorosis within 15 days Vegetative dry wt. reduced by 50% Reduced chlorophyll and protein content 48% reduction in grain wt. in cv Pak-81 31% reduction in grain wt. in cv Chakwal-86 19% reduction in vegetative dry wt. in cv Basmati-385 18% reduction in vegetative dry wt. in cv IRFU-6 57% reduction in grain wt. in cv. DRRI-6 Vegetative dry wt. reduced by more than 20% in Medicago sativa, Vigna radiata, Vigna mungo and Hibiscus cannabinus

~~~~

_

Reference

~

Singh and Rao (1982)

Agrawal et al. (1985)

Maggs (1996)

Maggs (1996)

Maggs (1996) Kasana (1988)

OZONE LMPACTS ON AGRICULTURE

45

earlier experiment with wheat, a major effect of ozone was to accelerate leaf sensescence, a phenomenon observed in many other studies of ozone on cereals, including the field chamber filtration study in Pakistan. A final, longer experiment, over 165 days, examined the effect of ozone alone on rice cv. IRRI-6. The mean 8 h concentration of ozone on fumigation days was 54 ppb (43 ppb over the entire experiment). This treatment caused a very large reduction, of 5796, in the total grain weight per plant. This was the result of a combination of reduced numbers of spikelets per plant, reduced panicle number per plant, increased spikelet sterility, and reduced 1000 grain weight. As in the other experiment, a major effect of ozone during the development of the crop was to accelerate leaf sensescence. The results of these experiments clearly demonstrate the sensitivity of the cultivars used to ozone, and indicate the potential for substantial yield reductions in the field. However, Maggs (1996) points out that the grain yield per plant achieved using these cultivars in the fumigation experiments was much lower than that in the field experiments in Pakistan, and thus extrapolation to field conditions must be made with caution. Table 111 also summarizes three studies of Indian crops or cultivars which have used realistic ozone exposures, in the range 50-80 ppb. Singh and Rao (1982) used a concentration of 80 ppb, for 2 h per day, to study ozone effects on gram (Cicer ariptinuni) plants, which were 90 days old. Within 15 days, chlorotic spots had appeared on the upper leaf surface, and both leaf number and root nodule number were significantly reduced, and plants harvested after 30 days showed large and significant reductions in both pod number and mean pod weight. Agrawal et ul. (1985) used a similar exposure regime in a longer-term study of Vicia faha. After 30 days of exposure, numbers of leaves and root nodules, protein and chlorophyll contents, and foliar nitrogen and phosphorus contents were all significantly reduced in the ozone treated plants. Whereas these two experiments were carried out in India, Kasana (1988) fumigated a range of Indian crops in an outdoor fumigation facility in the UK. The work focused on leguminous crops in view of their established sensitivity to ozone, and their importance as sources of both food and fodder in many tropical and subtropical countries. Exposure to 70ppb, for 7 h per day, for 6 weeks had relatively little effect on the vegetative dry weight of Cicer nrietinum, Cnjmus Cajun, Lens culinuris and Vigna unguicuhta. However, other tested species showed growth reductions of more than 20%. These were Medicagn sativa, Vigna rndiatu, Vigna mungo, in which the growth reduction was 60% and the fibre crop Hibiscus cannubinus.

B. FACTORS INFLUENCING OZONE SENSITIVITY IN THE FIELD

The experiments described in the previous sections are very limited, and also involve the growth of plants in pots, supplied with adequate water, and protected

46


from pests and diseases. In assessing the real influence of ozone on cropping systems under field conditions in tropical and subtropical areas, a number of other factors need to be taken into account, in addition to the actual ozone exposures at different locations. First, the time of year in which the crop is grown is of considerable significance. Many air pollutants have a typical seasonal cycle. In general terms, ozone levels tend to be higher in the summer, when conditions are more conducive to its formation. However, this will not apply in seasons with heavy rainfall, and in dry seasons, biomass burning may be an additional contributing factor. Furthermore, in the tropics, climatic conditions favourable to ozone production may continue almost throughout the year. The interaction of these factors with local cropping patterns needs further assessment. Secondly, the nature of the cropping systems may influence its response to ozone. The most obvious factor will be irrigation - in general, well-watered plants have a higher pollutant uptake and thus show greater sensitivity than rain-fed crops. However, there are a number of other factors, such as atmospheric humidity, temperature, salinity and fertilizer levels which may influence the responses of crops to air pollutants. A particularly important factor may be the known impacts of relatively low concentrations of air pollutants in influencing the performance of insect pests and plant pathogens (Bell et al., 1993). Finally, the choice of cultivar will also be important, since there is known to be a wide variation in sensitivity between different cultivars in pollution sensitivity. The issues of whether there are systematic differences between, for example, high-yielding cultivars and more traditional cultivars, and between cultivars bred in areas with high or low air pollution levels, are of considerable practical significance. The introduction of new, more tolerant cultivars, has proved effective in certain cases in the US in reducing the impacts of ozone; if the pollutant is indeed reducing crop yields in certain areas of the developing world, the ability to identify alternative cultivars with greater tolerance could offer the opportunity to reduce this yield loss more rapidly than might be feasible by addressing pollutant emissions.

VI. FUTURE CONCENTRATIONS AND IMPACTS OF OZONE There is now good observational data to suggest that the global background tropospheric ozone concentration is increasing as a result of human activities (Penkett, 1988; Hough and Dement, 1990). The production of ozone in the background troposphere is limited by emissions of nitrogen oxides, and should increase as these emissions increase. Thus, evidence of increased emissions of nitrogen oxides, and increases in nitrate concentrations in ice cores in remote areas, such as Greenland, supports the empirical evidence of a rising trend of background tropospheric ozone concentrations, linked to increased global emissions of nitrogen oxides.


47

Chameides et al. (1994) used a global model to examine in more detail the increases in ozone concentrations as a result of increased nitrogen oxide emissions, focusing on three ‘continental-scale metro-agro-plexes’. These areas, comprising eastern North America, Europe, and east China and Japan, are regions where currently high energy consumption, high population densities and intensive agricultural production coincide - the authors estimate they are responsible for 75% of global energy and fertilizer production, and 60% of global food production and export. The predicted rise in nitrogen oxide emissions by 2025 is particularly marked in East Asia. The increases in nitrogen oxide emissions predicted by Chameides et al. (1994) were then used to estimate increases in global ozone levels, which were then linked to data on ozone impacts on cereals. As discussed above, these suggest that the threshold for significant yield reductions in sensitive cereals, such as wheat, is 50ppb, whereas that for less sensitive cereals, such as rice, is 70ppb - both concentrations being expressed as 7 h seasonal mean concentrations. Using this model, the increase in nitrogen oxide emissions predicted for 2025 will increase ozone impacts significantly. Nitrogen oxide emissions are predicted to increase globally from 110 kT day-’ in the mid 1980s to 150-180 kT day-’ in 2025, depending on the economic scenario used. This change is predicted by Chameides et al. (1994) to increase the percentage of the world cereal crop exposed to ozone levels above the threshold of 50-70 ppb from 9-35% to 30-75%. Much of the increase in emissions predicted by Chameides et al. (1994) will occur outside North America and western Europe. Although the use of fossil fuels in power generation, transport and industry will be an important cause of the increased emissions, Chameides et al. ( 1994) also calculate that increased fertilizer use will be another important contributor to increased NO, emissions. Although the details may vary, other sources are in broad agreement with a scenario of significantly increased emissions of nitrogen oxides by 2020, concentrated particularly in Asia, resulting from nitrogenous fertilizer use, as well as fossil fuel usage, and that this will result in increased ground-level ozone concentrations (Galloway, 1989; Galloway el al., 1994, 1995; Houghton et al., 1996). Galloway (1995) emphasizes the importance of the increases in Asia, in particular, where the increases in fossil fuel and fertilizer use needed, under current scenario assumptions, to sustain 50% of the world’s population will be very large in absolute terms.

VII. CONCLUSIONS It is clear from this review that the amount of information available on rural ozone levels, and on the responses of local crops to these levels, is so small that no coherent analysis of the impacts of the pollutant on agriculture in developing countries is possible. Nevertheless, the evidence which does exist indicates that the effect of ozone in many areas of such countries could already be substantial.

48


Clearly, more research is urgently needed to assess the potential impacts of ozone more thoroughly. Enough information is available to identify the crops and regions globally where the problem might be of greatest concern. These are likely to be areas of the wet tropics, or the dry tropics where agriculture is based around irrigation, where emission densities of nitrogen oxides are relatively high. Key areas may thus be south and south-east Asia, parts of central and south America, and parts of north Africa. The evidence summarized in this paper indicates that ozone impacts on agriculture are no longer a problem only in North America and western Europe. It is clear that, in the future, this is likely to become more of a global issue, for two reasons. First, in many regions of the planet, local urban or industrial emissions of nitrogen oxides are likely to increase substantially over the next 30 years, producing polluted air masses bringing ozone into the surrounding rural areas. Secondly, the steady increase in global emissions of nitrogen oxides over the same period means that an increase in global background tropospheric ozone levels is likely. Where large local or regional increases are superimposed on the steadily increasing global background level, the impact may be considerable. It is thus possible that ozone concentrations are already, or could become, a significant constraint on national or regional agricultural production in a number of countries in which substantial increases in food production are needed to feed growing populations. Much of the attention concerning the issue of air pollution in developing countries is currently focused on the impacts on human health in large cities. However, air pollution impacts on agriculture in and around these cities could have significant economic and social impacts, and thus indirect impacts on public health. The importance of the issue has not yet been recognized by national or international agencies. Although this review has concentrated on ozone, as the most important pollutant on national or regional scales, the impacts of sulphur dioxide, in particular, at a more local scale also need more recognition. Air pollution monitoring has tended to focus on urban areas, and there has been little rural monitoring; this is a particular deficiency in the case of ozone, which may be of considerable significance in rural areas. There is a clear and urgent need to develop collaborative international experimental programmes to assess the current and future significance of ozone impacts on agriculture on a more global basis. The development of air quality guidelines for agriculture, which are related to local cropping patterns and climatic conditions, would also be helpful in guiding policy development in this area.

ACKNOWLEDGEMENTS We acknowledge the contribution of our colleagues Nigel Bell and Eleanor Milne to developing many of the ideas expressed in this paper. Our work on air pollution impacts on agriculture in developing countries has been supported by the Scientific

OZONE lMPACTS ON AGRICULTURE

49

Cooperation Programme of the European Community, and by the Environmental Research Programme of the Department for International Development.

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Signal Transduction Networks and the Integration of Responses to Environmental Stimuli

GARETH I. JENKINS

Plant Molecular Science Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Bower Building, University of Glasgow, Glasgow G I 2 8QQ, UK

I. Introduction .......................................................................................................... A. Networks Versus Pathways .......................................................................... B. Achieving an 'Appropriate' Response .........................................................

54 55 56

11. Interactions Within Signalling Networks ............................................................. A. Evidence of Negative Regulation ..... B. Evidence for Synergism ...............................................................................

57 57 63

111. Approaches to Identify the Mechanisms Involved in Interactions Between Signalling Pathways .............................................................................................

67

Conclusions .......................................................................................................... Acknowledgements ............... ........................................................... References ............................. ......

69 70 70

IV.

This paper is dedicated to the late Professor Harold W. Woolhouse, who will be remembered as a great enthusiast for plant science. Professor Woolhouse was the author's PhD supervisor.

The survival of plants is dependent on their ahiliy to sense and respond appropriately to (I wide range of environmental stiniuli, some of which fire potentially harmful. Such responses often involve the expression of .spec@- sub-sets of genes whose products are concerned with rnininzi,-ing cell duniage. Each response must be uppropriute in the context of other respnnses rind the necessary coordination and integration of responses i s uchieved through interaction or 'cross-tulk' between the relevont signal transduction pathways. Exuinples ure discussed both of negative regulation between signalling pathways and of partirulur contbinations of priniar?,stimuli together eliciting a hyper-response. For instance, negative regulation is observed in the repression of photosynthetic genes b y sugars, in defence gene regulation and beMJeen phytochronie signal trunsduction piithwvays. In cvntrmt. srrong

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synergistic interactions are observed between, jor example, ethylene and methyl jasmonate in stimulating speciJc defence genes and between blue, UV-A and UV-B light in the regulation of chalcone synthase gene expression. The combined application of biochemical, molecular and genetic approaches offers the most powerful means of dissecting the cellular and molecular mechanisms involved in these interactions.

1. INTRODUCTION Being sedentary, plants are at the mercy of their environment. Throughout their growth and development they are exposed to a wide range of environmental variables. Although some of these, such as daylength, are benign and may be used as cues to initiate developmental transitions, others, such as extremes of temperature, drought, UV-B and pathogen attack are potentially very harmful. Plants have, therefore, evolved protective responses which minimize the adverse effects of these abiotic and biotic stresses. Many of the protective responses of plants involve differential gene expression. That is, particular subsets of genes are expressed in response to specific external stimuli. Some gene products are induced by a range of stimuli whereas others are much more specific. Over the last ten to fifteen years numerous genes have been identified that are responsive to particular stimuli. For example, low temperature stimulates the expression of over thirty different genes (Hughes and Dunn, 1996). Similarly, pathogen attack induces a range of genes, some of which encode proteins, such as glucanases and chitinases, that have antipathogenic effects, whereas others encode enzymes that synthesize compounds, ‘phytoalexins’, that limit pathogen invasion in the affected tissues (Cutt and Klessig, 1992; Dixon and Paiva, 1995). As a further illustration, exposure of plants to damaging UV-B radiation triggers the expression of genes encoding enzymes that synthesize UV-absorbing phenylpropanoid and flavonoid compounds in the epidermal tissues (Stapleton, 1992; Jenkins et al., 1997). It is evident that the survival of plants is dependent on their ability to sense and respond appropriately to a wide range of environmental stimuli. Hence, plant cells possess mechanisms to detect specific environmental signals, to transduce the information within the cells and to effect the appropriate responses. In the case of gene expression responses, the end point of signal transduction is likely to involve the activation of specific transcription factors, unless of course the gene in question is regulated entirely via post-transcriptional mechanisms. Transcriptional control itself is very complex. In addition to the reversible modification of transcription factors by, frequently, phosphorylation, there is evidence that transcription factors may be stimulated to move into or out of the nucleus as a result of modification (Harter et al., 1994; Terzaghi et al., 1997). Moreover, the initial effect of the external stimulus may be to elicit the synthesis of a transcription factor or other effector, which in turn stimulates the transcription of other genes involved in the response. Thus several gene expression responses are prevented by inhibitors of protein synthesis (Lam et al., 1989; Green and Fluhr, 1995; Christie and Jenkins, 1996).

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Information on the signal transduction processes that mediate the effects of external stimuli on gene expression is gradually accumulating. Cell physiological techniques have identified events, such as transient increases in cytosolic calcium ion concentration, that are likely to be of key importance in mediating responses to, for example, mechanical stimulation (Knight et al., 1991), chilling (Knight et al., 1991, 1996) and light detected by phytochrome (Shacklock et al., 1992). Pharmacological approaches have further defined components of signalling pathways, for instance, initiated by UV-B and UV-Ahlue light (Chstie and Jenkins, 1996), phytochrome (Millar et al., 1994) and low temperature (Monroy and Dhindsa, 1995). In addition, the genetic approach has resulted in the identification and cloning of several novel signal transduction components, including those involved in light (Chory, 1993; Deng, 1994; Jenkins ef al., 1995), ethylene (Keiber, 1997) and abscisic acid (Merlot and Giraudat, 1997) signalling. These latter molecules, together with others such as jasmonic acid (JA) and salicylic acid (SA), are important in mediating responses to wounding and pathogen attack (ethylene, JA, SA) and low temperature and drought (abscisic acid). Research is now at the stage where the combined application of these complementary experimental approaches will result in a burgeoning of information on environmental signal transduction.

A.

NETWORKS VERSUS PATHWAYS

Signal transduction is often discussed in terms of ‘pathways’. Although this is a convenient and often appropriate way to refer to the processes through which a particular stimulus elicits a given response, it must be recognized that signal transduction may not proceed through a simple, linear sequence of events. Moreover, it is evident that multiple, potentially interacting, signal transduction pathways are present in cells. Clearly, the same stimulus may regulate several different processes and may do so via different or branching pathways. For example, the cold-induction of gene expression occurs through abscisic acid-dependent and -independent pathways (Nordin et al., 1991; Gilmour and Thomashow, 1991; Hughes and Dunn, 1996); also, phytochrome signal transduction involves distinct pathways (see section II.A.3). Furthermore, a particular process, such as the expression of a specific gene, may be regulated by a host of different stimuli. This is observed, for example, with the phenylalanine ammonialyase (PAL) and chalcone synthase (CHS) genes (van der Meer et al., 1993; Dixon and Paiva, 1995; Mol et al., 1996). It is therefore important to think in terms of ‘networks’ rather than isolated signalling pathways. There is increasing evidence that interactions occur within signal transduction networks and this will be discussed in more detail below. Thus signal transduction networks in cells contain overlapping, interconnected components. The perception of a given stimulus may therefore have an impact throughout the network, rather like the ripples formed when a stone hits a pond. Hence stimulus perception may

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potentially affect the responses to other stimuli, whether these are perceived at the same time or subsequently. A further important point is that signal transduction networks are not identical in all cells and that the capacity for signalling may change during development. For instance, many cold-induced genes are expressed in shoot and not root tissues (Hughes and Dunn, 1996), indicating that signalling andor effector components of these responses are spatially expressed. An illustration of a temporal change in responsiveness is provided by the CHS genes in parsley and white mustard (Batschauer et al., 1991; Frohnmeyer et al., 1992). In these plants CHS expression is regulated by phytochrome early in leaf development but by UVhlue photoreceptors later in development. Similarly in Ambidopsis, phytochrome induction is lost in older leaves (Kaiser er al., 1995; Jackson et al., 1995). Again, changes in the capacity for signalling are likely to be involved. Hence, a challenge for future research is to understand the regulation of expression of signal transduction and effector components.

B. ACHIEVING AN ‘APPROPRIATE’ RESPONSE

Plants are continually bombarded with environmental information. It is therefore vital that each stimulus elicits an appropriate response. There will be a ‘cost’, in terms of resource expenditure in each response, so cumulative unnecessary responses would be ‘expensive’ and hence potentially damaging. Moreover, the inability to curtail a response when it is no longer needed would also consume finite resources. On the other hand, failure to respond urgently to a potentially damaging abiotic or biotic stress may prove lethal. It is therefore vital, in terms of competitiveness, genetic fitness and survival, that the responses to external stimuli are appropriate and measured. More precisely, each response should exhibit the required sensitivity to the stimulus (threshold of the dose-response relationship), rapidity, magnitude, duration and specificity (with regard to which genes are regulated). Furthermore, each response must be appropriate in the context of other responses. To achieve this, responses must be integrated. Thus, information from one stimulus may affect the response to another, perhaps to curtail the response or to amplify it. For instance, sudden exposure to a potentially lethal stimulus may require the diversion of resources into a protective response which takes priority over other responses. In this case, a stimulus may switch off some genes while switching on others. Alternatively, the presence of several stimuli together may reinforce the ‘message’ that the plant is being exposed to a particular environmental situation, more so than any of the stimuli alone would do. In this case, the combination of stimuli may elicit a synergistic response involving the hyperexpression of a battery of genes. Thus it is evident that mechanisms are required for information transfer, or cross-talk, between stimulus-response pathways. Such integration enables appropriate responses to be made.


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This paper presents evidence for the existence of interactions between stimulusresponse pathways in plants. Examples are selected to illustrate particular points and it is not the intention to provide a comprehensive survey of the literature. The conclusion is that plant cells contain networks of potentially interacting signal transduction pathways and that these interactions enable the plant to integrate its responses to environmental stimuli to maximize its survival.

11. INTERACTIONS WITHIN SIGNALLING NETWORKS A.

EVIDENCE OF NEGATIVE REGULATION

In recent years evidence has been obtained that exposure to particular stimuli may result not only in the elicitation of a specific gene expression response, but the switching off of a separate response. Moreover, it has been shown that positive components of one signalling pathway may act as negative regulators of another. As discussed below, negative regulatory responses are of key importance both in the prioritization of responses to external stimuli and in achieving de-sensitization, the cessation of a response when it is no longer appropriate.

1. Negative regulation in the control of gene expression by metabolites It is vital for cells to regulate their metabolic activity in relation to both developmental and external factors. Particular organs and tissues constitute ‘sources’ or ‘sinks’ of metabolites and individual cells have a characteristic spectrum of metabolic activities commensurate with their roles in nutrient mobilization and biosynthesis. To illustrate, mature leaves function as net exporters of carbohydrates whereas developing seeds and meristems function as sinks. Metabolic activities within cells are regulated in the short term by the ‘fine control’ of enzyme activities, in particular through post-translational modification, and in the longer term by the ‘coarse control’ of gene expression. Environmental factors influence metabolism over both time scales through these processes. It is well established that the levels of particular metabolites, notably sugars, regulate the expression of various genes (Graham, 1996; Koch, 1996; Smeekens and Rook, 1997). Cells therefore possess mechanisms to sense the levels of specific metabolites and use this information to differentially regulate gene expression. However, many genes known to be regulated by metabolites are also regulated by environmental stimuli such as light or various stresses. Hence metabolic signalling processes must be integrated with environmental signal transduction pathways. A classic example of the metabolic regulation of gene expression is the repression of transcription of genes concerned with photosynthesis by soluble sugars such as glucose and sucrose. Sheen (1990) reported that the transcriptional activity of promoters of several genes encoding photosynthetic components was repressed by sugars in maize mesophyll protoplasts. Similarly, Krapp er al. (1993) found that the addition of glucose to a Chenopodium cell culture

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reduced expression of genes encoding the major chlorophyll-binding protein of thylakoids (CAB) and the small subunit (rbcS) of ribulose 1,5-bisphosphate carboxylase/oxygenase. Sugar repression of CAB transcripts was also observed in cultured Brassicu napus cells (Harter et al., 1993). Moreover, similar observations were reported when carbohydrate levels were artificially elevated in intact plants either by transgenic expression of apoplastic invertase (von Schaewen et al., 1990) or by cold-girdling to reduce source leaf export (Krapp and Stitt, 1995). The rbcS and CAB genes are induced by light in most higher plants and since, in the above examples, sugar repression was observed in the light, it can be concluded that the negative regulation by sugars overrides light signal transduction. Information is starting to accumulate regarding the signalling mechanisms involved in the sugar repression of gene expression. Experiments using glucose analogues and sugar metabolites led to the hypothesis that the phosphorylation of hexose sugars by hexokinase has a key role in sugar sensing (Graham el al., 1994; Jang and Sheen, 1994). Analogues of glucose which enter cells but are not metabolized by hexokinase, such as 3-O-methylglucose, did not repress rbcS expression in Chenopodium cells (Krapp et ul., 1993). Jang and Sheen (1994) reported similar effects on the repression of promoter-reporter fusions in maize protoplasts. The analogue 2-deoxyglucose, which can be phosphorylated by hexokinase but is not readily metabolized by glycolysis, was able to mediate repression. Furthermore, mannoheptulose, an inhibitor of hexokinase, blocked repression by 2-deoxyglucose. These findings point to hexokinase as the initiator of the signalling pathway resulting in sugar repression. Further support for this hypothesis was obtained in a recent study (Jang er al., 1997) with transgenic plants either under- or overexpressing hexokinase. Antisense reduction of hexokinase decreased glucose sensitivity of the plants in several responses whereas overexpression resulted in increased sensitivity. The hexokinase mechanism is not confined to photosynthetic genes, since sugar repression of genes encoding malate synthase and isocitrate lyase, which mobilize stored lipids following germination, also involves hexokinase (Graham et al., 1994). At present there is little detailed information on the sugar signalling pathway mediated by hexokinase and it is not known how this signalling pathway negatively regulates light signal transduction. Further information on sugar sensing is likely to be obtained from comparative studies with yeast, which also has a hexokinasemediated sugar sensing system. For example, a kinase termed SNFl is important in the yeast system and plant homologues of SNFl have been identified that can complement snfl mutants (Muranaka et al., 1994). There may also be parallels with sugar sensing in mammalian cells, which involves a glucokinase activity. Urwin and Jenkins (1997) reported that a promoter element involved in sugar repression of a Phaseolus vulgaris rbcS gene resembles elements concerned with the induction of mammalian genes by sugars. Although the above discussion has focused on the repression of genes by sugars, it should be mentioned that there are several examples of plant genes which are stimulated by sugars. These include CHS, nitrate reductase, patatin, sporamin and


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Phenylalanine PAL Cinnarnate

/

4-Cournaroyl-CoA

CHS

Naringenin chalcone

FLAVONOIDS ANTHOCYANINS

Fig. 1. Phenylpropanoid and flavonoid biosynthesis. The general phenylpropanoid pathway includes the steps from phenylalanine to 4-coumaroyl-CoA and is initiated by PAL (phenylalanine ammonia-lyase). CHS (chalcone synthase) catalyses the first step in flavonoid biosynthesis, the formation of naringenin chalcone. Further enzymatic steps (not shown) lead to the formation of specific flavonoids, anthocyanins, sinapic acid esters and furanocoumarins. For information on other branches of the pathways see Dixon and Paiva (1995).

P-amylase (Graham, 1996; Koch, 1996; Smeekens and Rook, 1997). In some cases (nitrate reductase: Jang et al., 1997; CHS: Urwin and Jenkins, 1997) there is evidence consistent with a hexokinase signalling mechanism. Therefore this sugar sensing system appears to be able to mediate both the repression and induction of gene expression.

2. Negative regulation in plant defence responses It is well established that genes encoding enzymes of the phenylpropanoid and flavonoid biosynthetic pathways are responsive to a range of environmental stimuli (van der Meer et al., 1993; Dixon and Paiva, 1995; Mol et al., 1996). PAL is the first enzyme in the general phenylpropanoid pathway and CHS is the first committed step in the branch from the pathway that leads to flavonoid biosynthesis (Fig. 1). Various products of these pathways have a key role in limiting the damaging effects of abiotic and biotic stresses. Both sinapic acid esters, derived from the phenylpropanoid pathway, and particular flavonoids function as UV-protectants in the epidermal layers (Stapleton, 1992; Jenkins er al., 1997). Compounds such as furanocoumarins, again derived from the phenylpropanoid pathway, have antimicrobial activity and are therefore important in defence against pathogens (Hahlbrock and Scheel, 1989; Hahlbrock et al., 1995; Dixon and Paiva, 1995).

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The regulation of transcription of PAL and CHS genes in response to various stimuli has been studied extensively in several species (van der Meer et al., 1993; Dixon and Paiva, 1995; Mol et al., 1996). In parsley, as in other species, both PAL and CHS genes are induced by UVhlue light. However, PAL expression is strongly induced by an elicitor derived from the fungus Phytophthora megasperma, whereas CHS expression is not (Lozoya et al., 1991). Furthermore, enzymes of the branch from the phenylpropanoid pathway that leads to furanocoumarin biosynthesis are stimulated by elicitor and not by light. Thus, it can be demonstrated that elicitor treatment of cultured parsley cells leads only to furanocoumarin, and not flavonoid, accumulation and that UV light has the opposite effect. Lozoya et al. (1991) demonstrated that flavonoid accumulation and the induction of CHS expression in response to UV light is in fact prevented by the fungal elicitor. In contrast, furanocoumarin accumulation and the induction of PAL expression by the elicitor was not switched off by UV light, although there was a significant reduction. The negative regulation of CHS expression by elicitor treatment was at the level of transcription. Although the mechanism of repression is not known, it most likely involves signalling events which affect the biogenesis and/or activation of transcription factors that associate with cis-elements in the CHS promoter, There is evidence that the UV light stimulation of CHS transcription requires both protein synthesis (Christie and Jenkins, 1996) and the posttranslational activation of relevant transcription factors (Harter et al., 1994). It is likely that the repression of CHS by the fungal elicitor serves to divert metabolites into the biosynthesis of compounds which are important in the defence response. Hence, negative regulation enables the plant to prioritize its protective responses to potentially damaging stimuli. It appears that pathogen attack requires a more urgent response than protection against UV radiation, although whether a different response would be observed to more severe UV-B exposure or to different pathogen signals is not known. Lozoya et al. (1991) suggested that negative regulation may be a common phenomenon in plant defence responses and cited several examples where elicitors differentially regulate biosynthetic processes. In fact recent work by Vidal et al. (1 997) illustrates that factors regulating defence gene expression may have mutually antagonistic effects. These authors studied the induction of several genes encoding pathogenesis-related (PR) proteins in tobacco in response to the soft-rot pathogen Erwinia carotovora. PR proteins are induced by a range of pathogens. They have been classified into various groups and several have been shown to have antipathogenic properties (Cutt and Klessig, 1992). Erwinia produces extracellular enzymes that generate elicitors by digestion of the host cell wall and the elicitors induce gene expression both at the local site of infection and systemically in other leaves of the plant. Hence a culture filtrate of Erwinia containing the enzymes is effective in inducing the defence response. In this case transcripts of a basic glucanase and of acidic and basic chitinase genes were induced rapidly in the plants in both treated and non-treated leaves. SA, which is known to be an important signalling molecule in the production of systemic resistance, induced the same

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genes more slowly and weakly, indicating that another signal was involved in their rapid induction in non-inoculated leaves. In contrast SA, but not the culture filtrate, induced the P R - l a gene. Thus, in common with other systems, there appear to be distinct pathways for the induction of defence genes in tobacco in response to infection by a particular pathogen. Vidal rt al. (1997) further showed that treatment with increasing amounts of the Erwinia culture filtrate negatively regulated the induction of the PR-la gene in response to SA. Similarly, SA inhibited the induction of the basic glucanase gene by the culture filtrate. The authors suggested that such reciprocal antagonistic effects may be mediated by signalling components common to different defence pathways which could function as either positive or negative regulators of gene expression. Such a mechanism would provide an efficient way for the plant to coordinate its local and systemic defences in response to attack by different pathogens. Identification of components of the pathways will enable this hypothesis to be tested. Negative regulation between phytochrome signal transduction pathways A further illustration of negative regulation is provided by the phytochrome signal transduction pathways controlling gene expression (Bowler and Chua, 1994; Millar et a1 1994). Phytochrome stimulates the expression of a range of plant genes, including the CHS genes and those encoding various chloroplast proteins, such as CAB. In recent years, information on the components of phytochrome signal transduction pathways has been obtained using direct microinjection of molecules into plant tissue and from pharmacological studies with cultured cells. Neuhaus et al. (1993) showed that microinjection of phytochrome (as phytochrome A) into hypocotyl subepidermal cells of the phytochrome deficient tomato nurea mutant restored both the phytochrome-mediated induction of chloroplast development and anthocyanin accumulation. Anthocyanin is a product of the flavonoid biosynthesis pathway (Fig. 1) and it was demonstrated that phytochrome microinjection stimulated expression of a gene fusion consisting of a CHS promoter ligated to the P-glucuronidase (GUS) reporter gene. Similarly, phytochrome injection stimulated the promoter of the CAB gene. By co-injecting various putative signal transduction components, Neuhaus et nl. ( 1993) and Bowler et al. (1994a) provided evidence that the phytochrome stimulation of both promoters required one or more heterotrimeric G-proteins. Furthermore, CHS promoter activity and anthocyanin accumulation were stimulated by cyclic GMP (cGMP). In contrast cGMP did not initiate chloroplast development. However, CAB promoter activity and the production of chlorophyll-containing plastids was stimulated by the injection of Ca'+ ions and the regulatory calcium-binding protein calmodulin. Neither Ca'+ nor calmodulin activated the CHS promoter or anthocyanin accumulation. These data therefore provide evidence for separate phytochrome signal transduction pathways with distinct target genes (Fig. 2). The pathways involve a G-protein as an early step and then bifurcate, one branch involving cGMP and stimulating

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7 GEN

Pfr

G Protein

f \

cGMP

CHS

FNR

CAB

Fig. 2. Interactions between phytochrome signal transduction pathways. Phytochrome Pfr activates a G protein and the pathways then bifurcate, one branch involving cGMP and stimulating CHS expression and the other involving cytosolic Ca2+and calmodulin (Cah4) and stimulating CAB expression. Both pathways are required to stimulate FNR expression. Inhibition of the cGMP pathway by genistein (GEN) is shown. The dashed lines indicate reciprocal negative regulation of the pathways. Modified from Bowler and Chua (1994).

CHS and one involving Ca2+/calmodulin and stimulating CAB. The authors further demonstrated that the production of chloroplasts with photosystem I and cytochrome b6f components, and also the expression of genes encoding particular PSI proteins (e.g. the ferredoxin NADP' oxidoreductase, FNR), required both signalling pathways (Fig. 2). That is, the production of chloroplasts with a complete set of complexes was stimulated by injection of cGMP and Ca2'/calmodulin together, but by neither compound alone. To complement the microinjection experiments, Bowler et al. (1994a) showed that cell-permeable cGMP analogues stimulated CHS transcript accumulation, but not CAB or FNR gene expression, in darkness in a soybean cell culture, Bowler et al. (1994b) provided evidence that the two distinct phytochrome signal transduction pathways were subject to reciprocal negative regulation. That is, components that had a positive function in one signalling pathway mediated a repressive effect on the other. Microinjection of high cGMP concentrations caused repression of CAB-GUS gene expression induced by Ca*+/calmodulin. Similarly, introduction of cGMP into soybean cells attenuated the level of CAB transcripts. The induction of CHS in soybean cells, and of the CHS-GUS fusion in injected tomato hypocotyl cells, was inhibited by the histidine/tyrosine kinase inhibitor genistein. The target of this compound was found to be downstream of cGMP in the signalling pathway. Genistein prevented the repression of CAB expression by cGMP, indicating that the component that interacts with the Ca2'/calmodulin pathway is likely to be downstream of cGMP and is either sensitive to genistein or downstream of the genistein-sensitive component. Interestingly, the light-induction of CHS expression in the soybean cells was transient, peaking after about 3 h. Thus there appeared to be desensitization to the stimulus during prolonged exposure. This phenomenon was not observed when cGMP was applied to dark-treated cells, indicating that transient levels of cGMP may be the cellular basis of stimulation followed by desensitization during normal illumination. cGMP therefore appears to regulate both phytochrome signalling pathways.


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Evidence that the Ca’+/calmodulin-dependent pathway could negatively regulate the cGMP pathway was obtained from experiments with inhibitors of CAB expression. Several compounds, including calcium channel and calmodulin inhibitors, prevented the light induction of CAB in soybean cells and the phytochrome A induction of CAB-GUS expression in microinjected tomato cells. However, these inhibitors additionally caused a hyperinduction of CHS transcript levels in the cell culture. In addition, Bowler et al. (1994b) found that injection of high concentrations of Ca”/calmodulin into tomato cells inhibited CHS-GUS expression stimulated by a constant level of co-injected cGMP. Thus Ca’‘ and calmodulin directly, or via a downstream component, function to negatively regulate the cGMP dependent phytochrome signalling pathway. The effect appears to be specifically on the extent of induction of CHS, because desensitization remains in the presence of the inhibitors of the Ca’+/calmodulin pathway. Direct measurements of the in vivo levels of cGMP and Ca2’ in the tomato and soybean systems are now required to complement the above findings. These will give more detailed information on the cellular mechanisms involved in the transduction of phytochrome signals. The above studies provide valuable insights into the cellular mechanisms of negative regulation of specific signalling pathways in plant cells. Bowler et a / . (1994b) speculated on the significance of these intriguing control mechanisms. They suggested that negative regulation could function to promote the synthesis of photoprotective compounds by the flavonoid biosynthesis pathway when plants are first exposed to light and to prevent the development of functional complexes in chloroplasts until the photoprotective mechanisms were in place.

B. EVIDENCE FOR SYNERGISM

In contrast to the above examples of negative regulation, there are several instances in the literature of synergistic interactions between signalling pathways resulting in hyper-responses to particular combinations of stimuli. In some cases, such as the interaction between NaCl and abscisic acid (ABA) in the regulation of Em gene expression in rice (Bostock and Quatrano, 1992). each stimulus elicits a significant response, although their combined effect is larger than the addition of their separate effects. In other cases, such as the interaction between methyl jasmonate (MeJA) and sugars in the expression of vegetative storage protein genes in soybean (Mason et al., 1992), the separate stimuli give negligible responses but hyper-responses when together. In some cases a stimulus which is not effective on its own may permit or enhance a response to another stimulus. For instance, blue light induces various responses in higher plants which are not elicited by red light, detected by phytochrome; however, in several cases illumination with far-red light prevents the blue light response, indicating that phytochrome in the Pfr form is required for blue light to be effective (Mohr, 1994). Synergistic responses reveal the existence of important mechanisms which

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ensure that plants respond appropriately to particular environmental conditions. Considering all the environmental information that impinges on a plant, it is important to have a means of discriminating, of being able to recognize when environmental conditions necessitate a particularly urgent, or especially large response. This is achieved by particular combinations of stimuli together eliciting much greater responses than those induced by individual stimuli. The examples discussed below illustrate the importance of synergism in the protection of plants against biotic and abiotic stresses.

I . Synergistic interactions regulate plant defence genes It is well known that exposure of plants to pathogens elicits the expression of a battery of genes. Prominent among the proteins synthesized are the PR proteins (see section II.A.2). Several of the signalling molecules involved in the regulation of particular PR genes have been identified and among these are SA (see section ILA.2), ethylene and JA. Xu et al. (1994) studied the regulation of two PR protein genes in tobacco, encoding PR- 1 and osmotin, a PR-5 protein. The latter protein owes its name to the fact that it accumulates in response to salt (NaCI) stress (Singh et al., 1987).In fact Osmotin gene expression is induced by a range of external and endogenous signals (Cutt and Klessig, 1992). Xu et al. (1994) reported that ethylene stimulated the Osmotin promoter, fused to GUS, in transgenic tobacco, whereas treatment with MeJA did not. However, a combination of ethylene and MeJA induced a very much larger increase than that seen with either stimulus. The increase was more than additive and therefore indicated a synergistic interaction between the ethylene and MeJA signalling pathways. Similar results were observed at the levels of mRNA and protein accumulation. The P R - l b gene showed minimal expression in response to either ethylene or MeJA, but again a large synergistic increase was observed in the presence of both compounds. Synergism in P R - l b expression was also observed between SA and MeJA. Interestingly, the extent of interaction between ethylene and MeJA in stimulating Osmotin gene expression differed between organs of the plant. In this case, a difference was observed between roots and cotyledons. Roots showed a much higher level of Osmotin promoter activity than cotyledons in control, non-treated seedlings. Both organs showed minimal stimulation of the Osmotin promoter by MeJA above the control level and a substantial ethylene stimulation. Evidence of synergism was observed between MeJA and ethylene in roots, but in cotyledons the hyperstimulation of the promoter was much greater than in roots, with approximately a 200-fold increase in GUS activity. Although differences in the endogenous levels of ethylene and MeJA in non-stimulated tissue may in part explain these findings, it is likely that there are genuine organ-specific differences in responsiveness. This emphasizes a point made earlier, that components of signalling networks may themselves be subject to spatial, temporal or environmental regulation. Xu et al. (1994) pointed out the significance of the hyperinduction of defence gene expression by combinations of signals. In the case of a gene (such as encoding


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osmotin) regulated by several, apparently unrelated signals, the particular combination of signals, rather than any one signal alone, may be the key inductive stimulus. The synergistic hyperinduction of various defence genes resulting from the combined presence of several defence-associated signals may therefore be of key significance in vivo. An illustration of the significance of such synergistic interactions in plant defence is provided by the recent work of Shirasu et al. (1997). These authors examined the role of SA in inducing local, as opposed to systemic, defence responses. Pathogen attack often elicits a localized ‘hypersensitive response’ (HR) which involves the production of superoxide radicals and hydrogen peroxide in the ‘oxidative burst’ and the activation of defence genes. Superoxide is a component of the signalling pathway that promotes defence gene activation (Jabs et al., 1997). Hydrogen peroxide has antimicrobial effects and, in addition, promotes oxidative cross-linking which strengthens the cell wall, causes localized cell death and stimulates the expression of several protective genes (Bradley el al., 1992; Levine et al., 1994; Jabs et al., 1997; Lamb and Dixon, 1997). The HR is a key element in plant defences against pathogen attack because it helps to prevent the spread of the pathogen. Exogenous SA, when applied alone, is often required at relatively high concentrations or for extended periods to induce defence responses. However, Shirasu et al. (1997) found that SA at low, physiological concentrations acted synergistically with a pathogen to induce a rapid localized defence response in soybean cells. In these experiments a strain of Pseudonionas syringae pv. glycinea was used that elicited a HR in the chosen soybean genotype. SA, in the presence of the pathogen, caused the production of high levels of hydrogen peroxide within two hours; levels much greater than those seen with either SA or the pathogen alone over the equivalent period. Moreover, the combination of SA and the pathogen accelerated hypersensitive cell death and the induction of genes encoding PAL and glutathione S-transferase. Thus SA, through its synergistic interaction with the pathogen, has an important role in potentiating the rapid localized response to pathogen attack.

2. Synergistic interaction between UV and blue light stimuli in the regulation of CHS expression Returning to the regulation of flavonoid biosynthesis genes, Fuglevand et al. (1996) have demonstrated that complex interactions between UV and blue light signal transduction pathways result in hyperstimulation of CHS expression in Arabidopsis. In Arabidopsis the phytochrome regulation of CHS expression is restricted to very young seedlings. In light-grown leaf tissue, induction is predominantly by separate UV-B and UV-A/blue light signal transduction pathways, the latter coupled to the CRY 1 photoreceptor (Fuglevand et al., 1996). The UV-B and UV-A/blue light signal transduction pathways are distinct from those involved in phytochrome regulation (Christie and Jenkins, 1996). Fuglevand et a/. ( I 996) reported experiments with transgenic Arabidopsis plants

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containing a CHS promoter-GUS fusion. Plants were grown for several weeks in a low fluence rate of white light that did not stimulate significant CHS-GUS expression. Subsequent exposure of mature leaves to either UV-B, UV-A or blue light resulted in approximately a 10-fold stimulation of CHS-GUS expression and similar increases were observed in the level of CHS transcripts. The authors categorized these responses to individual light qualities as ‘inductive’ responses. Synergistic responses were observed in specific combinations of light qualities. Exposure to either UV-B and blue light, or UV-B and W - A light, given simultaneously, resulted in approximately 4- to 8-fold greater stimulation of CHS-GUS expression than in the inductive treatments. UV-A and blue light gave an additive rather than a synergistic effect. Fuglevand et al. (1996) further demonstrated that blue light given before UV-B resulted in a synergistic response whereas UV-B before blue light did not. This indicated that blue light generates a signal or activates a component that interacts productively with components of the UV-B pathway to elevate the response. UV-B itself was unable to produce such an effect. When a dark period intervened between the blue and subsequent UV-B exposure, the ‘synergism signal’ was gradually lost. Nevertheless, the signal was sufficiently stable in darkness even after 15 h to give an enhanced response in subsequent UV-B compared to a control that had received no blue preillumination. In contrast, UV-A treatment prior to UV-B did not result in a synergistic enhancement of CHS expression and neither did UV-B exposure before UV-A. The fact that marked synergism was nevertheless observed with both treatments given simultaneously indicates that the signal generated by UV-A is not stable, but transient. Fuglevand et al. (1996) therefore concluded that the pathways producing the signals in blue and UV-A light were distinct. Consistent with this finding, they further showed that the CRY1 photoreceptor was not involved in the synergistic responses, because both synergisms were retained in the hy4-2.23N mutant, which lacks the CRY 1 photoreceptor. The different pathways involved are shown in Fig. 3. Further experiments showed that the distinct ‘synergism signals’ generated by blue and UV-A light could interact together with the UV-B signal transduction pathway to maximize the level of CHS expression. Plants were exposed to blue light and then to UV-A plus UV-B. In this case, the level of CHS-GUS expression observed was approximately double that produced with either synergistic combination alone. The results therefore indicated that the two synergistic interactions could function in an additive manner to maximize CHS expression. In fact, expression was 150-fold compared to the 10-fold increase observed with the single light qualities. This is the first report of two synergistic interactions together stimulating expression of the same gene. So what is the significance of the above synergistic interactions to the plant? Of course, plants are not exposed to separate UV and blue light qualities but to a complex spectrum. It is possible that seedlings growing under a leaf canopy may not experience UV-B radiation until they have been exposed to longer wavelengths, and some degree of potentiation of the response by blue light could therefore occur.


67

UV-Nblue blue

blue

I

I b

\stable \

UV-B

’.-.--’CHS /*

:tansient

I

I

UV-A

Fig. 3. Signal transduction pathways involved in the regulation of CHS gene expression by UV and blue light in Arubidopsis. Inductive pathways, involving either CRY I or the UV-B light detection system, are shown by solid lines. An additional, hypothesized inductive blue light signalling pathway is indicated by a dot-dash line. The distinct UV-A and blue light pathways that interact synergistically with the UV-B pathway and produce transient and relatively stable signals respectively, are represented by dashed lines. No information is available on the specific sites of interaction of the synergism pathways with the UV-B pathway. Reproduced from Fuglevand et al. (1996) with permission.

However, UV-B exposure should always be accompanied by UV-A and blue wavelengths. Hence the synergistic interactions will be the norm. The maximal stimulation of CHS expression will provide a basis for the synthesis of high levels of protective flavonoid pigments. However, it is not yet known whether other genes encoding flavonoid biosynthesis enzymes are subject to the synergistic regulation. Fuglevand el al. ( 1996) reported that the synergistic interactions do appear to have selective value. Aruhidopsis plants exposed to 24 h of UV-B radiation alone normally died whereas those exposed to combinations of UV-B and UV-A and/or blue light did not.

111. APPROACHES TO IDENTIFY THE MECHANISMS INVOLVED IN INTERACTIONS BETWEEN SIGNALLNG PATHWAYS To understand how plant cells regulate key genes in response to environmental stimuli, and how these responses are integrated, it is essential to identify components of the relevant signalling pathways and to define their specific functions. Furthermore, it is essential to identify the transcription factors that effect particular responses and to understand how terminal components of the signalling pathways, most likely protein kinases and phosphatases, regulate these effectors. Different signalling pathways may converge on the same transcription factor targets or, alternatively, different responses may be achieved through response-specific effectors. Establishing the specificity of function of particular components, such as transcription factors and kinases, will help us to understand how cells selectively stimulate transcription of specific genes in response to particular stimuli.

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The most powerful way to identify signal transduction and effector components is to use a combination of cell physiological, biochemical, molecular and genetic approaches. The cell physiological and biochemical approaches being used with cell cultures, microinjection systems and transgenic plants will be important in identifying components involved in cross-talk between signalling pathways. For example, pharmacological approaches combined with microinjection have already provided valuable insights into the mechanisms of negative control operating between phytochrome signalling pathways (Bowler el a/., I994b). Transgenic plants used in conjunction with the hy4 mutant have facilitated the analysis of synergism in the UV and blue light regulation of CHS expression (Fuglevand et al., 1996). Since calcium is a key second messenger in several of these responses, the use of transgenic plants expressing calcium-sensitive aequorin fusions targeted to particular cellular compartments (Knight et ul., 1996) will enable investigation of the roles of different calcium pools in the interactions between signalling pathways. In most cases, the functions of individual transcription factors in responses to external stimuli have not been defined. Although nuclear protein-promoter DNA binding activities have been described for various genes, it is usually unknown which member of a family of transcription factors interacts with a specific promoter element in vivo to effect a particular response. Moreover, relatively little progress has been made in identifying specific kinases, phosphatases and other components which couple second messengers to transcription factors. The genetic approach will be particularly important in this respect. The isolation and characterization of mutants altered in the environmental regulation of particular genes will enable the functions of specific transcription factors, protein kinases and phosphatases in these responses to be defined. This is proving to be the case in studies, for example, of ethylene and abscisic acid signal transduction, in which mutants have facilitated the isolation of novel signalling components (Bowler and Chua, 1994; Keiber, 1997; Merlot and Giraudat, 1997). Thus CTRl encodes a raf-like kinase (Keiber ef al., 1993), ABIl and AB12 encode protein phosphatases (Leung et al., 1997) and AB13 encodes a putative transcription factor (Giraudat et al., 1992). Furthermore, by the use of appropriate screens, it should be possible to identify components which mediate interactions between signalling pathways. To date, no plant mutants have been found that are altered in components which function specifically in cross-talk between signalling pathways. Transgene expression screens provide the best means of identifying components which couple specific signals to defined promoter targets. Several mutants altered in the light-regulation of CAB (Li et al., 1994, 1995; Millar et al., 1995) and CHS (Jackson el al., 1995) genes have now been isolated using this approach. The availability of these mutants will enable the corresponding wild-type genes to be cloned using the powerful techniques developed for this purpose in Ambidopsis. In addition, the construction of double mutants will enable the functional interactions between the corresponding genes to be investigated. This type of analysis has proved very effective, for example, in studies of seedling responses to light; good testable models have been

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developed hypothesizing the order of gene function and the relationships between pathways coupled to different photoreceptors (Chory, 1993). Interestingly, several of the putative signal transduction mutants isolated to date show essentially constitutive responses in the absence of a stimulus, for example, the cop/det@us mutants which are de-etiolated in darkness (Chory, 1993; Deng, 1994; MisCra et al., 1994) and c f r l , which has a constitutive response to ethylene (Keiber er al., 1993; Keiber, 1997). These mutants therefore identify negative regulators which constrain the response of the wild-type. The Ambidopsis icxl mutant also identifies a negative regulator, but is a different class of mutant in that it shows an enhanced response to the stimulus, in this case elevated expression of flavonoid biosynthesis genes in response to light (Jackson et al., 1995). The frequency of negative regulator mutants may reflect the nature of the screens used to isolate them or may indicate the widespread significance of such regulators in plant signal transduction. The latter point is emphasized by Bowler and Chua (1994). Negative regulators are certainly likely to be important in the interactions between signalling pathways, both in effecting repression and in synergistic responses. Although signal transduction pathways evidently generate positive signals which promote transcription, it is reasonable to hypothesize that the extent and speed of the response is often constrained by negative regulators associated with specific signalling pathways. Particular stimuli, or combinations of stimuli, may remove these constraints by inactivating specific negative regulators. Thus, synergistic interactions may represent the removal of negative regulation in parallel with the production of a positive signal. In other instances, for example in the interaction between the phytochrome signalling pathways or the effect of fungal elicitor on CHS expression, a signalling component that acts as a positive element in one pathway may function as a negative regulator of another.

IV. CONCLUSIONS Research is starting to elucidate the signal transduction processes in plants that couple external stimuli to transcription. It is evident that signals are not perceived and transduced in isolation. There are mechanisms for interaction or cross-talk within signal transduction networks which achieve the necessary coordination and integration of responses. There is evidence both of negative regulation between pathways and of particular combinations of primary stimuli together eliciting a hyper-response. In addition, responses to environmental stimuli must be integrated with other cellular activities; thus particular genes may be regulated by endogenous metabolic and developmental signals as well as by external signals. The cellular and molecular mechanisms which effect the different transcriptional responses to diverse stimuli are therefore likely to be extremely complex. In recent years the application of complementary experimental approaches has started to generate important information on the mechanisms of primary signalling processes as well as on the interactions between signalling pathways. In particular,

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the ability to make direct measurements of signalling processes, such as the activities of ion channels and transient increases in cellular calcium concentrations has resulted in major advances. So too has the development of systems enabling the direct introduction of signalling components by microinjection and the pharmacological analysis of signal transduction in cell cultures. In parallel, the genetic approach has identified novel components of signalling pathways. These approaches need increasingly to be integrated. Furthermore, the application of these approaches to the components that mediate specifically the interactions between signalling pathways will be instrumental in elucidating the cellular and molecular mechanisms involved.

ACKNOWLEDGEMENTS The author is indebted to the UK Biotechnology and Biological Sciences Research Council and the Gatsby Charitable Foundation for the support of his research on signal transduction.

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Mohr, H. ( 1994) Coaction between pigment systems. In “Photomorphogenesis in Plants” (R. E. Kendrick and G. H. M. Kronenberg, eds) pp. 353-373. Kluwer Academic Publishers, Dordrecht, The Netherlands. Mol, J., Jenkins, G. I., Schafer, E. and Weiss. D. (1996). Signal perception, transduction, and gene expression involved in anthocyanin biosynthesis. Critical Reviews in Plurit Science 15, 525-557. Monroy, A. F. and Dhindsa, R. S . (1995). Low-temperature signal transduction: induction of cold acclimation-specific genes of alfalfa by calcium at 25°C. Plunr Cell 17, 321 -33 1. Muranaka, T., Banno, H. and Machida, Y.(1994). Characterization of tobacco protein kinase NPKS, a homolog of Saccharomyces cerevisiae SNFl that constitutively activates expression of the glucose-repressible SUC2 gene for a secreted invertase of S. cerevisiae. Molecular and Cellular Biology 14, 2958-2965. Neuhaus, G., Bowler, C., Kern, R. and Chua. N-H. (1993). Calciudcalmodulin-dependent and -independent phytochrome signal transduction pathways. Cell 73, 937-952. Nordin, K., Heino, P. and Palva, E. T. (1991). Separate signal pathways regulate the expression of a low-temperature-induced gene in Arahidopsis thaliana (L.) Heynh. Plant Molecular Biology 16, 1061-1071. Shacklock, P. S., Read, N. D. and Trewavas, A. J. (1992). Cytosolic free calcium mediates red light-induced morphogenesis. Nature 358, 753-755. Sheen, J. (1990). Metabolic repression of transcription in higher pl,ants. Plunt Cell 12, 1027-1 038. Shirasu, K., Nakajima, H., Rajasekhar, V. K., Dixon, R. A. and Lamb, C. J. (1997). Salicylic acid potentiates an agonist-dependent gain control that amplifies pathogen signals in the activation of defense mechanisms. Plant Cell 9, 261-270. Singh, N. K., Bracker, C. A., Hasegawa, P. M., Handa, A. K., Buckel, S., Hermondson, M. A,, Pfankoch, E., Regnirr, F. E. and Bressan, R. A. (1987). Characterization of osmotin. Plant Physiology 85, 529-536. Smeekens, S. and Rook, E (1997). Sugar sensing and sugar-mediated signal transduction in plants. Planf Physiology 115, 7-1 3. Stapleton, A. E. (1992). Ultraviolet radiation and plants: burning questions. Plant Cell 4, 1353-1 358. Terzaghi, W. B., Bertekap, R. L. and Cashmore, A. R. (1997). Intracellular localization of GBF proteins and blue light-induced import of GBF2 fusion proteins into the nucleus of cultured Arahidopsis and soybean cells. Plant Journal 11, 967-982. Unvin, N. A. R. and Jenkins, G . I. (1997). A sucrose repression element in the Phaseolus vulgaris rbcS2 gene promoter resembles elements responsible for sugar stimulation of plant and mammalian genes. Plant Molecular Biology 35, 929-942. van der Meer, I. M., Stuitje, A. R. and Mol, J. N. M. (1993). Regulation of general phenylpropanoid and flavonoid gene expression. In “Control of Plant Gene Expression” (D. P. s. Verma, ed.) pp. 125-155. CRC Press, Boca Raton, Florida. Vidal, S., Ponce de L e h , I., Denecke, J. and Palva, T. E. (1997). Salicylic acid and the plant pathogen Envinia carotovora induce defense genes via antagonistic pathways. Plant Journal 11, 115-123. von Schaewen, A., Stitt, M., Schmidt, R., Sonnewald, U. and Wilmitzer, L. (1990). Expression of yeast-derived invertase in the cell wall of tobacco and Arahidopsis plants leads to inhibition of sucrose export, accumulation of carbohydrate and inhibition of photosynthesis, and strongly influences the growth and habitus of transgenic tobacco plants. EMBO Journal 9. 3033-3044. Xu, Y., Chang, P-F., Liu, D., Narasimhan, M. L.. Raghothama, K. G., Hasegawa, P. M. and Bressan, R. A. ( 1994). Plant defense genes are synergistically induced by ethylene and methyl jasmonate. Plant Cell 16, 1077-1085.


Plate 1 . Light micrographs of abaxial epidermis, stained with silver-rubeanate, of (A) Centuureu scubiusu and (B) Leonrodon hispidus grown with 15 mol rn-? calcium. The black deposits indicate the presence of calcium. The scale bar represents 100 pm. (from De Silva et al., 1996).

Plate. 2. Secondary ion mass spectrometry images of Ca and Mg isotopes in a corn root rip. Treatment was as described in the text with a 30 min labelling period and a 2 min ice-cold rinse to remove superficial and apoplastic label. The four isotope maps (tracers in right hand panels) were collected in a CAMECA IMS 4f operated in ion microprobe mode, utilizing direct detection on the electron multiplier and Faraday cup (Lazof et al., 1996a). The brighter the image the greater is the mass signal intensity. The labelled plant specimens were excised from the plant, quench-frozen, cryosectioned (1 0 p m thickness) and slowly freeze-dried. Employing the ‘depletion’ pre-treatment and these operating conditions, all of the 44Ca2+and 26Mg2+represents label which has arrived in the root tip during the labelling period. The three arrows point to the edge of the root, and the asterisk indicates an area where the section has split during freeze-drying. Bar = 80 pm.

Mechanisms of Na+ Uptake by Plant Cells

ANNA AMTMANN and DALE SANDERS

The Plant Luboratoiy. Biology Department. PO Box 373. University of York. York YO1 5yW; UK

1. Introduction

.......................................................................................................

A . Salinity Toxicity and Salinity Tolerance ................................................... B . Exclusion, Uptake and Sequestration of Na+ ...........................................

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I1 . Electrochemical Potential Differences for Na’ Across the Plasma and

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111. Carrier-Mediated Entry of Na’ .........................................................................

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IV. Channel-Mediated Entry of Na’ ..................................................... A . Ionic Selectivity of Ion Channels . B . Inward-Rectifying Channels ............................ C . Outward-Rectifying Channels ................................................................... ......................................... D . Voltage-Independent Channels ......... E . Co-residency of Different Channel Types ..............................

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Vacuolar Membranes

V. Contributions of Different Channel Types to Na+ Entry in Physiological Conditions ......................................................................................................... A . Semi-quantitative Dissection of Fluxes .................................................... B . Relative Activity of Different Channel Types Determines Rate of Na+ Uptake .......................................................................................... VI

.

Regulation of Monovalent Cation Influx Across the Plasma Membrane ......... A . Voltage ................... ........................................................ B. External Ca’* and p ......................................................... C . Cytosolic Ca” and pH ......................................... D . External and Cytosolic Na’ . .................................................... E. ATP ....................................................................... F. Other Regulators ...............................................................................

VII . Comparison of Salt-Sensitive and Salt-Tolerant Genotypes or Cell Lines ....

91 91 95 96 97 97 101 101 102 102

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Advaices in Buimical Research Vul . 29

incorporating Advances in Plant Pathology ISBN 11-12-00592’3-0

AII righla

Copyright 0 1999 Academic Press reproduction in any form rwerved

76 VIII.

A. AMTMANN and D. SANDERS

Future Work .................................................................................................... Acknowledgements ......................................................................................... References .......................................................................................................

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Soil salinity affects vast areas of land globally, with a particularly high impacr in some agricultural intensively used soils due to irrigation practice. A diverse range of plants is able to thrive on saline soils but all major crop species are intolerant to salt. Identijcation of pathways for Na+ transport across plant cell membranes has been highlighted as comprising a key gap in our understanding of salt tolerance in plants. During the last few years there have, however; been remarkable advances in this area as Na+ permeable ion channels in plant cells have been characterized. This review summarizes the present knowledge regarding Na' transport pathways across plant membranes. In particular; data on selectivity, conductance, abundance and regulation of the major cation uptake channel types have been collected an,d this information has been integrated into a simple model in order to address the following questions: (i) how much Nu+ enters the cell through an ensemble of different channel types in saline conditions? (ii) what is the relative contribution of each channel type to the total Na' inward current? (iii) how does modulation of the activity of the different channel types affect the ability of the plasma membrane to discriminate between K+ and Na' ? The model calculations underline the importance of voltuge-independent non-selective cation channels in Nu+-uptake and suggest that future reseurch in the field of salt tolerance in plants should include studies on the regulation oj' this channel type.

I. INTRODUCTION A. SALINITY TOXICITY AND SALINITY TOLERANCE

Soil salinity has a major impact on plant growth and affects about 6% of the total global land area (Flowers and Yeo, 1995). Increasingly, intensive irrigation practices are resulting in secondary salinization of agricultural soils, such that it has been estimated that 10 X lo6 ha per nnnum of irrigated land are abandoned due to salinization and alkalization (Szabolcs, 1987). Since crop productivity of irrigated land in many areas is much higher than that of non-imgated land the coincidence of irrigation and salinization threatens current agricultural productivity (Flowers and Yeo, 1995). Although all major crop species are intolerant of high levels of salinity, a taxonomically diverse range of plant species are able to grow and thrive on saline soils. In extreme cases, where growth is actually enhanced by the presence of NaCl, such species are said to be halophytic, although the spectrum between extreme halophytes and extreme glycophytes is a continuous one which embraces varying levels of salt tolerance. Studies of the mechanisms that underlie salt toxicity and salt tolerance of plants have revealed the involvement of many aspects of cellular, tissue and whole plant biology (for reviews see Rains, 1972; Flowers et al., 1977; Greenway and Munns, 1980; Munns el al., 1983; Cheeseman, 1988; Gorham, 1992; Munns, 1993; Niu et al., 1995; Serrano, 1996; Yeo, 1998). The two principal adverse effects of salinity

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in non-tolerant plants are osmotic stress and toxicity of Na' andor C1- (Serrano, 1996), whereas ion deficiencies (particularly of K+ and Ca2+;Lauchli et al., 1994), decrease of COz fixation and inhibition of protein synthesis probably follow as secondary effects (Marschner, 1995). Salt tolerance involves tissue- and whole plant integration of many different transport processes, as well as compartmenfation of ions and de n o w synthesis of organic osmolytes at the cellular level. In a seminal and critical review of processes limiting growth on saline soils, Munns (1993) points to a lack of research on the control of Na+ and CI- transport across the plasma and vacuolar membranes and concludes that 'advances in salt tolerance at the molecular level will lie in manipulating the expression and structure of proteins that control transport of salt across membranes'. Identification of pathways for plasma membrane Na' transport in plants has more recently been highlighted as comprising a key gap in our understanding of ionic homeostasis during saline stress (Niu el al., 1995). During the last two years there have, however, been some remarkable and significant advances emanating from research on Na' uptake mechanisms by plant cells, and this comprises the topic of the present review. B. EXCLUSION, UPTAKE AND SEQUESTRATION OF Na'

Plant cells in general, and halophytes in particular, face a dilemma in the context of Na' uptake from the soil. On one hand the absorption of Na+ is desirable as a metabolically cheap way of generating high internal osmotic pressure in response to high external osmotic pressure, thereby lowering cell water potential and sustaining turgor. On the other hand, Na' is cytotoxic at cytosolic concentrations in excess of about 100 mM. These cytotoxic effects of Na' are dual. First, the high charge:mass ratio (in comparison with K ' ) disrupts water structure and lowers hydrophobic interactions within proteins, thus reinforcing the overall destabilizing effect of high ionic strength on protein structure through decrease in hydrostatic forces within proteins (Pollard and Wyn Jones, 1979; Wyn Jones and Pollard, 1983). Second, Na+ can inhibit enzyme function more specifically, either directly by binding to inhibitory sites or indirectly by displacing K' from activation sites (Serrano, 1996). In both instances, competition between Na' and Kf is likely to be critical, and therefore the Na+:K' ratio in the cytosol is likely to be a more critical factor in determining Na' toxicity than the cytosolic Na' concentration per se. Two solutions to the dilemma of Na+ absorption are evident. A simple one, probably operational both in moderately halotolerant and halophytic species, is the efficient exclusion of Nab from the plant (Munns, 1985; Schubert and Lauchli, 1990). In addition, since halophytes exhibit a marked propensity for Na' accumulation, that Na+ which does enter the cell must be efficiently sequestered in the vacuolar lumen to prevent cytotoxicity. It is clear, then, that the regulation of Na* transport across the plasma and vacuolar membranes will comprise a critical factor in determining the specific manner in which plant cells handle extracellular Na' loads.

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11. ELECTROCHEMICAL POTENTIAL DIFFERENCES FOR Na’ ACROSS THE PLASMA AND VACUOLAR MEMBRANES Evaluation of driving forces is a central element in understanding transport processes. The driving force for transport of an ion across a membrane has two components, a chemical component established by the concentration difference between the cytoplasm and the extracytosolic compartment, and an electrical component consisting in the electrical potential difference between the two compartments. Quantitatively, the driving force or electrochemical potential difference can be described as

ASNa= zFV, + RT In

( [Na+l,,,IINafl,,,},

where V,, is the electrical potential difference across the membrane referenced to the extracytosolic side of the membrane, “a’] is the activity (“active” concentration depending on the ionic strength of the medium) of Na+, the subscripts cyl and e x t refer to the cytosolic and extracytosolic sides respectively and z, F, R and T have their usual meanings. At the plasma membrane, the chemical driving force for Na’ will obviously vary depending on the extent of salinity. At low salinity (100 mM), but it was not determined whether high Ca'+ levels were sustained or only transient. A rise of [Ca2t],,, was also found in Nitellopsis after salt exposure (100mM NaCI). Here, low resting Ca" levels were restored within 60min (Okazaki ei uf., 1996). Much shorter transient increases of [Ca'+],,, (90% recovery within 1 min) have been reported for Arubidopsis when exposed to 300 mM NaCl (Knight et a/., 1997). An increase in [Ca2+1,,, reduces currents through IRCs in guard cells. On the other hand, IRCs in root tissues appear insensitive to changes in [Ca2+lCy,.Contrastingly, a voltage-independent channel in mesophyll cells of Pisurn, which is more permeable to Nat than K' , has been found to be activated by cytoplasmic Ca" (for references see Table IV). It has yet to be verified whether this is a typical feature of non-selective cation channels. If so, a transient increase of [Ca2 Icy, would promote Naf-uptake through this channel type, whereas low resting Levels of [Ca2'Icy, would reduce non-selective channel activity while maintaining or even stimulating IRC activity. Cytoplasmic pH does not affect the IRCs in guard cells (Blatt, 1992) but is probably involved in the regulation of KATl (Hoshi, 1995). As for external pH, VICs have not yet been characterized in this respect. +

D. EXTERNAL AND CYTOSOLlC Na'

External Na+ inhibits K f inward currents in Cham and blocks the K + currents through the IRC in oat mesophyll cells (Tester, 1988; Kourie and Goldsmith, 1992). In protoplasts from barley xylem parenchyma cells external Na' was found to inhibit outward currents (tail currents) through open IRCs indicating either a reduction of the open probability or tight binding of Na' in the pore (Wegner and Raschke, 1994). In most cells blockage of IRCs in saline conditions would be problematic since it would further increase the Na ' :K' uptake ratio. In wheat root protoplasts and barley suspension cultured cells Na+ had no blocking effect on time-dependent K ' -inward currents, and neither were K' currents through KATl, when expressed in yeast, affected by Na' (for references see Table IV). However, exposure of another Arcrbidopsis K t inward rectifier expressed in yeast, AKTI, displayed larger K t currents when exposed to high external Na' concentrations and these currents were maintained for several minutes after wash-out of Na+ (Bert1 et al.. 1997). This indicates an up-regulation of AKTl by external Na', probably via tight binding of Na' to a modulation site in the channel.

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Cytosolic Na+, which blocks ORCs in guard cells (Thiel and Blatt, 1991) does not inhibit currents through IRCs or Na+-permeable channels. However, as in most patch clamp experiments, pH- and Ca2+-buffered pipette solutions were used in these studies; therefore an indirect effect of increased cytosolic Na+ via signalling pathways involving modulation of cytosolic Ca” or pH cannot be discounted.

E. ATP

Most patch clamp experiments on whole protoplasts include millimolar concentrations of ATP in the pipette (cytosolic) solution but very few studies have investigated whether ATP is directly necessary for channel activity. Wu and Assrnann (1995) observed in single channel experiments that the open probability of IRCs increased by a factor of five when ATP was added. Whole-cell currents through IRCs were still observed if the pipette solution did not contain ATP, but ATP-scavenging agents abolished the IRC currents. The authors concluded that only very low ATP-concentrations are needed for full activation of IRCs. In barley suspension cultured cells Amtmann et nf. (1997) detected no whole-cell IRC currents when ATP was absent from the pipette solution but large IRC currents in all experiments which included 2 mM ATP in the pipette. Washout of ATP from the pipette reduced IRC currents completely but slowly. In accord with the conclusions of Wu and Assmann (1995), this indicates that the critical ATP concentration for IRC stimulation is very low. In the same experiments about 50% of the VIC current disappeared very rapidly (A. Amtmann, unpublished results), suggesting that this current component is sensitive to millimolar, rather than micromolar, ATP concentrations. In single channel experiments a 7-pS, nonselective cation channel was indeed found to be activated by ATP (Amtmann et uf., 1997). None of the studies could distinguish between a direct effect of ATP on the channels and indirect effects via phosphorylation of the channel or membranebound regulators. Although the activities of both IRCs and VICs are dependent on ATP, modest changes or local depletion of cytosolic ATP (for example as a result of increased ATP-hydrolysis by plasma membrane and vacuolar proton pumps in high-salt conditions; see above) may selectively close VICs and thus increase the K+:Naf uptake ratio. Again, much more work is needed to test this hypothesis.

F. OTHER REGULATORS

Other factors such as protein phosphatases and G-proteins have been found to be involved in the regulation of IRCs (Table IV), but their effect on VICs remains to be elucidated. Reducing agents affect ORCs in Arubidopsis mesophyll cells (Spalding el ul., 1992), but until now no cation uptake channel has been reported to have such sensitivity.


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VII. COMPARISON OF SALT-SENSITIVE AND SALT-TOLERANT GENOTYPES OR CELL LINES Maintenance of a low Nat:Kt ratio in the cytosol of cells is a crucial aspect of survival for a plant in a saline environment. Some studies on the involvement of ion channels in salt-tolerance have therefore addressed the question of whether a certain channel type has a lower Na':K+ permeability ratio in salt-tolerant genotypes than in salt-sensitive genotypes (e.g. wheat: Schachtman et a/., 1991; Findlay et al., 1994). No such difference was found. Similar results emerge from the comparison of salt-adapted and non-adapted cell lines of suspension cultures (Murata et al., 1994; Amtmann et al., 1997). Whole-cell ORC currents of tobacco were decreased after adaptation (Murata et d., 1994). In barley, radiometric assays of fluxes revealed distinct uptake patterns in salt-adapted and non-adapted barley cell lines (S. Laurie and R. Leigh, personal communication). However, in both cell lines there was no significant difference either in the array of channel types present or in the basic attributes of these channel types (i.e. K ':Na+ permeability, Murata et al., 1994; Amtmann et al., 1997). As discussed previously, even a very low PNd:PKratio of IRCs would not essentially reduce Na' uptake as long as active VICs are present in the same membrane. Improvement of the selectivity of a certain channel seems not to be a reasonable way to achieve salt-tolerance, either in evolution or in biological engineering. A salt-tolerant plant will need both pathways for the uptake of Na' (which is the quickest and cheapest osmoticum in saline conditions) and other ions, and, in addition, adequate means to activate or de-activate these pathways in order to respond to the specific requirements of a cell at a given moment. The ability to compromise successfully between osmotic adjustment, ion nutrition, restriction of cytosolic Na' concentration and maintenance of energy pools is probably the key to salt-tolerance. Several different channel types facilitatjng cation uptake across the plant plasma membrane represent the necessary basis for such functional flexibility and seem to be present in most cells. Nevertheless, only those plants which can efficiently control NaC passage through these channels have a chance of survival in high-salt conditions. Analysis and comparison of channel regulation in salt-sensitive and salt-tolerant plants species must be a key issue of future studies on salt tolerance.

VIII. FUTURE WORK An integrative approach will be needed to further study Na' uptake across plant plasma membranes. So far, many of the transporters involved in Na'-uptake have been identified. Their voltage-dependence has been studied and their Na ':Kt permeability has been determined, albeit in most cases in relatively nonphysiological conditions. The following areas will need preferential attention in future experiments. The characterization of Na' uptake channels has to be extended to halophytic plants. Currents through all cation uptake channels have to be

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measured in physiological conditions (K', Na+, Ca2+ and pH). Interactive behaviour of relevant permeant ions has to be studied in order to design appropriate models for the calculation of the contribution of Na+ to the whole-cell current. Short-term and long-term effects of salinity on the membrane potential and putative regulators have to be determined. Special emphasis has to be put on the study of the regulation of non-selective cation channels. Once it has been revealed which are the determining factors for Na+-uptake across the plasma membrane, comparison between different cell types within the plant and between salt-sensitive and salt-tolerant species with respect to these factors should provide new insights into the complex field of salt-tolerance.

ACKNOWLEDGEMENTS We would like to thank Dr Frans Maathuis (University of York, UK) for valuable discussion and Dr Steve Qerman (University of South Australia, Adelaide, Australia) for providing us with manuscripts prior to publication. Experimental work in our laboratory was funded by the BBSRC and the EU. AA is supported by the EU.

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The NaCl Induced Inhibition of Shoot Growth: The Case for Disturbed Nutrition with Special Consideration of Calcium

DENNIS B. LAZOF' and NIRIT BERNSTEIN'

'Department of Chemistry, CB 3290, University of North Carolina, Chapel Hill,North Carolina, USA 21nstitute of Soil and Water; The Volcani Center, PO Box 6, Bet Dagan 50250, Israel

I.

Introduction to the Inhibition of Shoot Growth by Salinity ........................... A. Growth Inhibitions: General Considerations .. ............................. B. NaCI-induced Inhibition of Shoot Growth: Ge ypotheses .. C. A Nutritional Effect of NaCl on Shoot Growth ............................

11. Inhibition of Shoot Growth in Dicots and Monocots ..................................... A. The Timing of the Growth Inhibition ........................... B. Salinity Effects on Cell Extension .......................................................... C. Salinity Effects on Primordium Formation an ence ........... ............. D. Salinity Effects on Cell Division in Leaves ..

I I5 11s

121 121 123 123 12s

............................. 111. NaC1-induced Disruptions of Nutrient Transport . A. Influence of Some Experimental Conditions ................................. B. Effects on Whole Shoot Nutrient Accumulation. ...................................

126 126 128

IV. Nutrient Transport to Growing Shoot Tissue Under Salinity ......................... A. Protection of Growing Tissues ......................... B. Levels of Na and K in Young ...................... C. Disturbed Ca Status in Young .................................................... D. Other Nutrient Disruptions in E. Effects in Young Tissues Co F. Genotypic Salinity Effects i G. Lactuca sariva: a Model Dicot System ..... H. Summary: Salinized Nutrition of Growing Shoot Tissues .......

132 I33 133 137 138 139

V.

The Shoot Meristems: Special Nutrient Transport Challenges ...................... A. The Nutrition of Rapidly Dividing Cells: Possible Effects of Salinity .. B. Transport to Zones Proximal to the Meristem in Poaceae .....................

Advances in Botanical ReKidrch Vol. 29 incorporating Advances in Plan1 PdlhoIvpy ISBN 0-12-005920-0

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141 143 146 148 149

Copyright 0 19W Academic Press All nghts of reproduction in any form reserved

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VI. Phloem Transport and Ion Recirculation under Salinity ................, ,.............. A. Remobilization of Nutrients from Ageing Shoot Tissues, ‘Long-term Recirculation’ ...,.......,.............................................................,....,. .......... B. XylemPhloem Transfer, ‘Short-term Recirculation’ C. Calcium Recirculation in the Shoot ........................................................ D. Summary of Salinization and Recirculation ........................

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Nutrients ......................................

Nitrogen .................................................................................................. Micronutrients .............................................

157 157 157 158 158 160 162

Study of Nutrient Status and Transport on t Kinematic Growth Analysis and Elemental Microdissection .......................................... Specimen Preparation Considerations ........ Electron Probe X-ray Microanalysis .................................................... ... Secondary Ion Mass Spectrometry (SIMS) ............................................. Some Other Microanalytical Techniques ............................ ....................

162 163 166 166 167 168 170

VII.

Salinization and Shoot Nu

........

...........

B. Calcium ................................................................................................... C. Magnesium ..............................................................................................

E. F. VIII. The A. B. C. D. E. E

IX. Summary and Future Prospects A. Reassessment of Current St B. Model Systems ......... C. In Situ Elemental and sis ................................................. Acknowledgements ......................................................................................... References ............ ...............................................................................

151 154 155 156

171 171 173 174 175 175

The inhibition of shoot growth by NaCl salinization is reviewed from the perspective that determination of primary causes must involve evaluation of rapidly growing tissues specifically. Only within the minute volume of tissue comprising the zones of cell division and rapid cell extension can the direct causes of inhibited growth be found. Likewise, only there, can the events be identifed which allow the relevancy to be judged of more remote physiological changes. The hypothesis that a disturbance in mineral nutrition might be a primary cause of the NaC1-induced growth inhibition is evaluated within this framework. The review should be considered as an early evaluation of the hypothesis, given the paucity of data spec8ificallyrelevant to the minute zone of rapid growth and the similar paucity of anulyses for nutrients other than potassium, sodium and chloride. Data is reviewed und discussed which reflects on processes related to the maintenance of nutrient transport towards and into the meristem and rapidly extending cells, recognizing that this tissue is rather dissimilar to the whole, or mature shoot both in anatomy and transport properties. Recommendations are made fix the development of advanced methods of analysis towards the goal of quantibing alterations in nutrient transport and status within the minute zones of rapid growth.

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

I.

115

INTRODUCTION TO THE INHIBITION OF SHOOT GROWTH BY SALINITY

More than 80 countries suffer from water shortages each year (Gleick, 1993) and agriculture consumes more fresh water than any other human activity (Falkenmark, 1989). Regional water shortages are partially due to both the tendency of agriculture to degrade water quality and increasing agricultural demands (Pimentel et al., 1997). Salt-affected soils include about one third of the world’s irrigated soils presently and that portion is expanding (Downton, 1984; Chauhan, 1987). Diminishing supplies of fresh water for irrigated agriculture have increased competition for the resource and have led to abandonment of salinized agricultural land. A poor understanding of the mechanisms of the salt-induced inhibition of growth and of genotypic salt tolerance have hampered attempts to isolate specific genetic factors and develop new cultivars with improved salt tolerance (Cheeseman, 1988; Dracup, 1991; Munns, 1993). Crop scientists and farmers are interested in the development of new cultivars and in the modification of management practices in order to avoid the inhibition of growth. This growth inhibition is, during the first several days of stress, primarily restricted to the plant shoots (Munns et al., 1982; Munns and Termaat, 1986). A systematic approach in either of these endeavours requires an understanding of the underlying physiology. Despite the substantial genotypic variations in response to salinity which can be found in nature (e.g. Rozema et al., 1978; Venables and Wilkins, 1978; Cuartero et al., 1992) and among cultivars which were not intentionally bred for their salinity tolerance (Qureshi et al., 1980; Kingsbury and Epstein, 1984; Azhar and McNeilly, 1987; Ashraf and McNeilly, 1990; Taleisnik and Grunberg, 1994), it remains unclear what the primary physiological responses are which result in the inhibition of shoot growth and how these might best be determined (Munns, 1993). Provisionally, the simplistic answer might be that the primary responses can be identified by precise measurements at both the specific locale and time at which growth is affected. However, making precise measurements to show exactly when and where physiological processes occur account for most of the challenge in physiology. A.

GROWTH INHIBITIONS: GENERAL CONSIDERATIONS

Cell division and cell extension ultimately account for all shoot growth, although the tissue volumes in which they occur are usually merely a minute portion of the whole plant shoot. Where shoot growth inhibition occurs is usually quite some distance from the plant part which is directly exposed to salinization, namely the roots. So the concept of ‘primary response’ needs some qualification. For example, even if Na’ were to transport quickly to the growing tissue of the shoot, enter meristematic cells and inhibit a particular enzyme involved in cell division, the ‘primary response’ to salinization might be considered to be the increased transport of Na’ to the shoot apical meristem (SAM) and other processes in the plant might

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be affected before the “a] increases within the growing tissues. Still, the ‘immediate cause’ of the inhibited shoot growth would have to be found in the processes, regulatory events and metabolic changes occurring within these growing tissues. In this hypothetical example the primary and direct response of increased Na+ uptake and transport would bring about the immediate cause of the growth inhibition (the enzyme’s inhibition). However, the immediate cause may be more indirect. If, for example, photosynthesis immediately decreased by 90% upon salinization and remained at that level unless the plant were transferred to unsalinized medium, this would be an important response. However, to demonstrate that carbohydrate supply was critically limiting shoot growth it would still be necessary to show, at least, that transport of carbohydrate level to the growing tissues was affected following the photosynthesis effect. For either of these hypothetical cases, the crucial plant responses need to be discriminated from a number of organismal plant responses based on connection to the immediate cause within the growth zone of the shoot. The timing of the inhibition of shoot growth is as important to defining the mechanisms of the toxicity and of the tolerance-related response, as is the locale. Factors contributing to changes in cell division or cell extension (e.g. increased “a] or decreased carbohydrate supply) need to be established prior to the growth inhibition. Methods such as whole shoot growth analysis have on occasion demonstrated tendencies, at least, towards reduced growth within 1 to 2 days of initial salinization (e.g. Cheeseman and Wickens, 1986; Wickens and Cheeseman, 1988; Cramer et al., 1994b). However, it remains doubtful whether the precision in these methods allows evaluation of 1 day growth effects (see discussion in Wickens and Cheeseman, 1988). This doubt is founded not only on considerations of biological variability and the resulting statistical limitations, but also involves the necessity of bulking slowly growing and non-growing leaves (or, in the case of grasses bulking growing and non-growing portions of the same leaf) with the much smaller rapidly expanding leaves (or portions of leaves). Recent methodological developments such as the use of linear variable differential transformers (LVDTs) are discussed below with respect to their relevance in determination of immediate plant responses (section 1I.A). Reasonably, major physiological interest lies in those alterations which account for differential genotypic plant response. The increased Na’ accumulation or the reduced carbohydrate supply becomes much more interesting if they occur to a far greater extent in a species or a cultivar which are less tolerant of salinization, for in this case there are prospects for producing an improved cultivar. Experimental results bearing on genotypic differences are presented within most major sections of this review (sections I.C.3., III.B.3, 1V.F.) and summarized in section 1X.B. B. NaCI-INDUCED INHIBITION OF SHOOT GROWTH: GENERAL HYPOTHESES

Hypotheses for the mechanism by which salinity reduces shoot growth can be grouped into four general categories, each having several possible variations. One


I17

suggestion is that salinity reduces photosynthesis, which in turn limits the supply of carbohydrate needed for growth. A second is that salinity reduces shoot growth by reducing turgor in expanding tissues, which are not able to fully osmoregulate in response. A third is that roots sense salinity and down-regulate shoot growth via a long distance signal. Fourth, a disturbance in mineral supply to the shoot, either an excess (we have emphasized Na '- excess) or deficiency, might directly affect growth. The only hypothesis treated in great detail here is this last one. Each of the remaining hypotheses has some merit and may contribute in some way towards the long-term effects on growth. They are summarized briefly and can be explored more thoroughly in the reviews cited.

I . Disturbed Photosynthesis Photosynthesis may well be disturbed by salinization (e.g. Robinson et ul., 1983; Ye0 et al., 1985; Munns, 1993); however, it seems unlikely that reduced photosynthesis leads to reduced shoot growth. Increased starch accumulation has been reported for the whole shoot (Gauch and Eaton, 1942), mature leaves (Lechno et al., 1997) and for expanding leaf tissue (Rathert et af., 1981; Munns et al., 1982; Aslam et al., 1986) under salt stress. After weeks of salinization, photosynthetic activity per unit leaf surface area can be little affected by salinization, even though reduced photosynthetic leaf surface was exerting a large effect (Robinson et al., 1983; Kaiser, 1987; Munns, 1993; Cramer et al., 1994a), or even though the cells had accumulated high levels of salt (Fricke et al., 1996). In Zea mays, reductions in net photosynthesis did not occur during the first 5 h of salinization, even when the elongation of young leaves were inhibited in the same time frame (Cramer et al., 1994a). In Oryza sativa photosynthesis per leaf area did decline after 10 days of salinization, whereas the [NaILearsteadily climbed (Ye0 et al., 1985), but this might have been accounted for by accelerated leaf senescence (Ye0 and Flowers, 1989). In salt-sensitive Cirrus reficulata there was no reduction in net photosynthesis until 30 days after treatment with 50 mM NaCl (Walker et al., 1982). And in Triticum species there was actually an increase in I4CO2 fixation after 10 days at 2 5 m M NaCl even though net growth had been reduced by 20% (Passera and Albuzio, 1978). The inhibition of leaf growth, then, might be primarily responsible for reduced leaf area and a loss in photosynthetic capacity, not vice versa. Although primary carbon fixation is not a likely primary cause of reduced shoot growth, it remains possible that some other aspect of carbon utilization plays a more major role. A NaC1-induced disturbance in the supply of carbon to the growing zones of shoots might be associated with the increased starch accumulation in mature leaves. Further discussion of photosynthesis and salt stress is available elsewhere (Munns, 1993). 2. Reduced Turgor and IitsufJicient Osmoregulation The hypothesis that crop productivity may be limited during salt stress primarily by the requirement for increased turgor (e.g. Oertli, 1966, 1968; Flowers et al., 1991) and limitations of plant capacity for osmoregulation, has seen a great deal of

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criticism (Termaat et al., 1985; Munns and Termaat, 1986; Kramer, 1988; Munns, 1988). Turgor of growing tissues can remain unaltered in expanding leaves (Termaat et al., 1985; Cramer, 1992b), in the expanding regions of grass leaves (Matsuda and Riazi, 1981) and in whole shoots (Imamul Huq and Larher, 1984) experiencing a salt stress. Turgor may also return to near control levels after a transient decrease (Cramer and Bowman, 1991a; Arif and Tomos, 1995; Fricke, 1997). This is similar to the lack of enduring alteration in turgor found in plants experiencing water stress (e.g. Ackerson, 1981; Meyer and Boyer, 1981; Michelina and Boyer, 1982; Van Volkenburgh and Boyer, 1985). Indeed, an increase in turgor at the shoot apical meristem (SAM) has been indicated during water stress (Barlow et al., 1980). During the initial few minutes of exposing roots to a moderate salt stress, leaf expansion sharply decreases. However, such reductions seem to be largely restored within a few to several hours with adjustment of the cell wall’s yield threshold (Delane et al., 1982; Termaat et nl., 1985; Cramer and Bowman, 1991a; Cramer, 1992a). The reduction and recovery of leaf expansion may be coincident with the loss and recovery of cell turgor in the expanding leaves (Cramer and Bowman, 1991a; Cramer et al., 1994a). Where changes in turgor have been shown after transfer to salinized medium these have also been rapid, transient and reversible within 30 min of transfer (Fricke, 1997). Elsewhere, decreases in turgor of expanding leaves have been correlated with the initial changes in leaf expansion rate (Wilson et al., 1970b; Cramer, 1992a; Munns, 1993). These rapidly occurring changes in leaf expansion rate (minutes) were similar for salt sensitive and salt tolerant genotypes and so probably are not relevant to salt tolerance mechanisms (Wilson et al., 1970b; Cramer, 1992a). Several years ago Greenway and Munns ( 1980) suggested that osmoregulation was probably of little importance in the extent to which a genotype can cope with salinity stress, since virtually all plants seem able to osmoregulate, regardless of their relative sensitivity. Recently, it has been shown that in one case a single gene mutant with 20-fold increased sensitivity to NaCl actually accumulates 80% more proline than the wild type (Liu and Zhu, 1997). Results supporting an important role for cell turgor in salt-induced reductions of leaf expansion have been rare (Neumann, 1997). 3. A Signal from the Roots Several reports have suggested that a signal from the roots communicates with the expanding leaves and growing tissues of the shoot (e.g. Termat el al., 1985; Munns and Termaat, 1986; Rengel, 1992) and that this may be a similar process in water stress (Zhang and Davies, 1990; Ball and Munns, 1992). There is a good deal of circumstantial evidence that abscisic acid (ABA) may be involved in regulating the shoot response at the shoot (Munns and Cramer, 1996). For example, spraying ABA on the shoot of Lens culinarus partially ameliorated (74% recovery) the NaC1-induced inhibition of shoot growth (Bano and Hayat, 1995). However, it is unlikely that the levels of ABA transported to the shoot control the level in the growing tissues (Munns and Cramer, 1996). Although there is much circumstantial evidence that ABA is somehow involved, the long distance signal remains a


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mystery and there has been no progress in identifying any other factor which might be transported to the shoot and control ABA synthesis, turnover or compartmentation in growing shoot tissue (Munns and Cramer, 1996). The 'root signal hypothesises' remains to be thoroughly explored and tested. There is a limited similarity between this hypothesis and that of disturbed nutrition, as nutrients themselves might be considered long-distance messengers. After all, many nutrients have an essential role in the processes of cell division and cell extension and these would cease soon after the supply were halted, especially in tissues with little nutrient storage capacity. C.

A NUTRITIONAL EFFECT OF NaCl ON SHOOT GROWTH

Within the general hypothesis of a NaC1-induced disturbed nutrition, the dominant specific hypothesis has clearly been that of 'ion excess', i.e. the idea that Na' and/or C1 rise to toxic levels in the shoot, eventually to high levels in the cytoplasm leading directly to metabolic inhibitions. In a widely invoked version, the selectivity for K+ over Na becomes increasingly compromised with the severity of salinization and this leads to failure in maintaining an adequate [K] in the cytoplasm (e.g. Abel and MacKenzie, 1964; Lauchli and Wieneke, 1979; Flowers and Yeo, 1981; Jeschke, 1984). 1. Ion Excess and Selectivity Hypotheses Most nutritional physiological studies have assumed the idea of Na:K competition at the onset, by only considering Na' effects on K + transport and nutrition alone. In surveying 50 research articles published between 1980 and 1995 it was found that accumulation or transport data for Na' or C1, transport or accumulation of K+ was studied in 72%, whereas only 18% studied transport or accumulation of a nutrient other than K'. All reports which included transport of nutrients other than K', included K' as well and in most (60%) of these the focus was clearly on K+ effects. Besides the fact that salt-induced effects on other nutrients have rarely been considered, the idea that selectivity for K' over Na' in the shoot is of primary or overwhelming importance in salt tolerance has been carefully criticized (e.g. Munns et al., 1982; Cheeseman, 1988). One questionable aspect is the idea that high [Na] in the cytoplasm underlies the growth inhibition. However, high cytoplasmic and chloroplastic [Na] did not reduce photosynthesis per unit leaf area (Robinson et al., 1983; Schachtman et a/., 1989). Furthermore, high cytoplasmic [Na] has been reported for halophytes (Harvey et al., 1981), without any evidence that these have specially adapted enzymes (Flowers, 1972; Greenway and Osmond, 1972; Flowers et al., 1977; Yeo, 1981; Hajibagheri et al., 1985). Indeed, cytoplasmic enzymes of halophytes seem to be similarly sensitive to NaCl in vitro (Flowers et al., 1977), whereas cell wall enzymes from neither halophytes nor glycophytes show much salt inhibition (Banuelos et al., 1996). Evidence that Na' and C1 are more poorly compartmentalized in glycophytic shoot tissue remains tenuous (section VIII).

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2. Shoot N u f Accumulation: a Disadvantage? Although not an essential nutrient for all plants, Na’ is probably accumulated advantageously in the shoots of all plants under some conditions as an inexpensive omoticant (Yeo, 1983). Indeed, halophytes generally accumulate higher Na’ levels in their leaves, than do glycophytes (Flowers et al., 1977). This obvious challenge to the ‘ion excess hypothesis’, has been overcome, by inventing two classes of plant with respect to salinity response, ‘Na includers’ and ‘Na excluders’. However, even in the most salt-sensitive glycophytic genotypes, Na’ when applied at low levels (ca. 10% of levels causing 50% growth inhibition) has often stimulated shoot growth (e.g. Elzam and Epstein, 1969; Marschner et al., 1981a,b; Lazof and Cheeseman, 1988b). If researchers were to avoid the simplification of ‘excluder’ and ‘includer’ species, then perhaps, a much deeper criticism of ‘ion excess’ might follow (Cheeseman, 1988). Common physiological limitations to growth encountered by both halophytes and glycophytes with respect to salinization might emerge. Indeed there has been a great deal of study of physiological response in halophytes at growth-inhibiting levels of NaCl and there is no reason, a priori, for excluding these from a discussion of salt-induced growth inhibition. Indeed these are probably more enlightening than studies of halophytes at stimulating NaCl concentrations for comparison to the glycophytic growth inhibition. True, many halophytes possess specialized structures for compartmentation and secretion of salt, but not all halophytes possess such specialized structures. Those that do not, although mislabelled ‘pseudo halophytes’ by some (Breckle, 1995), might well be the more productively investigated group due to their lack of obvious morphological disparity with salt-sensitive glycophytes (Cheeseman et al., 1985). 3. N u f Exclusion and Genotypic Tolerance Several investigators have indicated that Na’ exclusion from the shoot might be correlated to genotypic tolerance, including studies in Z. mays, Glycine rnax and Triticum X Lophopyrum derivatives (Lauchli and Wieneke, 1979; Hajibagheri et al., 1987; Schachtman et al., 1989, respectively). However, other studies have shown a lack of clear correlation between shoot Naf accumulation and genotypic tolerance (e.g. Lessani and Marschner, 1978; Rush and Epstein, 1981b; van Steveninck et ul., 1982; Walker et al., 1982; Grattan and Maas, 1988; Ashraf el al., 1990; Alberico, 1993; Reimann, 1993; Botella et al., 1997; Davenport et al., 1997; Leidi and Sairz, 1997). Due to the tremendous diversity in how such correlations have been pursued, the general applicability of this model remains in doubt. In G. max although there was a great difference between genotypes in [Na],hooland [Cl],h,ol at high salinity (100 mM NaCl), the more sensitive cultivar was already fully growth inhibited (and the tolerant not at all) at just 10 mM NaCl. And at this or [K]~hoolof the two low level of salinity there was no difference in genotypes (Lauchli and Wieneke, 1979). Similarly for one genotype of Lnctuca sativa grown with either 0, 1, 10, 50, 100 or 150 mM NaCl, growth was inhibited first at 50mM (14%) and then much further (41 and 66%) at 100 and 150 mM NaC1, whereas [K]\hooldecreased 47% at 5 0 m M and then not further at the two


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higher salinities (Lazof and Cheeseman, 1988b). A more sophisticated formulation of the same general hypothesis might recognize that it would be nutritional disturbances within the growing zones in particular which may inhibit either cell extension or cell division. Owing to the paucity of existing literature relating to the condition of the zones of rapid cell division and extension subsequent to salinization, the existing literature relevant to the status of young (still growing) leaves and tissues is included in the following sections. First, a few additional details must be provided concerning the timing of NaCl induced shoot growth inhibition.

II. INHIBITION OF SHOOT GROWTH IN DICOTS AND MONOCOTS A.

THE TIMING OF GROWTH INHIBITION

The effect of salinization on leaf elongation is astonishingly rapid (0.1-2 h), under some conditions requiring no more than a few minutes after exposure of roots to salinized medium (Matsuda and Riazi, 1981; Cramer and Bowman, 1991a.b; Cramer, 1992a). This rapid response may, however, also be transient (Matsuda and Riazi, 1981; Delane et al., 1982; Cramer and Bowman, 1991a,b, 1994a) and rapidly reversible (Munns et al., 1982; Rawson and Munns, 1984; Cramer, 1992a). The elongation of Z. mays leaves decreased 65-85% within 2 min of adding NaCl to the external solution (Cramer and Bowman, 1991a). The leaf elongation rate (LER) readjusted within 1-35 min depending on the level of salinity. When the plants had been suddenly salinized to 80 mM NaCl or more the LER was reduced 25% after adjustment. Immediate restoration of LERs was reported following transfer of 2. mays from salinized media, even when salinized to 75 mM NaCl (Cramer, 1992a). The rapid decrease in LER may also be unrelated to genotypic sensitivity (Cramer, 1992a). although after longer adjustment to salinized medium the response correlated with the supposed genotypic sensitivities (5 h, Cramer et nl., 1994a). A varying capacity for adjustment and resumption of normal leaf elongation in three barley cultivars was reported using time-lapse photography, although their relative salt sensitivity was not established (Matsuda and Riazi, 1981). Similar to the subsequent work in Z. mays, Hordeum leaves ceased to elongate within 15 min of salinization to 9 or 1 1 bars of NaCl, reinitiating growth after 60-90 min. If the most rapid growth responses are both unrelated to sustained growth inhibition and to genotypic tolerance, then they may only mislead in the quest for the mechanism of the growth inhibition. The measurement of LERs by the LVDT method need not be restricted to the short term ( < 2 h). When a constant and sufficient driving force is provided as tension on the elongating leaf, larger reductions in LER with larger non-reversing components can be measured (Cramer and Bowman, 1991a). In 2. mays LERs were measured within 5 h of salinization which were in agreement with dry weight accumulation (2-7 weeks, Cramer and Bowman, 1991a; Cramer et al., 1994a). After a 5 h adjustment period the LERs of plants exposed to 80 mM NaCl remained

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Regulation of Growth-related Shoot Nutrition

r

Fig. 1. Flow diagram depicting the control over nutrient supply to the zones of shoot growth. Selected physiological effects and interactions, which could directly or indirectly affect shoot growth during salinization over the long term (several days to a few weeks, or more). Arrows in grey represent interactions (arrows cross behind other processes and parameters). A few processes occurring between blocks of the diagram are written adjacent to the blocks.

20-30% less than control rates, an effect similar to that for net 3 day leaf growth. Medium-term responses to salinity measured by LVDT have also appeared to be genotypically differential and in accord with NaCl sensitivity (Cramer et al., 1994a). The problem with studying physiological processes after several days or weeks of salinization is that initial responses trigger regulatory processes throughout the plant (Fig. 1). These processes occur at nearly every level of development and within each organ, each interacts and influences other and distant processes. The


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task of unravelling which events lead to the non-adjusting, genotypically differential plant responses becomes increasingly difficult as the time frame extends, because changes in metabolic rates, depletions and surplus of various resources are present within each component, while adjustments to reduced growth influence metabolism throughout the plant (e.g. Greenway and Munns, 1980). The time frame worthy of the most intensive research is probably that in which genotypically differential responses are seen to arise first (i.e. 3 h) and continuing to a time at which consistency to longer exposures in extent of stress and genotypic sensitivity can be evaluated.

B. SALINITY EFFECTS ON CELL EXTENSION

Rapid (3-24 h) measurable effects on growth are always effects on cell extension, since dividing cells do not contribute significantly to net leaf expansion until several cell cycles (20-25 h per cycle, Clarkson, 1969; Powell et al., 1986). This does not preclude important effects on cell division also, only that the latter could not be detectable so quickly by macro methods. Following this reasoning, there can be little doubt that NaCl stress results in an inhibition of cell extension. Indeed, more than 25 years ago a decrease in the rate of biomass accumulation of Gfycine species was reported after as little as 1 day salinization (Wilson et al., 1970b). By measuring the mass of very young leaves (those first apparent to the unaided eye following salinization) were reduced 70-75%, even though still growing but older leaves showed no decrease after 3 days of exposure. Additional reports of effects on LER within the 3 h to 3 days time frame were discussed above (section 1I.A). After an 8 day salinization to 80mM NaCl cell length of Hordeum vulgare leaves had decreased 30% compared to the 1 mM NaCl control (Lynch et al., 1988). At present very little is known about the biochemistry or cell biology of the inhibition of cell extension in leaves subsequent to salinization. As for the biophysics, apparently the effects on cell extension in the relevant time frame are exerted by changes in the yield threshold of the cell wall and not by turgor, since osmotic adjustment can be complete and rapid within the zone of rapid cell extension (section I.B.2, Cramer, 1992a). Experiments within a longer time frame suggest that ABA levels might regulate cell extension during salt stress (e.g. Ban0 and Hayat, 1995), but apparently no experiments have yet been carried out measuring specific levels within the most rapidly expanding leaf zones.

C. SALINITY EFFECTS ON PRIMORDIUM FORMATION AND LEAF EMERGENCE

Whereas leaf emergence rates can be precisely determined, the point at which a leaf is said to emerge is arbitrary and a matter of convenience. Often for dicots it is simply taken as the smallest leaf observable to the unaided eye. In Poaceae the presence of a leaf tip beyond the whorl of older leaf sheaths is most often

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considered the point of emergence. This is also the point of transition from heterotrophic to autotrophic tissue. A plastochron index can be developed in monocots, as in dicots, by applying an arbitrary length beyond the whorl as the point of emergence. Leaf initiation in dicots, as opposed to emergence, is limited to the SAM. It involves rates and patterns of cell division (Hay and Kemp, 1990). Immediate effects on dicot leaf initiation would also be located within the meristem, where the primordia form, or at the step when primordia launch into intensive cell extension. In grasses 6 to 7 leaf primordia have often already formed in the seed, so that treatments imposed on the seedling could not affect formation of the first leaves. The rate of leaves passing through the emergence point might be affected differentially from a more strictly defined rate of leaf initiation based on primordia formation. Primordia might accumulate on the apex of a salinized plant with a delay in cells entering rapid cell expansion. Such details of leaf initiation under salinity have never been reported, however such accumulation did not occur during cold stress of wheat (Hay and Kemp, 1990). Indirect evidence suggests that salinity may affect leaf development close to initiation. Expansion of Sorghum bicolor leaves was found to be more strongly inhibited for leaves still enclosed in the sheath than for visible leaves (Bernstein et al., 1993b). Similarly, in dicots the greatest effects on ultimate leaf development were found in leaves not yet in the phase of rapid expansion at the time of salinization (Rawson and Munns, 1984; Aslam et al., 1986; Lazof et al., 1991). Ultimate leaf formation (undefined developmental timing) was reduced 35% in Phaseolus vulgaris and for both leaves and flowers by 50% (salinization to 150 and 70 mM NaCl, respectively Lagerwerff and Eagle, 1961; Hussain and Ilahi, 1995). With the exception of a study in highly salt-tolerant Beta vulgaris (leaf fresh weight was not reduced by the treatment), leaf emergence rate has consistently been shown to be particularly sensitive to salinity (Papp et al., 1983). In Atriplex amnicola the numbers of leaves emerging per day decreased continuously with the severity of the salt stress from 20% to 68% to 40% at 200,400 and 600 mM NaCl respectively (Aslam et al., 1986). In L. sativa leaf emergence was 6% slower in salinized plants and this appeared to be effective after just 6 days of treatment (Lazof et al., 1991). Similar decreases were found for salt stressed Hibiscus cannubinus during 4 weeks of salinization (Curtis and Lauchli, 1985). In H. vulgarr leaf emergence was also delayed 5-6 days for leaves 17 days after stepwise salinization was initiated (Rawson et al., 1988), or delayed 4-5 days after plants had been salinized for 20 days (Jeschke and Wolf, 1985). In four Glycine species the total number of leaves was reduced by ca. 50% in the more salt-sensitive species (Wilson, 1967). In salt-stressed S. bicolor the plastochron index of shoot development (leaf emergence per day) decreased by half relative to that of the non-salinized control plants during the first 5 days of salinization (Bernstein et d., 1993a). In particular, leaf number four was delayed in development, a leaf which was still unemerged from the whorl but rapidly elongating at the time of salinization (Bernstein et al., 1993b). After 23 days of salinization 0. sativa leaf

THE NaCl INDUCED INHLBITION OF SHOOT GROWTH

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emergence was 2 days delayed specifically in the more salt-sensitive genotypes (Ye0 et al., 1991). Reduced whole shoot biomass of salt-stressed plants may result, in part, from either shorter periods or lower rates of leaf expansion (along with possible effects on stem growth). Although shorter periods and lower rates of leaf expansion are likely to be due to some complex effect of both cell division and cell extension, delayed primordia formation most likely represent effects on cell division specifically. It is thought that a critical number of cells needs to be formed within each leaf primordium before leaf initiation (Poethig and Sussex, 1985a,b; Dale, 1988). Microscopy has indicated that there is little cell expansion within leaf primordia (Esau, 1977; Lazof and Lauchli, 1991b), again suggesting that primordia form largely on the basis of cell numbers. The dominance of cell division during primordia formation is also suggested by 50-fold increases in the length of very young leaves coincident with increases in cell length of merely three- or four-fold (Sunderland and Brown, 1956; Dale, 1988). D. SALINITY EFFECTS ON CELL DIVISION IN LEAVES

In dicot leaves cell division continues, to some extent, up until 95% of the final leaf size is attained (Maksymowych, 1973; Dale, 1988) occurring largely within pockets of cells surrounding minor veins (Sachs, 1989). Even though some cell division continues through much of leaf expansion, the rate of cell production falls exponentially arriving at a low limit by the middle of rapid leaf expansion (Maksymowych, 1973; Lamoreaux and Chaney, 1978; Sachs, 1989). In Lactucu sativn, leaves gained fresh weight exponentially from emergence until they reached 540mg fresh weight but continued expansion for another week at less than one third the earlier rate, finally reaching about 3.Og (Lazof and Bernstein, unpublished). If the steady exponential expansion rate, as determined during the first few days of visible growth, were continuous from the instant at which primordia ‘launch’ into intensive cell extension (and exponential leaf expansion), then it would require about 1 day for primordia to reach the easily visible 5 0 m g fresh weight (FW). If the leaf development pattern in L. sativa is consistent with the general pattern in dicots (Maksymowych, 1973; Dale, 1988), then effects detected before reaching 270mg FW, would have occurred during a period in which cell division dominated leaf development. Ultimate cell numbers in grass leaves were significantly reduced by salinization (Munns and Termaat, 1986). Two studies, one in H . vufgure and one in I? vulgaris measured interstomatal distance, as an indication of cell expansion and judged that there was a major effect on cell extension (Brouwer, 1963; Munns et al., 1982). Such methodology is inconclusive, however, since a reduced rate of meristematic activity would not only affect the final number of cells per leaf, but could also (or exclusively) affect the number of primordia formed and leaves initiated. It is uncertain also whether final epidermal cell size reflects the size of underlying mesophyll cells, which account for most leaf cells. were studied during salinization and Leaf expansion rates in Heliunfhus ~~rznuus

126


during either the 0-24 or 24-96 h period after removal of plants from salinized medium (Rawson and Munns, 1984). Leaf expansion rates increased (on average for all leaves) 15%and 59%over the rates in saline solution, during the initial 24 h after removal from media salinized to 50 or 100mM NaCI, respectively. The expansion rates then decreased 21% and 28% from that initial 24 h rate during the next three days. The leaf expansion rates in salinized plants was increased over that of control plants by 10% and 8%, suggesting that cell extension was repressed during salinization and derepressed during the initial recovery (see section 1I.B). Although effects on cell extension may have been generalized, acting on all expanding tissues within the shoot, younger leaves (numbers 14 to 19) had the most severe reduction in expansion rate during salinization, whereas older leaves (numbers 8 to 14) had the largest increase in rate during the recovery period. In the younger group of leaves (or fully formed primordia) the pool of cells ready for rapid cell extension, then, either had not increased over the control level during the 10 day period of salinization, or they were present in primordia which were repressed from launching into intensive cell extension. This young group of leaves was barely or not yet emerged at the time of initial salinization (just barely measurable with a ruler, personal communication R. Munns). Hence, in these leaves cell division was still a dominant process during salinization. If the repression of cell extension is indeed general throughout expanding shoot tissues, then the younger leaves in showing the largest irreversible growth inhibition suggests a strong effect on cell division specifically, since cell division was the dominant process during the treatment. Furthermore, if cell division had not been inhibited, there would have been a higher potential for increased expansion in these younger leaves upon derepression. Some biochemical work has also had a bearing on effects of salinization on cell division. Protein synthesis was reduced in Nicotiana tabacum by 50% after 20 h of salinization (Ben-Zioni et al., 1967). This reduction was only slightly greater in young leaves (not defined specifically) than in older leaves (57 vs. 48%reduction). Rapid effects of drought or salt stress on polyribosome formation in the whole shoot might also be taken as circumstantial evidence that protein synthesis may be inhibited crucially in meristematic cells, where rates of protein synthesis are most intensive (Rhodes and Matsuda, 1976). Decreases in root tip protein synthesis upon salinization were found for Pisum sativum and Glycine m x (Rauser and Hanson, 1966; Kahane and Poljakoff-Mayber, 1968).

111. NaCI-INDUCED DISRUPTIONS OF NUTRIENT TRANSPORT A. INFLUENCES OF SOME EXPERIMENTAL CONDITIONS

Complex and variable plantlmicrobe, /soil, and /climate interactions are the norm for most environments in which plants grow. Simplification, however, is necessary to isolate factors and observe the resultant responses. The experimentalist typically


127

attempts to restrict the extent of departure from common natural conditions. A compromise between these inherently conflicting objectives is usually sought. However, enhanced statistical significance can be achieved by employing overly simple, unrealistic systems. This becomes especially troubling when extreme conditions are not properly discussed within a report. Two such cases are of special importance to salinity research. First, more often than not, salinized plant response has been compared to that of plants growing in the absence of Nat (nominal 'absence', actually an undetermined micromolar level). Second, much experimentation has considered effects of salinity with enormous Na to Ca molar ratios in the treatment solution. The critiques and implications of these two design flaws are discussed below. Several additional aspects of experimental design, however, although important in understanding the salinity response, are not discussed nor involved in the remaining chapter. These include, most notably, salinity effects of salts other than NaCl (e.g. Na2S0,), specific chloride effects and effects on the nutrition of plant roots. I . Control Levels of Na Low levels of Na' have been found to be growth-stimulating to some glycophytes (section I.C.2). This could possibly prove valid for a wide range of species. In all cases the plants accumulated significant Na' at growth stimulatory levels. Metabolism is undoubtedly altered at these levels of exposure, at the very least growth-related metabolism. It is therefore completely incorrect to ascribe all metabolic alterations at inhibitory levels of Na' to deleterious NaCl effects without examination of plant status under conditions of Na-induced growth stimulation. For example, consider that the Na:K ratio in treatment solutions can be varied over an enormous range while maintaining Na' well below inhibitory levels (Marschner er al., 1981a). Growth was stimulated by the increased Na' levels which never reached 5 m M (inhibition commences at levels close to 100 mM, Marschner et al., 1981b). Not only have less salt-tolerant crops shown growth stimulation by low levels of NaC1, but also in monocots increased Na:K ratio of the nutrient medium gave rise to more than a doubling in shoot tissue Na:K even though shoot growth inhibitions were either nil or 50% (Z. mays, Botella et al., 1997). Increased Na and decreasing levels of the major mineral osmoticant (K) are logical consequences of increasing [NaImedlum, independent of whether growth is inhibited. The importance of the effect of salinization on (NaIrhoo,and [K]shoo,has been artefactually exaggerated by the practice of using a '0 Na ' ' control treatment. This conclusion is further supported by hundreds of reports on Naf uptake and translocation in halophytes, many of which have been conducted at growth stimulating levels of NaCl (e.g. Flowers er al., 1977; Lauchli. 1986; Breckle, 1995).

2. Na:Cu Ratios There is nothing new about the concept that the Na:Ca ratio of the soil solution affects the extent of NaCl stress. The ability of increased soil Ca to protect plants from salinity stress was reported almost 100 years ago (Kearney and Cameron,

128

D.9.LAZOF and N. BERNSTEIN

1902; Kearney and Harter, 1907). Sodium absorption ratios (SARs), where SAR = Na/[(Ca

+ Mg)/2Ios (elements in mequivA)

are specific mineral forms of the Kerr Gapon equation for the equilibrium between cations in solution and held on a colloidal matrix. Explicit warnings have appeared previously against conducting salinity studies at unrealistic SARs (Maas and Grieve, 1987) with plant growth inhibition being heavily dependent on the solution Na:Ca ratio (e.g. Grieve and Fujiyama, 1987; Grieve and Maas, 1988; Gorham and Bridges, 1995). Ratios of Na':Ca2+ > 100 are rare in nature and seawater has an SAR of about 85. A 0.25X strength Hoagland solution (1 and 0.25 mM in Ca and Mg, respectively) would need to approach 170mM in Na' to reach an SAR of 150. Irrigation waters with SARs in the 20s have been classed as 'unsatisfactory for use in imgation', since much higher SARs result as the soil begins to dry (Hawkes et ul., 1975). Most physiological salinity work has been conducted within a more realistic range for salt-sensitive plants using Na:Ca ratios < 80, and/or SARs < 150. The following sections of this review have been based almost entirely on studies, or on treatments within such a range. Although any such limits are somewhat arbitrary, establishing some liberal limits allows a more meaningful interpretation of the literature. Explicit comments on Na:Ca ratios and SARs have also been included for many of the studies below to this same end. Where the nature of the treatment has been properly discussed and the results interpreted within the framework of their relevance for natural environments, some studies with extreme Na:Ca ratios have been retained, often with qualification of the experimental conditions. It might also be noted that effects of salinization at constant Na:Ca ratios have been studied and that growth inhibitions do occur under these conditions (e.g. Eaton, 1942; Lagerwerff and Eagle, 1961; Abel and MacKenzie, 1964; Pitman, 1965; Bernstein et al., 1969; Shannon, 1978; Ehret et L J ~ . , 1990). The major emphasis of the present review is phenomena within growing shoot tissues. Although there seems little reason for assuming that increasing or decreasing [ K]ahoothave anything to do with growth inhibitions or with a particular genotype's ability to cope with salinity (section I.C.l), there may be value in evaluating the evidence for inhibitions of nutrient supply to the whole shoot (sections 111 and VII). It is undeniable that all nutrients which are supplied to growing shoot tissues are subject first to the control of general translocation to the shoot. Furthermore, the discussion of whole shoot nutrition provides a framework for discussion of the less extensive literature available on the nutrition of growing shoot tissues under salinity. B. EFFECTS ON WHOLE SHOOT NUTRIENT ACCUMULATION

There are several points in the pathway of nutrient supply to growing shoot tissue, where transport might be disrupted, including: root uptake, radial transport, transendodermal transport, xylem loading, long-distance transport, reabsorption by xylem parenchyma, partitioning within the shoot, whole shoot retranslocation.


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inter-leaf retranslocation and leaching through leaf cuticles. Most of the work on disturbed transport to the shoot has followed the suggestion that salt tolerance in glycophytes would be evidenced as lower shoot accumulation rates for Na', with the frequent inclusion of the hypothesis that K + shoot accumulation rates would also be better maintained. These studies have shown universally that Na' accumulation in the shoot is much greater and K' accumulation much decreased under conditions of high NaCI. This finding is neither surprising nor in dispute (section 1.C). nor are the important metabolic roles of K + in doubt. However, these roles may be fully preserved at relatively low [KIshoot(section I.C.1). More important questions for the hypothesis of disturbed nutrition might be: (1) whether the level of K + becomes so low as to inhibit growth or metabolism; ( 2 ) whether any similar disruptions in mineral nutrient supply occur for other nutrients; and (3) whether any such disruptions correspond to the locale and time frame of well documented growth inhibitions. Calcium is a particularly interesting nutrient for evaluating these questions, considering that it is known to be both essential and closely regulated during the processes of both cell division and extension. It has also been studied more extensively with regard to salinity than any other nutrient excluding Na' , CI- and K'. 1. Translocation of Ca2' to the Shoot: Comparison with K' Translocation of Ca" to the shoot seems to be more drastically affected by salinization than is the root accumulation of Ca2+ and often even more reduced than that of K + . In Gossypium hirsutum [CaIshoo,decreased 60% more than [Ca]rool (Na:Ca of 20, Kent and Lauchli, 1985). In an unimproved line of Trifolium prutense the [CalShoo,decreased almost 60% with salinization to 100 mM NaCI, while [Ca],,,,, was unchanged with similar results for unimproved lines of both Medicago sativa and Trifolium alexandrinum at moderate SARs (Ashraf et al., 1986). In L. sativa [CaIshootwas decreased 67% but [CaIrootonly 40% (SAR of 113, Cramer and Spurr. 1986). In a salt-sensitive cultivar of Hordeum vulgare 32 h root accumulation of Ca2+decreased 11% whereas shoot accumulation decreased 55% (Lynch and Lauchli, 1985). Also in H. vulgare exposed to 75 mM NaCl for 7 days, the 4 h root accumulation of 4sCa2+was decreased by 63%, while the translocation to the shoot was reduced 94% (Na:Ca > 150 and SAR > 140, Cramer et al., 1989). Although this latter salinity treatment was rather extreme, greater shoot than root reductions (56 vs. 30%) were found with a more moderate salinity treatment also (Na:Ca of 7.5 and SAR < 70). In two lines of each of four grass species [Ca]roolwas decreased very little, whereas the [Callearwas reduced 60-85% in all four species at the most severe salinities (Ashraf er al., 1990). In a relatively salt-sensitive wheatgrass, Agropwon intermedium, [CaIshootdecreased twice as much as did [Ca]roolwhen salinized to 20 mM NaCl (Elzam and Epstein, 1969). In salt-stressed G. hirsuturn [Ca]shooldecreased significantly more than [KIshw, (87 vs. 57%. for a NdCa of 20, Kent and Lauchli, 1985). Although this may have been partially due to luxury consumption of Ca2+ in the '0 Na' solution with l0mM Ca, even when compared to the I mM Ca control, the [CaIbhoo, had been

130


reduced 83% by 200mM NaCl. In G. mux a 27% decrease in [CaIshoorwas accompanied by a 30% increase in [KIshoo,at increased salinities (Na:Ca< 16, Grattan and Maas, 1988). In 7: pratense (the most salt-sensitive species of the three tested) [Ca]shooland [KIshootdecreased 56 and 14%, respectively (Ashraf et al., 1986). In L. sativa [Ca]sl,oo,decreased slightly more than [KIshoot(Cramer and Spun-, 1986). In salt-stressed Persea americnna [Ca],,,, and [KIlcf decreased 65% and 8%, respectively, in the most salt-sensitive genotype (Downton, 1978). In Phaseolus vulgaris [Ca]shootdecreased 46% whereas [KIshoolincreased 38% after salinization for 21 days ( I mM Ca, Kawasaki and Moritsugu, 1978b). In Lupinus luteus (only two spectra shown) the vacuolar [Ca] of the spongy mesophyll decreased by more than half with no decrease in the K level (van Steveninck et al., 1982). Even in the case of salt-stressed halophytes, well adapted to osmotic replacement of K+ with Na+, the [Call,,- has sometimes decreased much more than the [KIleaf(88% vs. 76%, Ye0 and Flowers, 1986). As for monocots, in moderately salt-stressed Zea mays [Ca],,,, was decreased 54% and [K],,,, not at all after 8 weeks (Benes et al., 1996). The [CaIshoo,of a relatively salt-sensitive Zeu muys cultivar decreased 12-fold more than did the [K]slloo,6 or 18 days after salinization (Na:Ca = 80, Cramer et al., 1994b) and nine-fold more than the decrease in [K]ahool (Na:Ca = 40, Kawasaki and Moritsugu, 1978b). In H. vulgare [CaIshool was reduced 49% more than [K]shoo,after 21 days of salinization, although in 0.sativa [Ca]sl,ool increased while [K]shooldecreased 60% (Kawasaki and Moritsugu, 1978a). In the four grass species Holcus lanatus, Lolium perenne, Dactylis glumerata and Festucu rubra reductions in [CaIshootwere greater than those for [KIshm, (ca. 600, 400, 50 and 50% greater reductions, Ashraf et ul., 1990). In salt-sensitive Agropyrun decreased 87% and [K]ehoo, not at all with a salinization intermedium the [Ca]sl,oot which reduced growth 90% (low Na:Ca, Elzam and Epstein, 1969). Although, NaC1-induced K deficiency has received intense study, it is rare that [CaIshmtand [KIshoot were both determined and [KIshooldecreased more than [CaIshoot.In the more sensitive of two corn cultivars the decrease in [KIshool exceeded that of [CalShoat and occurred only in one of the two treatments (100 mM NaC1, Fortmeier, 1995). In H. vulgare the [CaIshootdecreased 25%, while the [K]nhootdecreased 45%, but the salinized treatment solution had been increased fourfold for Ca and not at all for K (Gauch and Eaton, 1942). Perhaps, the weak reductions of [CaIshootin these two reports were, in part, due to the rather low Na:Ca ratio and SAR (13 and 14, respectively). The reductions in [K]shoor exceeded the reductions in [CaIshootin just two of seven crops after salinization for 23 days to 100 mM NaCl (Lepidium sativum and Capsicum annum, Lessani and Marschner, 1978). In 0. sativa while the Na:Ca ratio of the medium was directly conelated with the whole shoot Na:Ca ratios, neither of these were correlated with the relative inhibition of shoot growth at moderate SARs (Ye0 and Flowers, 1985). 2. Ca2+translocation: comparison to A@+ Calcium and magnesium are divalent, cationic, essential nutrients. They both typically accumulate in shoot tissue at the level of a few to several pmol (g fresh


131

weight)-’. One important difference is that whereas Ca2’ is widely considered to be phloem immobile and incapable of transport symplastically, Mg2+ is thought not to be similarly restricted. In 7: pmtense [Cali,,,. was decreased 34% on average with the greatest reduction for the most salt-sensitive genotype, whereas decreases in [MgIleaf were about 20% with little variation (Ashraf et al., 1986). In L. sativa salinized for 20 days to 120 mM NaCl the reduction in [CaIshoo, was ca. 25% greater than was the reduction for [Mglsh0,,,(Cramer and Spun, 1986). In salt-stressed F! americana [CaJleafdecreased 65%, whereas [Mg],, decreased just 23% in the most saltsensitive F! americarza tested (Downton, 1978). In Lupinus albus the [Mg] of the xylem was increased less than was the [Ca] (2.8-fold vs. 3.8-f0ld), and the petiolar [Mglphloem was reduced only half as much a [Ca]phlcKln (Jeschke et al., 1986). In P. vulgaris [Ca]shoordecreased 46% after salinization for 21 days, whereas [MgIshoo, decreased only 20% (Kawasaki and Moritsugu, 1978b). The comparison of changes i n [Ca] and [Mg] in shoot tissues appears to be much the same for monocots. The [Cali,,, in Triticuin aestivum and H . vulgare at a Na:Ca of 5 and an SAR < 8.5 were decreased 49% and 72%, respectively, after 3 weeks of salinization, despite the fact that [Ca]mrd,um was 2.6 times greater in the salinized treatment (Ehret et al., 1990). The [MgIledincreased 3.5-fold and two-fold for the two species although it too was increased 30-fold in the salinity treatment. Shoot growth was inhibited 52% and 17%, respectively. Doubling the [CaImedium at the time of salinization had no ameliorative effect on the growth inhibition and only attenuated the reduction in [Ca]l,,f (by 3.5% and 30%, respectively, in the two species). When H. vulgare was salinized at a low SAR (loo% increase in the same leaves of plants grown at a non-stressing control level of NaCI. Although the youngest leaves doubled their 32Pcontent during the chase, the 32Pcontent of older


I55

leaves did not increase appreciably. Although '?P was moving also from the root during the chase, the dissimilarity in distribution during the labelling and chase periods must have been due to short-term recirculation from older to younger leaves. Exact estimates of immediate arrival and extent of redistribution will require more detailed time courses of translocation during both pulse and chase periods. It was also shown that transport from the root to the shoot during the chase was also less than 10% of that found in the control plants. Also in moderately salt-stressed cotton (SAR = 140) 3 h recirculation of 32Ptowards the apex (into two leaves weighing only 130 mg together and accounting for 7.5% of the shoot mass) was not reduced in salinized plants. although a more extreme salt treatment (SAR of 300) did cause a 55% reduction in "P recirculation (Martinez and Lauchli, 1991).

C. CALCIUM RECIRCULATION IN THE SHOOT

Calcium is the major nutrient most restricted from phloem transport and, therefore most likely to be critically affected on any inhibition of recirculation. In the past Ca' ' has been characterized as non-recirculating due to virtual immobility in the phloem (Hanson, 1984). However, it is clear that, although phloem immobility may be generally true for senescing leaves (e.g. Norton, 1963; Hill, 1980). even this is not absolute (see below). I n the present context, smaller exchangeable pools of Ca'~' associated with parenchyma cells of major and minor leaf veins and traces may be more crucial to short-term recirculation (Biddulph et nl., 1958; Biddulph and Nakayama, 1961; Norton, 1963; Millikan, 1967). Somehow, of course, even the most problematic of nutrients must be continuously supplied to the dividing cells and tightly regulated in both these and the cells undergoing extension, although the latter do have capacity for storage of mineral surplus. Transport of Ca" in the phloem does occur, at least in some plants (see reviews Lauchli, 1972; Pate. 1975; Bangerth, 1979). This has been demonstrated directly in leaves of Avrnu (Ringoet and Sauer, 1968) and in the shoot of Phmeolus (Biddulph and Nakayama, 1961). It has also been shown indirectly in a number of other dicots (Bukovac and Wittwer, 1957; Jeschke and Pate, 1991a,b). Indeed in some plants the [Ca]phlmmhas been greater than [Ca] in the xylem (Pate and Sharkey, 1975). Transport of Ca' ' through the phloem may also be important in particular locations within the plant, particularly where the xylem cannot supply the nutrient requirement, including non-transpiring fruits, storage organs and meristematic and expanding tissue (Pate, 1975). In the first 4 days after leaf emergence phloem transport of Ca' was 80% as great as xylem transport into young leaves of Ricinirs communis, whereas 8 days later, there was no net increase of [Ca],,,, from phloem transport (Jeschke and Pate, 1991a). It is likely that the phloem is involved in acropetal Ca' ' transport in dicots, probably by receiving Ca' ' from the xylem in petiolar leaf traces and transporting Ca2 basipetally in the leaf and petiole, at least for a short distance. The nutrient might then either continue transport through the

156


phloem or be transported back into acropetally flowing xylem once more for movement towards the apex, although in the latter case it would again have to circuit through the dominant transpirational sinks superior to the leaf from which it is exiting. Abundant xylem phloem transfer cells have been found in leaf traces, although direct evidence is lacking implicating these in Ca2’ transport (Pate, 1969). Strong arguments and a mechanism have been provided by which transport through the phloem can be explained for some ‘non-phloem-mobile’ micronutrients (Udo and Scholz, 1993). Application of exogenous chelators has also been shown to promote the retranslocation of Ca2+from older to younger leaves (Millikan, 1965). Although Ca2* may be the most likely candidate for restricted transport towards the meristem due to disruption of recirculation, some research has also suggested a role for Ca2” in the maintenance of recirculation capacity in general (Martinez and Lhchli, 1991). Backflow of Ca” through the xylem has also been suggested as a means by which Ca” recirculates out of the rapidly transpiring mature leaves towards the stem and up towards the apex (Martin, 1982). The driving force would probably be created by cell expansion. Studies have suggested that xylem backflow may operate on a diurnal schedule, with the transport out of the transpirational sinks in the dark and at increased humidity (Palzkill and Tibbitts, 1977; Wiebe er al., 1977; Bangerth, 1979; Tibbitts, 1979). Humidity increases the water potential of the mature leaves, so that less negative water potentials at the meristem would be effective in driving acropetal transport. Apparently a relative humidity of 30% might be adequate to allow this nightly flux (Bangerth, 1979). Despite the strong logical arguments for a possible effect of salinity on the recirculation of Ca” there are apparently no available pertinent data. The decrease in 40% of [Ca]phlue,n in petioles of L. albus (twice the decrease found for K or Mg) was discussed above (section 1V.D). This decrease in [Ca]p,llorm also occurred simultaneous to a threefold increase of [Ca] of the xylem. It is likely that ion levels in petiolar phloem sap reflect rates of transport out of leaves.

D. SUMMARY OF SALINIZATION A N D RECIRCULATION

Most long-term recirculation studies have been limited by consideration of only Na’ and C1- and by the absence of a non-stressing NaCl control treatment. Of the pulse+hase studies, the one case in which nutrient, K’ (“Rb+), transport was considered, indicated no effect of salinization (Yeo, 1981). Additionally, the one study which included a non-inhibitory NaCl control, indicated that salt tolerance might be associated with an increase in maintained recirculation towards the apex, not a decrease as would be expected on the basis of the ‘young tissue protection hypothesis’ (section IV.A, Wieneke and Lauchli, 1980). Results of applying tracer to older leaves also did not support the protection hypothesis. Of the five studies in H. vulgare using whole organ and xylem/phloem analysis, only one study presented unequivocal evidence indicating that recirculation might be restricted by saliniza-


157

tion with a large decrease in K+ import from recirculating pools after salinization from 1 to 100 mM with NaCl (Wolf et al., 1990). In the L. albus studies a decrease in recirculation within the shoot due to salinization was indicated and not only for K', but also for N and more strongly for Cazt and Mg2+ (Jeschke et al., 1986). Besides the logical increase in Na ' recirculation following a 40-fold increase in [Na],ne,,lum, the recirculation of SO:- and P, were also increased. Taken together the studies on long-term recirculation present conflicting and weak indications for either the hypothesis that recirculation capacity regulates protection of the youngest shoot tissue, or that salinization affects nutrient recirculation. The few short-temi recirculation studies which have been carried out under saline and control conditions have indicated a large effect on nutrient recirculation of salinization (Martinez and Lauchli, 1991 ; Martinez ef al., 1996). Unfortunately it appears that only P, has been considered thus far, although an inhibition of recirculation has been shown for both young leaves and towards the apical meristem. There are reasons to suspect that Ca' ' recirculation might be the most critically affected of any major nutrient by restricted recirculation under salinization.

VII. SALINIZATION AND SHOOT NUTRITION: SPECIFIC NUTRIENTS A. POTASSIUM

It is generally recognized that K ' uptake to the plant, and deposition in both growing and non-growing tissues is reduced by salinization. Whether any shoot tissue approaches a deficient, metabolically limiting level of K during salinity stress remains open (section 1.C, I.C.2). Potassium is highly mobile in both apoplast and symplast, as well as in both phloem and xylem with no special transport restrictions relative to meristematic tissues ever having been suggested. Despite these reservations about the importance of disturbed K nutrition, discussion of K ' transport has been central to the discussion in most sections of this review, largely due to the great body of salinity related research which has considered such effects and the value of comparison to other nutrients. B. CALCIUM

Many studies conducted with reasonable Na:Ca ratios in the solutions have suggested that Ca' ' uptake, translocation and distribution may be critically affected by salinity. Transport of CaZf towards meristematic cells and cells in the earliest phase of extension may be reduced by salinization (sections 1V.C V). This may in part be due to a relatively low phloem mobility and the consequent difficulties in recirculating Ca2+ from transpirationally dominant mature shoot tissues to weakly transpiring growing tissues (section VI). The challenge of Ca' ' transport towards the meristem is heightened by its 'phloem immobility' and symplastic restrictions

158

D. B . LAZOF and N. BERNSTEIN

(sections V, VI). To maintain a critical nutrient level in actively dividing cells the rate of supply must occur at a rate equal to the product of the average rate of cell division and the total [Ca] of the meristem (primary cell wall included). Assuming a 30 h cell doubling time and a meristematic volume of 16 pl,the 4 pmol ( g F W - ’ concentration (Figs 5 and 7 in Lazof, 1991b). the meristem of a rapidly growing lettuce plant would require a net supply of about 50 nmol day- I . C. MAGNESIUM

Magnesium has been discussed above mainly as a point of comparison to the nutrition Ca (section III.B.2). Less study has been made of the effects of salinization on Mg nutrition, however, than has been made on Ca nutrition. This may be more due to lack of an appropriate radioisotope than to any intrinsic lesser importance of an effect. Among other essential metabolic roles, Mg” is required in mitosis, specifically in microtubule assembly, at specific and well-regulated levels. This means that the supply of Mg2+ to meristematic tissues, must be carefully maintained if shoot growth is to be continuous. In most saline soils (and in seawater) the [Mg] is relatively high (at least sevcral millimolar). Nonetheless, several investigations have included consideration of salinity effects on Mg nutrition (e.g. Lazaroff and Pitman, 1966; Downton, 1978; Ashraf et al., 1986, 1990; Cramer and Spurr, 1986; Jeschke et al.. 1986; Lynch et a/., 1988; Ehret et al., 1990; Omelian and Epstein, 1991). Only a few studies considering Mg nutrition have contributed towards an improved understanding of nutritional effects within the zones of cell division and rapid cell extension (e.g. Aslam et al., 1986; Grieve and Maas, 1988; Wolf et a/., 1990; Jeschke et al., 1992; Bernstein et al., 1995). These were discussed above (section IV.D), where it was judged to be still unclear whether [Mg] was generally low in young shoot tissues of plants subjected to salinity. Magnesium is not generally considered to be particularly prone to restriction either in phloem or symplastic transport. The consistency of data pointing towards reduced [Ca] in young tissues in the absence of reduced [Mg] supports the concept that some component of the recirculation process may be most severely limited under saline conditions (section VI). D. PHOSPHORUS

There has been considerable study of NaC1-induced effects on P nutrition. Several laboratories have reported that [P]aho,l,can be greatly increased by salinity. However, it has been suggested that this ‘P toxicity’ effect may be an artefact of the unrealistically high solution levels of P used in such studies (e.g. Bernstein et d., 1974; Nieman and Clark, 1976; Grattan and Maas, 1984. 1985; Martinez and Lauchli, 1991; Martinez et al., 1996). This is supported also by studies in soils where salinization did not lead to increased (e.g. Francois et id., 1984). Exactly what the minimum solution level of P is, however, which can result in


1.59

excessive P levels in the shoot, or which can exacerbate the salt-induced inhibition of shoot growth is not clear. Often such levels have been submillimolar levels. For example, concentrations as low as 120, or 100 p M in soybean (Grattan and Maas, 1984, 1988) or 100 pM in L. lzrteus (Treeby and Steveninck. 1988) were found to produce excess P accumulation. Concentrations as low as 20 pM P were found to produce increases in [P]ahcx,i in Brnscica olemcea, with trends towards the same in L. sntivn, Daucus cnrotn and other B. olerncen (Bernstein rt al., 1974). There have also been studies of the effect of salinization on P nutrition indicating decreased P transport. Some of these were carried out at high [PImrdlum. In R. coninzunis growing with 1.3 mM Pi there was a decrease in Pi exudation from excised roots of 90-9596 (Jeschke and Wolf, 1988). In Z . mays the [PIrhoo,decreased 50% with 0.17 mM P in the solution (Maas and Grieve, 1987). In G. hir.vutum there was a decrease in 32P translocation to the shoot even when growing at I mM Pi (Martinez and LPuchli, 1991). Several laboratories have investigated salinization effects on [P],ll,,c,i with attention to a possible association with relative species or genotypic salt tolerance. The more salt-sensitive Glycine fomentella had a greater decrease in [P]sl,,~ot, both at lower levels of salinization and after shorter periods at high salinity (as short as one day) (Wilson CT a/., 1970b). Also, in a study of five Glycine species the most salt-tolerant species had an increase in [P]ledl of 5% to 7% (at two harvest dates), while four species with greater salt sensitivity averaged decreases in [PIl,,, of 7%. to 20% (Wilson rt al.. 1970a). However elsewhere, of 22 accessions of G. wigktii the four most salt tolerant had the lowest decreases in [P]slloL,, (Gates et d.,1970). In that study all accessions decreased in although grown with 1 mM PI.Four genotypes of C. i i z u clearly segregated into two accumulating high and two accumulating only moderate P in their leaves during salinization when grown at 0.3 mM IPInledlum (either 600 or 300 nrnol (gDW)-l, Grattan and Maas, 1984). The two with higher [P].IIo,,,were also 15% more inhibited in growth than was the latter at this highest [P]mcdiu,,,. In four other G. mar lines the two genotypes which accumulated high [P]~llo,,i, were also almost twice as inhibited in growth as the more sensitive lines (Grattan and Maas, 1985). However, at low IP]ll,edi,i,nthe more sensitive lines neither attained higher (PIshoo,nor exhibited greater inhibition. The most salt-sensitive 2. tnoys genotype also had the greatest increase in [P]hlloo,with 0.17 mM [P]llledlum (Maas and Grieve, 1987). whereas in Agropyron ehngarum the most tolerant lines accumulated the highest [P]shou,(Shannon, 1978). In both G. Iiirsutunz and L. sativm short-term transport of ."P to young tissues and shoot apices were reduced by salinization (Martinez and Liiuchli, 1991; Martinez ef d., 1996). Decreases in young tissue [PI (or that of recirculating P) can even occur while P accumulation has been increased in the whole shoot (Jeschke et al., 1986). Similarly, in L. s u f i w grown with P, at 0.09 mM, salinization led to increascd [P]l,,l in the bulk shoot and most of the leaves, but to reductions averaging 9% of in the three youngest leaves (Fig. 5). This was a modest decrease in the youngest leaves, especially considering the much greater decrease in [S]lcaf.These data suggest that there might be ;L specific effect of salinization on the supply of P to

160


7

8

9

10

11

12

13

Leaf number (younger-

14

1

Fig. 5 . Concentrations of P and S in leaves numbered 7 to 14 of salinized L. surivu 18 days after transferring seedlings to solution culture. Treatments, means and errors are as for Fig. 2.

young shoot tissue (section V1.B. 1). Rather than contradicting the ‘P toxicity’ concept, it is possible that a NaCI-induced P deficiency in young shoot tissue is physiologically linked with excessive [P]hhOO,. Perhaps a localized P deficiency within the growing zones elicits a signal to the root calling for increased uptake of P or increased allocation to the shoot. And, perhaps, at least in the case of phosphorus nutrition, this regulation at the shoot meristematic zones is not affected by ‘toxic’ P levels in the largest transpirational sinks. In general, the results of salinity effects on total [P]ahoo,have been highly variable and will need a good deal of careful experimentation with consideration of effects in growing tissue specifically, before anything of substantial value can be derived relative to the shoot growth inhibition.

E. NITROGEN

There has been only modest consideration of a possible role for N in the salt-induced shoot growth inhibition. Many studies treating salinity effects on N uptake and metabolism have been focused on uptake effects at the root plasma membrane, following the hypothesis of protein release from the root plasma membrane by osmotic shock (e.g. Klobus er al., 1988). This hypothesis is not relevant to the focus of the present review on nutrition of the growing shoot. Other studies of plant N nutrition and salinity have focused on effects of particular N sources on the shoot growth inhibition and on whole plant N uptake. Apparently NH: nutrition, may enhance the shoot growth inhibition (Bourgeais-Chaillou and Perez-Alfocea, 1992; Speer et al., 1994; Speer and Kaiser, 1994),however this may sometimes be an effect of amendment with NH4CI in particular (Speer et al., 1994). Alternatively, the enhanced growth inhibition under NH; nutrition, may be related to the dark period recirculation of NH: to younger shoot tissues (Ouny at al.,


161

1996), or a specific salinity effect on recirculation (section VI). Although unrelated to salinity stress, a recent review of NO; effects on plant development may prove valuable to any reader pursuing an interest in N effects in young and meristematic shoot tissues (McIntyre, 1997). In the more salt sensitive of two Glycine species [N] of young leaves decreased much more than did the [N] of the same leaves in its more tolerant relative (Wilson et d . ,1970b). This occurred, however, only at the most severe level of salinization (160 mM NaCI). In another study of five GIycine species the most salt-tolerant species had a reduction in [N]plu,,t of 2 I %, which was greater than the reduction in any of the other five, which together averaged a 12% reduction (Wilson et al.. 1970a). Of 22 accessions of Glycine wighrii the [N] in the four most salt-sensitive decreased 47%, whereas the four most tolerant averaged only a 21% reduction after 2 weeks of salinization (SAR = 68, Gates ef id., 1966). In two Citrus species the more salt tolerant had only a 8% reduction in IN]lra,, whereas the more salt sensitive had 26% reduction after four weeks of salinization and this occurred despite the much greater reduced leaf mass (18% vs. 3%) of the more sensitive species ( 1: 1 NH: to NO; supply, Lea-Cox, 1993). Interestingly, in the new shoot tissue the IN1 was only reduced in the sensitive species ( 3 I %), whereas it increased in the more tolerant by 27%. In 2. riinys salinity had no effect on [NIlelr.,however in salt-sensitive varieties of T aestivum and H. iwlgare lower levels of NOj supply (2 mM) led to enhanced reductions of grain yield due to salinization (Bernstein et al., 1974). There is also some evidence in conflict with the concept of NaCl disturbed N nutrition. In six vegetable crops the [N],hr,l,,was not reduced by salinization, but actually increased for the lowest levels of N supply in four of the crops (Bernstein et d.,1974). Reductions in N supply to the youngest leaves and to the apex (30%and 32%) in moderately salinized L. d b u s were only proportional to the reduction in NO; uptake (Jeschke ef al., 1992). Under similar salinity conditions R. rnmmunis had exactly the same reduction in NO, uptake as salinized L. alhus (32%), however net N shoot accumulation was more than twice as reduced in Ricinus and this was even greater (with a 75% reduction) for NH: fed plants (Peuke et al., 1996). Unfortunately, the earlier work from this same laboratory with great detail on N flow in the shoot of salinized R. commurzis did not attempt to separate ontogenetic and salt treatment effects (Jeschke and Pate, 1991b, 1992). The paucity of work on N nutrition and salt-induced shoot growth inhibition is astonishing given the importance of N in growth and development. Certainly some of this poverty is due to the lack of an easily handled radioisotope. The use of I3N may be especially prohibitive for studies involving dissection of the shoot and isolation of the growing regions, however application of stable isotope "N techniques has been made with root dissection (e.g. Lazof ef ol., 1992) and this should be relatively straightforward to apply to the plant shoot. In what seems to have been the only study of salinity effects on N transport to very young shoot tissue, it was reported that a moderate salinization did not decrease N deposition rates in any region (5 mm zones) of a wheat leaf, although growth effects were uncertain (Hu and Schmidhalter, 1997).

162

D. 9. LAZOF and N. BERNSTEIN F. MICRONUTRIENTS

With the exception of iron, there has been slow progress in the understanding of micronutrient nutrition in recent years (Kochian, 1991). Very few studies of micronutrients have been related to environmental stresses, other than that of micronutrient levels. Some of the recent work with iron has highlighted the necessity for recirculation of the nutrient within the shoot for transport towards young tissues (e.g. Zhang et d.,1996). This work may imply some restriction of iron nutrition of young tissues, if the hypothesis of inhibited recirculation and phloem transport has merit (section VI). A report that supplemental Mn could ameliorate the NaCIinduced inhibition of growth in H. vu1,gm-e seems to be unique in suggesting that a salt-induced growth inhibition could be ameliorated by addition of a micronutrient (Cramer and Novak, 1992). The work was preceded by evidence that [MnIshoo,in H . vulgctre decreased due to salinization (Cramer el nl., 1991). However, both reports suffer from serious flaws. In the first place, there was no clear statistical difference shown for [Mn]a,,oo,even in the most extreme treatment (>300 Na:Ca), although a trend towards reduction of [Mnlahoo1 was apparent for 2 out of 7 sampling dates. In the more moderate salinization (125 mM NaCl with 10 mM Ca) there was even less indication of a trend for reduced [Mn]bhUI)l occurring at any level of salinization (Cramer et al., 1991). Secondly, beyond the results for [Mn],h,,,, only analysis of [Ca]sh,,,,land total cation concentration were presented. The more detailed work on salinity amelioration by Mn, was conducted with a moderate salinization and showed that the mass of salinized plants could be doubled with either a few micromolar Mn added to the nutrient solution, or a few millimolar added as a foliar application (Cramer er al., 1991). Reductions in plant mass due to salinization were still 68% and 6.5% (nutrient solution and foliar treatments, respectively) with the amended Mn, as opposed to reductions of 84% and 79% without amended Mn. Under the conditions of the later study 25% of the growth inhibition (biomass attained after 23 days) was recovered by amending with Mn. There have been a few reports that plants evolved for optimal growth in saline environments may also be especially effective in excluding heavy metals, including the micronutrients Cu, Ni and Zn, when these appear in soils at high concentration (Fernandes and Henriques, 199I ; Otte et al., 1993). Logically, there is some reason to suspect that there might be an association of enhanced micronutrient regulation with salt tolerance, since retranslocation in the shoot and mobility through the phloem may be both particularly affected by salinity and crucially restricted for several micronutrients (Fe, Cu. Zn and Mn, Bukovac and Wittwer, 1957).

VIII. THE STUDY OF NUTRIENT STATUS AND TRANSPORT ON THE MICROSCALE It has been argued in the preceding pages that immediate causes of growth inhibition are best sought within the minute tissue volumes in which growth


163

processes are most intensive. In the case of the disturbed nutrition hypothesis this would involve chemical analysis of nutrients and ions, and quantification of their deposition rates within precisely defined dividing and expanding zones, as well as compartmentation and transport studies such as net ion flux, isotope flux studies and recirculation studies. All of these would have to be adapted to the microscale. in order to associate alterations directly with effects o n growth. Even among researchers who have assumed that ‘ion excess’ (Na and CI levels) were key parameters in understanding salinization effects growth, it has long been recognized that compartmentation of ions was of prime importance, although the scarcity of appropriate methods has often been lamented. For example, Ye0 ( I98 I ) summarized that the only two existing methods for looking at ion compartinentation, at that time, were EPXMA and compartmental analysis by efflux. Only the former of these two methods inspires the least confidence and will be discussed in any detail. As for compartmental analysis by efflux, the assumptions and limitations have been detailed previously from mathematical and physical perspectives (Cheeseman, 1986). That critique contained precise determinations of the inherent statistical limitations, even under ideal conditions. Little serious defence of the method has appeared during the subsequent 10 years. Of greater interest are the emerging methods for the study of nutrient status and transport at the microscale. Two of these methods are discussed in some detail below. Kinematic growth analysis quantifies tissue growth intensities along the protile of elongating tissue, such as a growing monocot leaf. The method allows examination of: ( I ) correlations between localized intensities of tissue growth and growth inhibition under stress and ( 2 ) localized levels and deposition rates of nutrients and other factors suspected to take part in the growth inhibition or maintenance processes. Indirectly, the method can also suggest alterations in the compartmentation of elements. since cells become progressively dominated by the cytoplasmic component as sampling approaches the leaf base. Secondary ion mass spectrometry (SIMS) is an isotope sensitive microanalytical method which is being used to directly determine localized isotope enrichment patterns and so to investigate alterations in nutrient compartmentation and nutrient flux through tissue on a cellular scale. In this case the results are obtained through direct imaging (isotopes are released from the first few nanometres of surface) of freeze-dried cryosections following short-term labelling and freeze fixation.

A.

KINEMATIC GROWTH ANALYSIS A N D ELEMENTAL DEPOSITION RATES

The study of elemental distribution profiles of elements in growth zones of graminaceous leaves is a powerful method allowing discrimination of those alterations in ion composition which are most closely associated with growth. Several such studies have been discussed in earlier sections of this review (Bernstein et d., 1993a, 1995; Hu and Schmidhalter, 1997). Several other instances of applying the method to study the effects of environmental stress, or to study the

164


deposition of factors other than minerals can also be found in the recent literature (e.g. Schnyder and Nelson, 1987, 1988; Beemster and Made, 1996; Dodd and Davies, 1996). The first step in such an analysis is a kinematic analysis of growth. Growth kinematics analysis (GKA) allows quantitative measurements of parameters reflecting cell division and cell expansion specifically in unidirectionally growing organs such as developing graminaceous leaves. Although the growth zone of the grass leaf is concealed inside the whorl of older leaf sheaths and therefore not readily accessible for direct measurements, it is well suited for the study of leaf growth processes because of the relatively simple organization of its elongation zone (Volenec and Nelson, 1981; Schnyder and Nelson, 1988) and its distinct location. The existence of a ‘development gradient’ in the graminaceous leaf has long been employed to advantage in physiological studies. Less often specified are the temporal aspects implied by the spatial patterns. If growth is steady, then the ‘growth trajectory’ (a plot of distance from the leaf basal meristem which a particle traverses over time) allows inference of the time course of developmental properties (Silk and Erickson, 1979; Gandar, 1983). These processes include the accumulation of energy and mineral resources. A deposition rate is a useful characterization that combines experimental measurements of the spatial distribution of growth velocity and resource concentrations within the leaf growth zone. The observed concentration of a substance in growing tissue is, of course, the result of several processes. Local metabolism and transport can increase concentration, whereas growth-associated water uptake (often called ‘growth dilution’) can decrease it. The relative contributions of deposition and growth to the concentration profile can be evaluated with a onedimensional form of the continuity equation of fluid dynamics (Silk and Erickson, 1979; Silk, 1984). This equation provides the basis for evaluation of physiological factors suspected to be important in growth maintenance and inhibition (i.e. organic osmolites, carbohydrates, proteins, wall-loosening enzymes, solutes, etc.). Studies of S. hicolor leaves have revealed that [K], [Mg] and [Ca] in the growth zones of salt-stressed leaves were lower than in leaves of control plants and so was their deposition rate (sections 1.V.A-1.V.C). This occurred despite the lower expansion rates and the longer period of time that an elongating cell remains within the elongation region. The approach, however, may not be conclusive enough to determine a role in affecting a specific growth process (cell division or extension), nor may it be applicable to dicots, nor universally to monocots. The technique does not yield information on cellular compartmentation (thus, ‘microscale’, rather than microanalytic). After completion of such a study it remains essential to investigate subcellular concentration profiles along the expanding region and how these may be altered by stress, before any conclusion can be drawn about inhibition of a growth process. Microlocalization of several mineral elements has been performed by collection of vacuolar sap of growing cells of a S. bicolor leaf throughout the elongation zone (N. Bernstein, D. Tomos and W. Fricke, unpublished). This was done using a modified pressure probe sampler (Malone et al., 1989), as earlier reported in

165


TABLE 111

Effect of salinization on unicellular vacuolar sap concentrations oj expanding cells in the Sorghum bicolor leaf growing zone Distance from leaf base (cm) 1.5

3.5

Element

Control

Salt

Control

Salt

Na CI K

36 t 8 I1 + 16

91 +- 18 141 ? 31 130 ? 23

35 2 10 12+ 1 93+ I

90 2 6 141 30 112 ? 42

9 6 2 10

+

Leaf 6 was sampled on the first day after emergence above the whorl of older leaf sheaths ( 5 days after salinization). Plants were grown in I or 100 m M NaCI.

non-growing leaf tissue (Fricke ef nl., 1996). Ion concentrations in subsamples of unicellular sap were then determined by EPXMA. Vacuolar sap concentration in two locations (1.5 and 3.5 cm from the leaf base) are presented in Table 111. The length of the elongation zone of sorghum leaves is 3.0 and 2.4cm in the non-stressed and salt-stressed tissue, respectively (Bernstein et al., 1993a). Cells located 3.5 cm from the base are therefore young cells which have just ceased elongation growth. At location 1.5 cm from the leaf base, local growth intensity (extension per unit length) is at its maximum with a 35% reduction in local growth rate induced by the salinization. Vacuolar "a], [Cl] and [K] increased with salinization at both locations (Table 111). The increase at the 1.5 cm location was similar to that at the end of the growing zone. Together with published results indicating that in the bulk tissue of the 1.5 cm zone [Na] and [K] were higher than at the terminus of the growth zone (Bernstein et nl., 1995). the vacuolar data suggest that cytoplasmic and/or cell wall concentrations Na and K may be lower in the zone of most intensive expansion. Conclusions regarding deposition of elements into specific cell compartments and the effects of changes in such deposition rates which might affect growth under salt stress, will require microanalysis, or sampling of cell compartments other than the vacuole. A final step in combining kinematic growth analysis. elemental distribution profiles, and cell compartmental data would be determination of cell compartmentation along the leaf profile (i.e. morphometric analysis). Digital images of cross-sections throughout the growing zone of salt stressed and control maize leaves were acquired and relative volume fractions for wall, cytoplasm and vacuole determined (Bernstein, Neves Piastun, Yeo, Flowers and Thorpe, unpublished). As expected, the relative volume fraction of the vacuoles increases between the same two developmental locations described above (Table IV). Salinization led to a decreased relative volume of the vacuole in both locations, the reduction being larger in the younger cells (16% vs. 9%), whereas the cytoplasmic and cell wall volume fraction both increased. Since the cells of the stressed leaf, mature closer to the leaf base than in control leaves and their

166


TABLE IV Morphometric analysis of expanding Iecrf tissue of Z. mays

Distance from leaf base (cm) 2.3

I .3

Control .

Salt

Control

Salt

0.61 i- 0.03 0.39 ? 0.03 0.41 2 0.06

0.82 ? 0.01 O.l8? 0.01 0.34 ? 0.17

0.76 ? 0.01 0.24 2 0.01 0.36 ? 0.014

.-.-

0.73 -+ 0.02 Vacuole 0.27 ? 0.02 Cytoplasm Cell wall X lo3 0.38 2 0.02

Leaf was sampled 4 days after plants were salinized to 100 mM NaCl (salt), or left at 1 mM NaCl (control).Errors are standard errors of the mean (n = 30).

displacement from the base is slower, then at each location the salt-stressed cells are more developed. The reduced relative vacuolar fraction in the salt-stressed leaf is opposite to that which would be expected on the basis of development and certainly affects compartmentation of the tissue. B. MICRODISSECTION

Microdissection is an approach applicable to both dicots and monocots, including the less accessible growth zones such as the graminaceous SAM. A cryostat equipped with a trimming tool could be used to dissect and isolate structures of interest such as leaf primordia and shoot meristems of defined dimension. Any such operation should be conducted only with frozen samples and under a light microscope, possibly employing micromanipulators for the dissection. Combination of this approach with a strictly microanalytic approach would allow the combination of radioisotope techniques, cellular compartmentation, improved quantitation of microanalytic imaging methods. as well as isolation of small zones of interest for preparation towards microanalysis, which are later cryosectioned or cryofractured. The method suggested here should not be confused with relatively crude attempts at dissection performed without the aid of a microscope, on fresh, injured, hand-held tissues at room temperature with disregard for current microtechniques and cryological principles. Although such studies can be found in the recent literature, they are easily identified by the lack of precise definition, both developmentally and dimensionally, of what has been isolated and analysed, as well as by the absence of any details on the conditions during dissection. C. SPECIMEN PREPARATION CONSIDERATIONS

With any microanalytical technique, the method of tissue preparation is of paramount importance. Artefacts introduced from the redistribution of analytes invalidate any interpretation. Specimen preparation can also influence the limits of


167

spatial resolution, background noise levels, signal suppression and other factors depending on the particular microanalytical method. There is little basis for interpretation of intracellular localization in samples which are chemically fixed and embedded in plastic. Fortunately, reports of such methods for ion microlocalization research seem to be published only rarely. Partial chemical methods, however, continue to be used. including freeze substitution protocols. In this method, the tissue is freeze fixed, but cell and apoplastic water are replaced with organic solvent, while the tissue supposedly retains the ionic species of interest within the respective cellular compartments (e.g. Harvey et nl., 1981; Hajibagheri et nl., 1987; Flowers et al., 1991). Serious challenges to this idea have been detailed elsewhere (Morgan et al., 1975; Morgan, 1980; Chandra and Morrison, 1992; Zierold, 1992; Steizer and Lehman, 1993; Chandra and Morrison, 1995). Such preparation methods are still widely practised for ion microlocalization, despite the criticisms and controversies and the availability of more purely cryological alternatives. More complete explorations of the controversies and underlying principles can be found in the literature (Morgan, 1980; Echlin, 1992). Although a pure cryological preparation, determination in freeze dried tissue blocks is also of limited value. Protoplasm from adjacent cells dries against each cell wall making any determination of cellular compartmentation unlikely (Echlin and Taylor, 1986). Furthermore, depending on the particular method employed, the volume of specimenheam interaction might be quite large (section VII1.D). Both of these difficulties might be largely overcome by cryosectioning quench frozen specimens and freeze drying the cryosection (section VII1.D). Such an approach might be followed for either EPXMA or SIMS. for example. A far superior method for EPXMA analysis in almost every respect is the analysis of frozen hydrated specimens, especially with regard to the virtual elimination of any opportunities for analyte redistribution. D. ELECTRON PROBE X-RAY MICROANALYSIS (EPXMA)

EPXMA has been used to monitor nutrient levels in meristematic cells and in young leaves (Storey et af.,1983; Seeniann and Critchley, 1985; Lazofand Lauchli, 199lb). Although such investigation is still infrequent, EPXMA undoubtedly has valuable contributions to make in investigating nutrient relations of young and meristematic tissue. The chief advantages of the technique, especially as practised with frozen hydrated cryofractured (FHC) samples, are that with proper standardization it allows quantitative estimates of nutrient concentrations on a microscale, at times allowing subcellular compartments to be analysed directly. Together with proper quench freezing, cryofracturing and transfer equipment the FHC EPXMA system allows analysis of tissue preserved nearly in the in v i w state, although several crucial limitations need be kept in mind. Chief among these is the fact that EPXMA is not strictly a surface analytical method, although the exact depth of analysis will vary with the substrate density and accelerating voltage of the primary beam.

168

D. B . LAZOF and N. BERNSTEIN

The volume of specimenheam interaction (the volume of tissue from which the characteristic elemental signal originates) for EPXMA is approximated by a teardrop 2 M O pm in diameter in a block of freeze-dried plant tissue (Goldstein er al., 1981; Lazof et al., 1996a). The nanometre resolution so impressive in the secondary electron image (SEI) bears only an imprecise relation to the size of dots an ‘X-ray dot map’, or the pattern dots superimposed over the SEI. Improvement in lateral resolution results when the samples are analysed frozen hydrated, rather than freeze dried, as increased specimen density leads to both a shallower penetration of the primary electron beam and a much shorter distance over which characteristic X-rays can escape from the sample (tenfold improvement in resolution, Boekestein and Stolo, 1980; Echlin and Taylor, 1986). The greatest lateral resolution in EPXMA can be obtained with ultrathin sections and when these remain frozen hydrated for analysis artefactual redistribution can also be avoided. However, obtaining ultrathin botanical cryosections remains impracticable (Michel and Hillmann, 1990; Stelzer and Lehman, 1993). A second major limitation of EPXMA, especially with the superior preparation, FHC EPXMA, is that concentrations must usually be in the millimolar range for any estimation of concentration to be made (Lazof and Lauchli, 1991a). This high detection limit means that use of an elemental analogue as a tracer (e.g. S?‘ for Ca”), although theoretically allowing detection by EPXMA, will not provide sufficient instrumental sensitivity for short-term studies. Furthermore, for particular elements there may be severe peak overlaps in EDS EPXMA. Of particular importance with regard to the material of this review, a severe peak overlap occurs between the major Ca peak (Ca ka) and the secondary K peak (K kp, Lazof and Lauchli, 1991 #2038). Although spectra can be corrected for the peak overlap, this would be more difficult for a characteristic X-ray map, requiring correction at each pixel (Lazof and Lauchli, 1991a). Use of a wavelength dispersive detector may also obviate this difficulty. With respect to sensitivity there can be great advantage in use of freeze-dried specimens with the resultant sampling of much greater tissue volumes, possibly complementing freeze-dried analysis to FHC EPXMA (Lazof and Lauchli, 1991a). Many FHC EPXMA studies with relevance to salinity, but irrelevant to the issue of shoot growth zones, have not been included in this review. The interested reader might begin with the review by Stelzer and Lehman (1993).

E.

SECONDARY ION MASS SPECTROMETRY (SIMS)

Biological applications of SIMS are recent, as the technique has been mainly limited to applications in materials sciences. Active programs in biological SIMS exist in France, Germany, Japan and the US. Both of the US laboratories with on-going active in situ biological SIMS programs insist on cryological preparation and exclude even freeze-substitution as inappropriate for preparing specimens to be analysed at the high spatial resolution and sensitivity of which SIMS is capable (Chandra et al., 1992b; Lazof et al., 1994b). The major disadvantage of using


169

freeze-dried cryosections is the opportunity for subcellular redistribution during freeze-drying. however movement of solutes and protoplasm will be primarily downward against the underlying substrate for sections of several micrometre fresh thickness (Waisel et nl., 1970; Lazof et al., 1994b, 1996a,c). Recently, SIMS has been applied to localization studies of aluminium in plant roots (Lazof et al., 1994a,b, 1996c) and to nutrient tracer studies in plant physiology (Goldsmith et al., 1996). Sophisticated short-term SIMS experiments involving 44Ca'+ labelling of either animal cell culture, or whole organ within a living organism have been carried out (Chandra et al., 1990, 1992a). Doublelabelling SIMS experiments with "Ca2"' and r6Mg2' have been conducted with intact roots of corn (Plate 2), and additional technical details can be found in recent reviews (e.g. Chandra and Morrison, 1995; Lazof er nl., 1995, 1996a). Transport of Ca". and Mg2* was followed in 400 pm longitudinal root sections of corn. Intact roots were exposed to the tracers 44Ca2+and 26Mg2' for times ranging from 0.5 to 5.5 h. Directly determined isotope distributions are shown in Plate 2 for the tracers and the two naturally dominant isotopes, 40Ca2+and 24Mg2t, following the 0.5 h exposure. Enrichment of the tracers after 30 min exposure to the tracer isotopes was quantitatively determined within defined regions of the 0.4 mm root tip from images acquired from replicate roots, and was found to be lowest in the most apical regions (Lazof, unpublished). Kinetic analysis of average enrichment in the 250 pm root tip demonstrated a more rapid exchange of 44Ca2' than 2sMg2t 3 h respectively, Fig. 6). The kinetic and distributional studies together suggested that 44Ca' was transported into the 400 pm tip from more basal regions of the root. Mass spectra collected in the SIMS instrument indicated that there were no serious limitations due to mass interferences during collection of the Ca and Mg isotope images (major potential interferences were separated by 0.03 atomic mass units). The j4Cari~and "MgZt were already enriched 30- and 2.5-fold, respectively, after 30 min in the tracer solution over levels in unlabelled depleted tissue. In cryofractured cell cultures exchangeable Ca pools could be resolved to Golgi (Chandra et al., 1994). These results demonstrate a powerful new approach to in siru elemental microanalysis and short-term nutrient transport studies at the tissue and cell level. The root images shown here were collected with the instrument in 'ion microprobe' rather than in the 'ion microscope' mode, although the latter was used earlier in A1 toxicity studies (see Lazof et al., 1996a). In this mode of operation the linear detection range for incoming mass signals is increased a further 4 orders of magnitude up to lo9 counts per second. The primary beam current can be turned up allowing detection of lower levels of isotope or trace metal, without resulting in an off-scale mass signal for more abundant isotopes being ratioed. Lateral resolution in ion microprobe mode is 0.5 pm, perhaps less, on a perfect sample, but in practice is largely a function of specimen preparation (Lazof et al., 3996b). Detection limits for Na, K, Rb, Ca and Mg are all favourable due to high ion yield and limited mass interferences. The five elements listed should be detectable in freeze-dried cryosections within the range of pmol (gFW)-', on the basis of published factors +

170

D. B. LAZOF and N. BERNSTEIN c

C

E

f

.-o 30 L

C

w

El0

a,

2 a, a

0

1

2

3

4

5

6

Time (hours) Fig. 6. Tinie dependency of average enrichment for 44Caand '"Mg in the apical 250 Fni of corn root tips, grown in a complete nutrient solution with Ca and Mg concentrations of 0.2 and 0.4 mM. respectively. The roots were depleted to less than 0.2% AE for both isotopes prior to the labelling period. The isotope composition for Ca and Mg was the only variation between growth, depletion and labelling solutions. The enrichment values were determined by SIMS.

of relative sensitivity and limits already determined in plant cryosections (Ramseyer and Morrison, 1983; Ausserer et al., 1989; Lazof et al., 1996a). The great potential SIMS applications in plant physiology have been assessed more broadly elsewhere (Lazof et ul,, 1992).

F. SOME OTHER MICROANALYTICAL TECHNIQUES

Fluorescence has been used for monitoring fluctuations in cytosolic and vacuolar Ca' ', The technique can be applied to thick sections of tissue in a confocal system, allowing in vivo analysis in some specimens. Apparently, there has not yet been a study of Ca2+ relations within shoot meristems, or very young shoot tissues using fluorescent techniques. A primary limitation of fluorescence relative to the issues under discussion is the inability of the technique to discriminate isotopes and therefore to shed any light on issues of short term in siru ion transport. Beyond this, there are limitations to confocal fluorescent microscopy: ( I ) the technique is rarely quantitative, since numerous assumptions about quenching of the fluorescent signal, competitive binding and background need to be made in each cell phase; ( 2 ) there are depth limitations and it is necessary for excising a tissue such as a shoot menstem (Sandison et id., 1994), whereas frozen specimens are unsuitable. Microautoradiography is another potentially very useful microanalytical technique. It has been only sparingly applied to major issues in plant physiology and apparently never to nutrient transport to shoot meristems. This is probably due to the difficulty and time-consuming nature of the method. Certainly, excellent microautoradiography has been carried out on plant tissues (e.g. "Ca' , Luttge and


171

Weigl, 1962). It should be noted, however, that even though 45Ca2+is a relatively good isotope for this purpose the average track length is 230 pm through Ilford G5 (Lazof et al., 1992). Double-labelling experiments are extremely difficult, worse yet are studies of elements which do not have suitable radioisotopes (e.g. Mg).

IX. SUMMARY AND FUTURE PROSPECTS A.

REASSESSMENT OF CURRENT STATUS

Out of necessity physiologists and breeders tend to cast the most favourable light on the practical achievements and outcomes of their work. In a recent review of breeding for salt tolerance it was claimed that ‘remarkable achievements have been made in improving different agronomic traits through artificial selection during the past’ and that with regard to salt tolerance, in particular there has been great achievement in the breeding of alfalfa, barley, rice, pearl millet, maize, sorghum and other grasses (Ashraf, 1994). The optimism expressed in this particular review was not unusual. However, a greater measure of scepticism might be more helpful in providing a framework for future research needs. In examining the six crops and 17 improved lines in this review little evidence was found of remarkable achievements (Table V). Admittedly a multitude of criteria and methods have been used in the current literature to evaluate salt tolerance and the measure used here would not be universally the most relevant. However, the inhibition of vegetative shoot growth is certainly one valid measure and usually results in increased seed production for grain crops. Critical assessments need to be carried out with regard to additional criteria. If anyone working in salinity research were to take a few dozen salinity research articles from their files more or less randomly (as we have done) they would find that in the introductions, at least 75% mention the necessity for research based on the encroaching salinization of agricultural land or the increasing scarcity of high quality water for irrigation. This framework, however, is rarely applied to evaluation of the significance of breeding programmes. For only two of three legume crops was there even a minor improvement in the range of salinity tolerance, whereas for the third, M. sarivu, there was a decrease when plotted as a percentage of control growth (Fig. 7, Ashraf et ul., 1986; Ashraf, 1994). For the ‘hypothetical crop’ a case is shown wherein selection has resulted in a significant increase in the range of salinity tolerated by the crop species (70% increase, from 83 to 142mM NaCl). Indeed greater variations have often been found between genotypes of a crop species, as for example that reported in wheat, where the increase in salinity level could be estimated as severalfold (Table V, Qureshi et ul., 1980). This variation, however, is usually found among existing cultivars and reflects ‘natural’ variation rather than any achievement of breeding. The data for the four grass species was similar (Ashraf, 1994). In order to reduce growth by 50% in the four lines selected as tolerant, an additional 28,38, 18 or 0 mM NaCl was added

172

D.B. LAZOF and N. BERNSTEIN TABLE V The improvement of salt-tolerance as reported in 13 articles for 10 crops

Crop

Reference

~

Dobrenz et al. (1983)

Alfafa

No

Noble et al. (1984)

Alfafa

No

Ashraf et al. (1987)

Alfafa, two clover spp., rape

No

Epstein and Norlyn

Barley

No

Akbar (1986)

Rice

No

Rana ( 1977)

Wheat

No

Francois et al. (1984)

Sorghum

Yes

Rush and Epstein (1981a)

Tomato

Yes

Qureshi et a/. (1980)

Wheat

Yes

Kingsbury and Epstein

Wheat

Yes

( 1977)

(1984)

Comments'

Range"

~

~

~~~

..

Improved line has 7.5% increase in forage over the unselected. Fls with 'some improvement' in numbers of leaves damaged at 250 m M NaCI. Fls with improvements of 62, 35, 57 and 30% over most sensitive individuals of parent generation. No growth data and no comparison of grain yield for those lines selected for Na-tolerance. No data comparing any lines in any terms relevant to extent of improvement. No growth data. Comparison of available cultivars only (not bred for salinity-tolerance). Comparison of two cultivars. Not result of selection programme for salt-tolerance. All lines show fruit production reduced by 50% at lowest salt treatment (vegetative growth not shown). All 'improved' lines show less fruit production than the unimproved salt-sensitive cultivar at this salinity level. Selections from existing cultivars, not bred for improved salt-tolerance. By extrapolation 30% reduction in growth varied from an EC of 38 to 15 1 mmhos/cm. Constant Na to Ca. All extreme levels of salinity save one. No testing of progeny. Most resistant line only inhibited 59% at EC = 10.6, two developed lines improved but at EC = 10.6 inhibited 59 and 90%.


173

TABLE V Contd. Reference

Crop

Range’

Al-Khatib er 01. (1993)

Alfafa

Yes

Ashraf and McNeilly

Corn

Yes

(1990)

Commentsh

F1 improved up to 30% in terms of salinity which could be tolerated at 50% growth inhibition. Selected line improved up to 20% in salinity tolerated at 50% growth inhibition.

Salt-tolerant lines of crops developed either through selection or breeding. Whether a quantitative comparison o f vegetative growth for selected and non-selected genotypes over a range of salinities was included. ’Additional comments critical to judging the extent of the improvement in salt-tolerance.

over the level of NaCl which had inhibited the non-selected lines by 50% (15, 30, 14 and 0% increases in salinity). By comparison the natural variation among crops presented in the same article was eightfold. Many reports concerned with crop improvement for salt tolerance specify establishing whether salt tolerance is heritable as their main objective. Certainly most of the cited reports have indeed demonstrated that heritability exists for many species and, thus, that future breeding would seem to hold promise. However, the ability to withstand 40 rather than 30mM NaCl before a given level of growth reduction results may not much extend the cropping range or ability to use low quality irrigation water. All levels of inhibition do not serve equally well for comparison. Differences in maintaining high production (say 70% control growth) is more important than the point at which a genotype might decline from 80% inhibition to 95%. Although our analysis of the literature in this respect is incomplete, it suggests that if the literature were critically reviewed, then even after decades of salinity research one might continue to wonder about any extension in crop range with respect to salinity. B. MODEL SYSTEMS

All four general hypotheses (section LB), with the possible exception of the turgor/osmoregulation hypothesis, need to be considered within complex tissues and morphologies and within minute volumes of undifferentiated, rapidly dividing cells. Although each hypothesis suggests a particular mechanism for salinity induced growth inhibition, they involve integrated synthesis, partitioning, longdistance transport and regulation of metabolism. The processes are organismal and poorly modelled as ‘average’ cellular accumulations within a whole plant or whole shoot. If the osmoregulatory/turgor hypothesis is not of major major importance in genotypic salt tolerance (section I.B.2) and if the ‘ion excess’ hypothesis is to be


174 I

'

I

'

I

~

I

*

I

'

I

.

Trifolium alexandrinum

60

-

= 40

-

E

+

a-,

-

Q

c 20-

Selected

0

'

I

'

-

-.

__

0 Non-selected

Hypothetical crop -

Trifolium pratense

60

I

Medicago sativa

-

40-

20

-

1 I

0

,

I

50

,

I

100

,

I

150

"a]

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0

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I , , , , , , .I. , . I

50

. . , . * I ,

100

!.I

I

.

,

,

,

,

,

150

mot m-3

Fig. 7. The effect of salinization on growth of two lines of each of four crop species, one of each selected for salt tolerance. Data are replotted for three legumes as percentage change from the non-selected line (replotted from Ashraf et al., 1987). Also shown are data for a hypothetical crop which has been improved for salinity tolerance by 7 1%, measured as the level of salinity that can be tolerated while maintaining 70% of control growth. Dashed lines show differences in salinity that can be tolerated by the two lines of each species while maintaining 70% of control growth.

questioned (section I.C.1) in its most generalized form (applied to tissue status throughout the shoot), then the value of cell culture physiology and selection becomes highly suspect. The usefulness of testing salt tolerance of angiosperms in cultured undifferentiated cells (e.g. Blum, 1985; Niu et al., 1995; Kuznetsov and Shevyakova, 1997; Winicov and Shirzadegan, 1997) should be carefully reconsidered as a model for tolerance mechanisms. Similar recommendations have been made in previous reviews (Munns, 1993), apparently without any response, nor apparent effect on the course of research.

C. IN SITU ELEMENTAL AND ISOTOPIC ANALYSIS

Within almost every section of this review the dearth of relevant literature on the basic physiology of shoot growth and development as affected by salinization has been remarked upon. Studies of primordia formation, leaf initiation and direct analysis of cell division have not been conducted, despite a great deal of evidence that these are affected by salinity. Nutrient supply to growing leaf tissues


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(elongating regions of grass leaves or entire very small dicot leaves has been poorly studied, only to be outdone in rarity by data on nutrition of the meristematic zones. Vague developmental definition of organs and tissues from which samples have been collected and broad categories such as ‘young leaves’ and ‘growing leaves’ also severely limit meaningful interpretation. Development and utilization of modem microscale and microanalytical methods will involve precise definition of leaf development and size, the study and analysis of minute structures, the necessity of linking elemental and isotopic analyses within cells which are rapidly dividing and extending. Such physiological study is, in turn, requisite to understanding the processes contributing to growth inhibitions from environmental stress.

ACKNOWLEDGEMENTS The SIMS application to plant physiology and the work presented were achieved in collaboration with J. G. Goldsmith, R. W. Linton and G. Gillen (respectively of the Department of Chemistry, University of South Carolina, Aiken, South Carolina, 29801, USA; the Chemistry Department, University of North Carolina, Chapel Hill, N Carolina 27599 3290, USA; and the Surface and Microanalysis Science Division, Bldg. 222, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA). Support for the microanalytic approaches to plant salinity has been supplied by USDA CRCR 87 1 2462 and USDA NRICGP 96 35100 3245.

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Wilson, J. R. (1967). Response to salinity in Glycine 3. Dry matter accumulation in three Australian species and G. javanicu. Australian Journal of Experimental Agriculture and Animal Husbandy 7 , 50-56. Wilson, J. R., Haydock, K. P. and Robins, M. F. (1970a). Changes in the chemical composition of three Australian species of G. wightii (G. juvanica) over a range of salinity stresses. Australian Journal qf Experimental Agriculture and Animal Husbandry 10, 156-165. Wilson, J. R., Haydock, K. P. and Robins, M. E (1970b). The development in time of stress effects in two species of Clycine differing in sensitivity to salt. Australian Journal of Biological Science 23, 537-55 I . Winicov, I. and Shirzadegan, M. (1997). Tissue specific modulation of salt inducible gene expression: callus versus whole plant response in salt tolerant alfalfa. Physiologiu Plantarum 100, 314-319. Wolf, 0. and Jeschke, W. D. (1987). Modeling of sodium and potassium flows via phloem and xylem in the shoot of salt-stressed barley. Journul of Plunt Physiology 128. 371-386. Wolf, O., Munns, R.,Tonnet, M. L. and Jeschke, W. D. (1990). Concentrations and transport of solutes in xylem and phloem along the leaf axis of NaCI-treated Hordeum vulgare. Journul of Experimental Botativ 41, 1 130- 1 141. Yeo, A. ( 1983); Salinity resistance: -physiologies and prices. Physiologia Planfururn 58, 2 14-222. Yeo, A. R. (1981). Salt tolerance in the halophyte Siiaedu mnritimu L. Dum.: Intracellular cornpartmentation of ions. Journul of‘ Experimental Botany 2. 487497. Yeo, A. R. and Flowers, T. J. (1982). Accumulation and localisation of sodium ions within the shoots of rice (Oryza sutivu) varieties differing in salinity resistance. Physiologiu Plantarum 56, 343-348. Yeo, A. R. and Flowers, T. J. ( 1985). The absence of an effect of the NdCa ratio on sodium chloride uptake by rice (Oryza sativa L.). New Phytologist 99, 81-90. Yeo, A. R. and Flowers, T. J. (1986). Ion transport in Suaeda maritima: its relation to growth and implications for the pathway of radial transport of ions across the root. Journal of Experimental B O ~ U F37, Z ~ J143- 159. Yeo, A. R. and Flowers, T. J. (1989). Selection for physiological characters - examples from breeding for salt tolerance. h i “Plants under Stress” (H. G . Jone, T. J. Flowers and M. B. Jones, eds) pp. 217-234. Cambridge University Press, New York. Yeo, A. R., Caporn, S. J. M. and Flowers, T. J. (1985). The effect of salinity upon photosynthesis in rice (OQW sutiiw L.): gas exchange by individual leaves in relation to their salt content. Joirrnul of Experirnenral Botany 36, 1240-1248. Yeo. A. R., Lee, K. S., Izard, P., Boursier, P. J. and Flowers, T. J. (1991). Short and long term effects of salinity on leaf growth in rice (0ry:u sativa L.). Journal of Experimental Botany 42, 88 1-889. Zhang, C., Romheld V. and Marschner, H. (1996). Effect of primary leaves on 59fe uptake by roots and 59fe distribution in the shoot of iron sufficient and iron deficient bean (Phaseolus vulgaris L.) plants. Plant and Soil 182, 75-8 1. Zhang, J. and Davies, W. J. (1990). Does ABA in the xylem control the rate of leaf growth in soil dried maize and sunflower plants’? Journal (?f E.xperimental Botany 41, 1 125-1 132. Zierold, K. ( 1992). Comparison of cryopreparation techniques for electron probi microanalysis of cells as exemplified by animal erythrocytes. Scanning Microscopy 6, 1137-1 145.


AUTHOR INDEX Numbers in italic refer to pages on which full references are listed

A Abdel-Aal, M. M., 38, 50 Abdel-Hay, E A.. 38, 50 Abel, G. H., 119, 128, 175 Ackerson, R. C., 118, 175 Adams, R. M., 32, 35, 49 Agrawal, M., 44, 45, 49 Ahmad, R., 115, 171, 172, 186 Ahmed, D. M., 37, 38,50 Akbar, M., 172, 175 Akohoue, S., 119, 176 Alberico, G. J., 116, 117, 118, 120, 121, 122, 116, 130, 132, 175, 178 Alexander, M., 20, 28 Alien, S., 172, 179 Al-Khatib, M.,173, 175 Allen, G.J., 79, 83, 89, 104 Alpert, S., 115, 186 Altschmied, L., 68, 72 Amthor, J. S., 33, 49 Amtmann, A., 80, 83, 84, 86, 87, 88, 91, 92, 95, 97, 98, 99, 100, 102, 103, 104 Amundsen, R. G., 41, 51 Anderson, J. A., 84, 85, 99, 104, IOS, I10 Andersson. M. E., 9, 10, 25 Andre, R., 37, 38, 50 Aniol, A., 8, 26 Aragules, R., 130, 176 Arif, H., 11 8, I75 Armstrong, F., 99, 104 Arnon, D. I., 9, 26 Ashenden, T. W., 18,28 Ashmore, M. R., 33, 34, 36, 39, 40, 42, 43, 49, 50, 51, 52 Ashraf, M., 115, 120, 129, 130, 131, 158, 171, 172, 173, 175, 176 Aslam, Z., 115, 117, 124, 133, 134, 136. 139, 140, 158, 171, 172,176. 186 Assmann, S. M., 98, 99, 102, 106. 108, 112

Astruc, S., 84, 108 Aswathappa, N., 134, 176 Atkins, C. A., 136, 152, 153, 157, 158, 159,182 Atkinson, C. J., 16, 17, 26, 29 Ausserer, W. A., 168, 169, 170, 176,I77 Austin, R. B., 130, 176 Ayah, F., 100, 104 Azhar, F.M., 115, 176 Azimov, R. R., 86, 104

B Bachelard, E. P., 134, 276 Baldwin, I. T., 20, 26 Ball, M. C., 118, 124, 176, 186 Ballarin-Denti, A., 97, 105 Baluska, F., 133, 176 Barnbawale, 0. M., 40, 41, 49 Bangerth, F., 155, 156, 176 Banno, H., 58, 73 Bano, A., 118, 123, 176 Banuelos, G. S., 119, 176 Barkla, B. J., 79, 104 Barlow, E. W. R., 118, 176 Barlow, P. W., 133, 176 Barrett-Lennard. E. G., 117, 124, 133, 134, 136, 139, 140, 158,176 Barsky, T.,115, 186 Bartlett, R. J., 6, 8, 26 Barz, W., 58, 71 Basset, M., 84, 108 Batschauer, A,, 56, 70, 71 Beaton, C. D., 8, 26 Becker, D., 84, 85, 98, 99, 105, 107, I09 Beemster, G. T. S., 164, 176 Bell, J. N. B., 39, 40, 42, 43, 46, 49, 50, 52 Benes, S. E., 130, 176 Bennett, A. B., 100, 110 Bennett, R. J., 6, 8, 26. 27 Benton, J., 36, 49

192

AUTHOR INDEX

Ben-Zioni, A., 126, 176 Berestovskii, G.N., 86, 104 Bernstein, L., 91, 105, 128, 158, 159, 161, 176, 177 Bemstein, N., 124, 134, 137, 138, 139, 140, 141-2, 144, 145, 147, 149, 150, 154, 157, 158, 159, 163, 164, 165, 177, 183, 184 Bertekap, R. L., 54, 73 Bertl, A., 85, 86, 87, 99, 102, 105, 110 Bhatti, A. S., 152, 154, 182 Biddulph, O., 155, 177

Biddulph, S . , 155, 177 Binzel, M., 78, 79, 100, I05 Blatt, M. R., 85, 87, 98, 99, 102, 104, 105, 109, 111 Block, A., 60, 72 Bloebaum, P., 120, 178 Bloom, A. J., 119, 120, 187 Blum, A., 174, 177 Blumwald, E., 79, 104, 105 Boekestein, A., 168, 177 Bogner, W., 17, 18, 26 Bonetti, A., 79, 105 Bonneaud, N., 84, 110 Bonte, J., 33, 49 Booth, C. E., 34, 51 Bosseux, C., 83, 84, 85, 98, 111 Bostock, R. M., 63, 70 Botella, M. A., 120, 127, 177 Boucaud, J., 160, 186 Bourgeais-Chaillou, P., 160, 177 Boursier, P. J., 125, 135, 189 Bowler, C., 61, 62, 63, 68, 69, 70, 73 Bowling, D. J. F., 78, 97, 105 Bowman, D., 118, 121, 178 Boyer, J. S., 118, 147, 184, 185, 188 Bracker, C. A., 64, 73 Bradley, D.J., 65, 70 Bradshaw, A. D., 120, 129, 130, 131, 171, 172, 175, 176 Brady, C. J., 118, I76 Brauer, D. K., 8, 30 Braun, Y., 79, 100. 105 Breckle, S . W., 120, 127, 177 Bregante, M.,85, 98, 107 Bressan, R. A., 64, 73, 76, 77, 78, 79, 100, 105, 109, 110, 174, 185 Bridges, J., 128, 180 Broekman, R. A., 162, 185 Brouwer, R., 125, 177 Brown, B., 56, 68, 69, 71

Brown, P. H., 163, 181 Brown, R., 125, 188 Brown, V., 34, 49 Brown, V. C., 46, 49 Brownlee, C., 14, 28 Briidem, A., 98, 100, 111 Brune, A., 160, 188 Brunet, J., 9, 10, 25 Buckel, S., 64, 73 Bukovac, M.J., 155, 162, 177 Busch, H., 84, 85, 98, 99, 105. 107, 109 Bush, D. R., 81, 105 Bush, D. S., 14, 16, 26, 85, 105 Bytnerowicz, A., 41, 50 C Caetono-Anolles, G . , 20, 26 Cakirlar, H., 78, 97, 105 Calba, H., 6, 7, 18, 26 Calheiros, R., 37, 38, 50 Callander, B. A., 47, 50 Cameron, F. K., 127, 182 Cameron, R. S . , 5, 26 Campbell, A. K., 55, 68, 72 Canvin, D. T., 136, 182 Cao, Y.,84, 85, 111 Caporn, S. J. M., 117, 189 Caradus, J. R., 8, 26 Carre, I. A., 68, 72 Carter, C., 135, 136, 140, 148, 167, 188 Cashmore, A. R., 54, 73 Cerana, R., 84, 87, 105 Cerda, A., 120, 127, 177 Chameides, W. L., 47, 49 Chandra, S., 167, 168, 169, 170, 176, 177 Chaney, R. L., 24, 27 Chaney, W. R., 125, 182 Chang, P.-F., 64, 73 Chapin 111, F. S., 19, 26 Chauhan, Y. S., 115, 177 Cheeseman, J. M., 76, 80, 97, 105, 108, 115, 116, 119, 120, 121, 154, 163,

178. 183, 188 Chory, J., 55, 68, 69, 70, 72 Chrerel, I., 84, 106 Christiansen-Weniger, C., 9, 26 Christie, J. M., 54, 55, 59, 60, 65, 70, 71 Chua, N.-H., 54, 55, 61, 62, 63, 68, 69, 70, 72, 73 Claringbold, P. J., 161, 180 Clark, R. A., 128, 158, 159, 161, 176, 177, 185

AUTHOR INDEX

Clarkson, D. T., 5, 26, 123, 147, 178 Cocucci, M., 97, 105 Colavito, L., 41, 51 Collier, G. F., 146, 178 Colling, C., 65, 71 Collins, J. C., 173, 175 Colls, J . J., 34, 49 Colmer. T.D., 77, 108 Colombo, R., 79, 84, 87, 105 Conejero, G., 84, 108 Conti, F., 98, 107 Cory, R., 155, 177 Craig, S., 8, 26 Cramer, G. R., 97, 106, 116, 117, 118, 119, 121, 122, 123, 129, 130, 131, 132, 158, 162, 178, 185 Creelman, R. A., 63, 72 Critchley, C., 167, 187 Cuartero, J., 115, 178 Culligan, K., 68, 72 Curnniing, J. R., 33, 49 Curtis. A. V., 2. 13, 21, 27 Curtis, P. S., 124, 133, 134, 136, 138, 140, 141, 145, 178 Cutt, J. R., 54, 60, 64, 70 Cvitas, T., 38, SO Czempinski, K., 86, 88, 106

D Dahlgren, R. A., 20. 21, 28 Dale, J. E., 125, 178 Daram, P.,84. 106 Davenport, R. J., 100, 106, 120, 178 Davies, M. S., 22, 26, 123. 186 Davies, T. W., 167, 185 Davies, W. J., 118, 164, 179, 189 Dawson, P. J., 41, 50 de Bauer, L. I., 37, 38, 40, 41, 51 De Boer, A. H., 86, 88, 98, 99, 100. 106, 112

De Felice, J., 18, 27 De Silva, D. L. R., 16, 17, 26, 27 De Temmerrnan, L., 32, 35, 50 Delane, R., 115, 117. 118, 119, 121, 125, 134, 137, 140, 179, 185

Delhaize, E., 8, 9, 22. 24, 26. 30 Denby, K. J.. 58, 71 Denecke, I., 60, 61, 73 Deng, X.-W., 55, 69, 70 Derwent, R. G., 46, 50 DeWald, D. B., 63, 72 Dhindsa, R. S., 55, 73

193

Dias, P. L. S., 37, 38, 50 Dietrich, P., 84, 85, 98, 99, 107, 109 Ding, L., 81, 82, 106, 112 Dixon, R. A., 54, 55, 59, 60, 65, 68, 70, 72, 73 Djordevic, M. A., 20, 29 Dobrenz, A. K., 172, 179 Dodd, C., 164, 179 Dodd, W. A., 80, 106 Doering, H.W., 117, 186 Donovan, T., 158, 172, 179 Downton, W. J. S., 115, 117, 119, 120, 130, 131, 132, 158, 179, 187, 188

Dracup, M., 115, 179 Dreyer, I., 84, 85, 98, 105, 107 Dunn, M. A.. 54, 55, 56, 71 DuPont, F. M., 79, 106 Durand, M., 134, 136, 179 Dvorak, J., 119, 120, 187

E Eagle, H. E., 124, 128, 182 Eaton, F. M., 117, 128, 130, 179, 180 Echlin, P., 167, 168, 179, 180 Ecker, J . R., 68, 69, 72 Ehlig, C. F., 128, 176 Ehmann, B., 56, 70, 71 Ehret, D. L., 128, 131, 158, 179 Ehrhardt, T., 86, 88, 106 El-Bahnasaway, R. M., 37.49 Ellenberg, J., 84, 85, 98, 99, 109 Elzam, 0. E., 82, 106, 120. 129, 130. 132. 179

Elzenga, J. T. M., 83, 87, 90, 98, 106 Emmler, K., 56, 71 Enkoji, C., 120, 178 Epstein, E., 82, 97, 106, 115, 120, 129. 130, 131, 132, 158, 162, 172, 178, 179, 182, 185, 187 Erasmus, D. A., 167, 185 Ericson, R. 0.. 164. 187 Esau, K., 125, 147, 179 Eshel, A., 169, 188 Esser, J. E., 99, 108 Ezz El-Din, M. R. M.,37, 38.50

F Fairley-Grenot, K., 98, 99, 106 Falkengren-Grerup, U., 9, 27 Falkenmark, M., 115, 179 Fan, T. W.-M., 77, 108 Fang, H., 115, 186

194

AUTHOR INDEX

Fangmeier, A., 33, 49 Farag, S. A., 37, 49 Favelukes, G., 20, 26 Feldmann, K. A., 68, 69, 72 Felle, H. H., 81, I06 Fernandes, J. C., 162, 179 Fernndez, J. A,, 81, 82, 108 Fernandez, J. M., 83, I10 Fewtrell, C., 169, 177 Findlay, G. P., 78, 84, 86, 87, 89, 90, 91, 92, 95, 97, 98, 99, 100, 103, 106, 108, I l l Fiori, C., 168, 180 Fisher, D. B., 152, 153, 185 Flowers, T. J., 76, 78, 79, 91, 106, 107, 115, 117, 119, 120, 125, 127, 130, 135, 137, 151, 165, 167, 178, 179, 181, 189 Fluhr, R., 54, 71 Fortmeier, R., 130, 134, 138, 140, 141, 145, 179 Fox, D., 86, 109 Foy, C. D., 24, 27 Francis, D., 123, 186 Francois, L. E., 158, 159, 161, 172, 177. I 79 Freijsen, A. H. J., 115, 187 Fricke, W., 117, 118, 164, 165, 179, 180 Frohnmeyer, H., 54, 56, 50, 71 Fuglevand, G., 54, 5 5 , 56, 59, 65, 66, 67, 68, 69, 71 Fuhrer, J., 33, 34, 36, 49, 50, 51 Fujii, T., 79, Ill Fujiyama, H., 128, 181 Fullmer, C. S., 169, 177 Furuya, M., 56, 71 G Caber, R. F., 84, 85, 99, 104, 105, 109, I10 Gajon, A., 24, 30 Galaup, S., 33, 49 Galloway, J. N., 47, 50 Gambale, F., 85, 98, 107 Gandar, G. W., 164, 180 Garbarino, J., 79, 106 Garciadeblas, B., 78, 107 Gardner, P. A., 135, 149, 185 Garrill, A., 84, 86, 87, 89, 90, 91, 92, 95, 98,99, 100, 103, 106, 111 Gassmann, W.,5, 27, 81, 84, 85, 98, 106, 107, 110, I l l Gates, C . T., 159, 161, 180

Gauch, H. G., 117, 130, 180 Gaymard, F., 83, 84, 85, 98, 106, 108, 109, 110, 111 Gebauer, G., 17, 18, 27 Geiger, M.,24, 30 Geissler, P. A., 34, 49 Geletyuk, V. I., 86, 104 Gelli, A., 79, 105 Gibbs, J., 115, 117, 118, 119, 121, 125, 134, 137, 140, 179, 185 Gigon, A., 17, 18, 27 Gilmour, S. J., 55, 71 Gilroy, S., 5 , 6, 28 Gimeno, B.S., 36, 49 Giraudat, J., 55, 68, 71, 72 Glaab, J., 24, 30, 161, 186 Gleick, P. H., 115, 180 Glyer, I. D., 32, 35, 49 Goldsmith, J. G., 168, 169, 170, 180, 183 Goldsmith, M. H. M., 85, 86, 87, 99, 102, 108, 110 Goldstein, J. I., 168, 180 Gonzalez-Fontes,A., 24, 30 Goodman, H. M., 68, 71 Gorham, J., 76, 107. 128, 180 Grabov, A. M., 86,107 Gradmann, D., 80, 83, 85, 86, 87, 97, 98, 100, 102, 104, 105. 107, 109, 110, 111 Graham, I. A., 57, 58, 59, 71 Grandjean, A., 33, 50, 51 Grattan, S. R., 120, 130, 159, 176, 180 Green, P. J., 54, 72 Green, R., 54, 71 Greenway, H., 76, 107, 109, 115, 117, 118, 119, 121, 123, 124, 125, 133, 134, 136, 137, 139, 140, 145, 151, 158, 176, 179, 180, 185 Grieve, C. M., 128, 132, 134, 137, 138, 139, 140, 141, 145, 158, 159, 181, 184 Grignon, C., 84, 108, 110 Grime, J. P., 2, 6, 10, 12, 13, 21, 27, 28 Groneman, A. F,, 9, 26 Grunberg, K., 115, 188 Gug-Ortega, R., 6, 29 Gunn, A., 134, 137, 151, 180 Gustafson, J. P., 8, 26 Gusten, H., 38, 50 H Haarsma, M. S . , 162, 185

195

AUTHOR INDEX

Hahlbrock, K., 56, 59, 60, 65, 71, 72 Hajibagheri, M. A.. 78. 79, 91, 106, 107, 117, 119, 120, 167, 179, 181

Hall, J. L.. 119, 167, 181 Halloran, G. M., 172, 185 Hamill, 0. P.. 82, 107 Handa, A. K., 64, 73 Hansen, U. P., 83, 107 Hanson, J. B., 126, 147, 155. 181, I87 Harmon, A. C., 14, 29 Haro, R., 78, 107 Harris, N., 47, 50 Harter, K., 54, 56, 58, 60. 71 Harter, L. L., 128, 182 Hartung, W., 133, 134, 136, 138, 139, 140, 152, 154, 158, 161, 182

Harvey, B. L., 128, 131, 158, 179 Harvey, D. M. R.. 78, 79, 91, 107, 119, 120, 167, 181

Hasegawa, P. M., 64, 73, 76, 77, 78, 79, 100, 105. 109, 110, 174, I85 Hassan, G . K. Y., 37. 38, 50 Hassan, 1. A,, 40, 42, 50 Hassidim, M., 79, 100, I05 Hauge, B. M., 68, 71 Hauskrecht, M., 133, 176 Havill, D. C., 17, 18, 27 Hawkes, G . R., 128, 181 Hay, R. K. M., 124. 181 Hayat, S., 118, 123. 176 Hayatsu, M., 25, 28 Haydock, K. P., 118, 123, 135, 136, 139,

Hilpert, A., 152, 154, 182 Hiramoto, Y.,101, 109 Hirsch, R. E., 84, 109 Hoagland, D. R., 150, 188 Hodgson, J. G., 6, 12, 27 Hoffen, A,, 169, 188 Hofmann, B., 57, 58, 72 Hope-Simpson, J., 2, 27 Hoshi, T., 98, 99, I07 Hoth, S., 84, 85, 98, 99. 105, 109 Hough, A. M., 46.50 Houghton, J. T., 47, 50 Houlden, G., 46, 49 Houser, J., 115, 186 Hu, H., 163. 181 Hu, Y., 137, 138, 139, 161, 181 Huber, J. J. L., 115, 187 Huffaker, R. C., 160, I82 Hughes, M. A., 54, 55, 56, 71 Hunter, D., 5 , 27 Huntington, V. C., 146, 178 Huprikar, S. S., 84, 104 Hussain, F., 124, 181 I Ichida, A. M., 84, 88, 98, 109 Ilahi, I., 124, 181 Ilyas, M., 115, 171, 172, 186 Imamul Huq, S. M., 118, 181 Itai, C., 126, 176 Iwasaki, N., 101, I09 Izard, P., 125. 135, 189

140, 141, 145, 159, 161, 180. 189

Heck, W. W., 32, 35, 50 Hedrich, R., 82, 83. 84, 85, 98, 99, 105, 107, 109, 110

Heime, R., I I , 27 Heino, P.,55, 73 Heinrich, G., 37, 38, 50 Heller, R., 16, 28 Hemmingsen, S., 78, 107 Henriques, F. S., 162, 179 Hepler, P. K.. 17, 27 Hermondson, M. A., 64,73 Hess, F. D., 78, 79, 105 Hether, N. H., 12, 27 Hetherington, A. M., 14, 17, 26. 27, 30 Hewitt, E. J., 4, 27 Higashi, R. M., 77, 108 Hill, J., 155, 181 Hille, B.. 82, 83, 107 Hillniann, T., 168, 184

J Jabs, T., 65, 71 Jackson, I. L., 12, 27 Jackson, J. A., 55. 56, 65, 66, 67, 68, 69, 71

Jacoby, B., 79, 100, 107. 109. 112 JaffrC, T., 11, 27 Jagadish, V. C., 8, 26 Jager, H. J., 32, 34, 35, 50, 51 Jaillard, B., 6, 7, 18, 26 Jang, J.-C., 58, 71 Jefferies, R . L., 16, 27 Jeftic, J., 38, 50 Jenkins, G. I., 54, 55, 56, 5 8 , 59, 60, 65, 66, 67, 68, 69, 70, 71, 73

Jeschke, W. D.,, 117, 119, 124, 133, 134, 135, 136, 137, 138. 139, 140, 141, 152, 153, 154, 155, 157, 158. 159, 161, 176, 181, 182, 186. 189

196

AUTHOR INDEX

Jia, Z.-P., 78, 107 Johannes, E., 83, 107 Johnson, C.M., 9,26 Johnson, J., 6, 29 Johnson, P. A., 6, 27 Jones, D. L., 5, 6, 27, 28 Jones, R. L., 85, 105 Joy, D. C., 168, 180 Jurgens, G., 69, 72

K Kahane, I., 126, 182 Kaiser, T., 56, 71 Kaiser, W. M., 91, I l l , 117, 160, 161, 182, 186, 187, 188 Kakutani, T., 86, 88, 103, 109 Karlsson, P. E., 34, 51 Kasamo, K., 79, 111 Kasana, M. S., 44, 45,50 Kashibhatia, P.S., 47, 49, 50 Kats, G., 41, 50 Katsuhara, M., 78, 97, 99, 107, 108 Kattenberg, A., 47, 50 Kawasaki, T., 130, 131, 182 Kay, S. A., 68, 72 Kearney, T. H., 127. 128, I82 Keiber, J. J., 55, 68, 69, 72 Kelly, W. B., 99, 108 Kernp, D. R., 124, 181 Kent, B., 119, 167, 181 Kent, L. M., 129, 182 Kern, R., 61, 73 Kielland, K., 19, 26 Kikuyama, M.,101, 109 King, P., 41, 51 Kingsbury, R. W., 115, 172, 182 Kinraide, T. B., 5, 8, 28, 30 Kinzel, H., 16, 28 Kircher, S.,54, 60, 71 Kirchhoff, V. W. J. H., 37, 38, 50 Kirst, G. 0.. 76, I09 Kjellbom, I?, 65, 70 Klagges, S., 152, 154, 182 Klasine, L., 38, 50 Klessig, D. F., 54, 60, 64, 70 Klobus, G., 160, 182 Knight, H., 5 5 , 72, 101, 108 Knight, M. R., 55, 68, 72, 101, I08 Kobayashi, K., 41, 50 Koch, K. E., 57, 59, 72 Kochian, L. V., 5, 6, 7, 8, 27, 28, 29, 84, 104, 162, 182

Kohut, R. J., 41, 51 Kosuge, N., 25.28 Kourie, J. I., 78, 85, 97, 99, 108 Kramer, P. J., 118, 182 Krapp, A., 57, 58, 72 Krenz, M., 54, 60, 71 Kretsch, T., 56, 71 Kristiansen, L., 34, 49 Krysan, P. J., 82, 108 Kubica, S., 133, 176 Kuch, A., 98, 107 Kuhn, W., 146, 150, 156, 188 Kuiper, P.J. C., 120, 127, 132, 184 Kumar, R., 82, 110 Kuznetsov, V. V., 174, 182 Kylin, A., 120, 127, 132, 184 1 Lacan, D., 134, 136, 179 Lacroute, E, 84, I10 Lado, P., 79, 105 Lagarde, D., 84, 108 Lagares, A,, 20, 26 Lagerwerff, J. V., 124, 128, 182 Laguette Rey, H. D., 40, 41, 51 Lam, E., 54, 72 Lamart, A., 16.28 Lamb, C.J., 65, 70, 72, 73 Lamoreaux, R. J., 125, 182 Larher, E, 118, 181 Lass, B., 81, 108 Liiuchli, A., 77, 96, 97, 101, 106, 108, 110, 119, 120, 123, 124, 125, 127, 129, 130, 132, 133, 134, 135, 136, 137-8, 139, 140, 141-2, 143, 144, 145, 146, 147, 148, 149, 150, 152, 154, 155, 156, 157, 158, 159, 162, 163, 165, 167, 168, 177, 178, 182, 183, 184, 188 Lauer, M., 24, 30 Laurence, J. A., 41, 51 Laurie, S., 83, 84, 86, 87, 88, 91, 92, 95, 97,98, 99, 102, 103, 104 Laycock, D., 16, 27 Layzell, D.B., 136, 182 Lazaroff, N., 131, 158, 183 Lazof, D., 80, 108 Lazof, D. B., 120, 121, 124, 125, 134, 136, 138, 141-2, 143, 144, 145, 146, 147, 148, 149, 154, 158, 161, 167, 168, 169, 170, 171, I83 Le Gales, Y., 16, 28

AUTHOR INDEX

Lea-Cox, J. D., 161, 183 Leaver, C. J., 58, 71 Lechno, S., 117, 183 Lee, J. A., 11, 13, 17, 18, 21, 22, 24, 27, 28 Lee, K. S., 125. 135, 189 Lee, R. B., 25, 28 Lehman, H., 167, 188 Lehnen, M., 84, 105 Leidi, E. 0.. 120, 183 Leigh, R., 83, 84, 86, 87, 88, 89, 90. 91, 92, 95, 97, 98, 99, 100, 102, 103, 104, I l l . 117, 164, 165, 180, 184 Lemaillet, G., 85, 98, I09 Lemtiri-Chlieh, F., 87, 90, 92, 98, 100, 112 Lenherr, B., 33, 51 Leon, P., 58, 71 Lepetit, M., 84, 108 Lerner, H. R., 79, 100, 105 Lessani, H., 120, 130, 131, 152, 184 Leung, J., 68, 72 Levine, A,, 65, 72 Levy 11, H., 47, 49, 50 Li. H.-M., 68, 72 Li, W., 99, 108 Li, W. W., 99, 108 Libbenga, K. R., 86, I l l Lifshin, E., 168, I80 Ling, Y. C., 170, 176 Linton, R. W., 149, 168, 169, 170, 171, 180, I83 Liu, D., 64, 73 Liu, J., 118, 184 Logemann, E., 59, 71 Lois, R., 60, 72 Long, J. C.. 55, 71 Long, M. J., 124, 135, 187 Lowendorf, H. S., 20, 28 Lozoya, E., 60, 72 Luan, S., 99, 108 Lucas, W. J., 84, 85. 104, 110 Luckhardt, R. L., 128, 181 Liittge, U., 80, 108, 170-1, 184 Lynch, J.. 97, 101, 106, 108, 123, 129, 132, 134, 137, 138, 141, 145, 158, 184

M Maas, E. V., 120, 128, 130, 132, 134, 137, 138, 139, 140, 141, 145, 158, 159, 180. 181. 184 Maathuis, F. J. M., 81, 82, 84, 86, 87, 88. 90. 91, 92, 98, 108, 109. I12

197

Macduff, J. H., 160, I86 Machida, Y., 58, 73 Machler, F., 33, 51 Mackay, A. D., 8, 26 MacKenzie, A. J., 119, 128, 175 Maggs, R., 39, 40, 41, 43, 44, 45, 51. 52 Mahmoud, A., 10, 28 Maksymowych, R., 125, 184 Malone, M., 164, 184 Mansfield, P. J., 46, 49 Mansfield, T. A,, 16, 17, 22, 26, 27, 29 Mariano, M. M.. 37, 38, 50 Marinho. E. V. A., 37, 38, 50 Marrt., E., 97, I05 Marschner, H., 12, 14, 28, 29, 77, 79, 109, 120, 127, 130, 131, 132, 152, 162, 184, I89 Marsh, E. L., 82. 110 Martel, R., 78, 107 Marten, I., 85.98, 109 Martin, P., 156, 184 Martinez, V., 120, 127, 134, 139, 140, 149, 154, 155, 156, 157, 158, 159. 177, 184

Marty, A., 82, 107 Maruyama, S., 147, 184 Maskall, K 47, 50 Made, J., 164, 176 Mason, H. S . , 63, 72 Mass, E. V., 158, 172, 179 Mathy, P., 32, 35, 50 Matsuda, K., 121, 126, 184, 187 Matyssek, R., 147, 184 McAinsh, M. R., 14, 28, 30 McCarl, B. A,, 32, 35, 49 McCullough, N., 78, 107 McGrath, R. B., 55, 61, 72 McIntye, G . I., 161, 184 McKendree, W. L., 84, 109 McNeill, S., 46, 49 McNeilly. T., 115. 120, 129, 130, 131. 158, 171, 172, 173, 175, 176 McVickar, M. H., 128, 181 Meira Filho, L. G., 47, 50 Meleigy, M. I., 37, 49 Mendoza, N. M., 40,41, 51 Mennen, H., 79, 109 Meriot, S., 55, 68, 72 Mesnick, L., 115, 186 Meyer, R. F., 118, I84 Michaels, A., 47, 50 Michel, M.,168, 184

AUTHOR INDEX

Michelina, V. A., 118, 184 Millar, A. J., 55, 61, 68, 72 Millard, P. J., 169, 177 Miller, P., 37, 38, 51 Millhouse, J. A.. 117, 119, 187 Millikan, C. R., 155, 156, 184 Mimura, T., 99, 108 Minet, M., 84, I10 MisBra, S., 69, 72 Mohr, H., 63, 73 Mol, J., 55, 59, 60,73 Mol, J. N. M., 59, 60, 73 Mollanen, L., 19, 26 Monnich, E., 37, 38, 50 Monroy, A. F., 55, 73 Moran, N., 86, 109 Moran, O., 98, 107 Morcuende, R., 24, 30 Morecroft, M. D., 17, 18, 28 Morgan, A. J., 167, 184, 185 Morgan, S. M., 18, 28 Modtsugu, M., 130, 131, 182 Morrison, G. H., 167, 168, 169, 170, 176, 177, 186 Miiller, A. J., 69, 72 Muller-Rober, B., 84, 85, 86, 88, 98, 99, 106. 109 Mullet, J. E., 63, 72 Mummert, H.,80,109 Munns, R.. 76, 77, 107. 109. 115, 117, 118, 119, 121, 123, 124, 125, 126, 135, 137, 146, 149, 152, 153, 157, 158, 174, 176, 180, 185, 187, 188, 189 Munns, S.,118, 121, 134, 140, 179 Muranaka, T., 58, 73 Murata, Y., 86, 88, 103, I09

Neuwinger, K., 98, 107 Neves Piastun 165 Newbury, D. E., 168, 180 Nieman, R. H., 158,185 Niu, X., 76, 77, 109, 174, 185 Noble, C. L., 172, 185 Noguchi, M., 86, 88, 103, 109 Nolasco, A. Q., 37, 38, 51 Nomoto, K., 12, 30 Nonami, H., 147, 185 Nordin, K., 55, 73 Norlyn, J. D., 172, 179 Northup, R. R., 20, 21, 28 Norton, R. A., 155, 185 Novacky, A., 81, 111 Novak, R. S., 162, 178 Nurnburger, T., 59, 71

N Nagata, T., 25, 28 Nagatani, A., 56, 71 Nagy, E, 54, 60, 71 Nakajima, H., 65, 73 Nakamura, R. L., 84, 109 Nakamura, Y., 37, 38, 50 Nakayama, F. S., 155, 177 Nandi, P. K., 44.45, 49 Narasimhan, M. L., 64,73 Neher, E., 82, 84, 85, 86, 107, 110 Nelson, C. J., 164, 187. 188 Neuhaus, G., 61, 62, 63, 68, 70, 73 Neumann, I?, 118, 185 Neumann, P. M., 100, 112

P Paiva, L. A., 54, 55, 59, 60, 70 Palme, K., 84, 98, 105. 107 Palmer-Brown, D., 36, 49 Palva, E. T., 55, 73 Palva, T. E., 60, 61, 73 Palzkill, D. A., 150, 156, 186 Papernik, L. A., 8, 29 Papp, J. C., 124, 186 Parcy, F., 68, 71 Pardines, J., 120, 127, 177 Pardo, 5. M., 76.77, 109, 174, 185 Parker, D. R., 5, 28 Parniske, M., 59, 71 Passioura, J. B., 118, 188

0 Obermeyer, G.,87, I09 Obi, I., 86, 88, 103, 109 Oertli, J. J., 117, 185 Okazaki, Y.,101, 109 O’Leary, J. W., 100, 104 Olsen, C., 16, 29 Olsen, R. A., 12, 27 Olson, R. K., 20, 26 Olsson 13, 25, 30 Olszyk. D., 41, 50 Olszyk, D. M., 35, 51 Omelian, 3. A., 131, 132, 158, 185 Osmond, C. B., 80, 108, 119, 180 Otte, M. L., 162, 185 Ourry, A., 160, 186 Ownby, J., 6, 29

199

AUTHOR INDEX

Pate, J. S., 133, 136, 152, 153, 155, 156, 157, 158, 159, 161, 181, 182, 186 Pei, Z.-M., 88, 112 Penkett, S. A., 46, 51 Pereira, E. B., 37, 38, 50 Perez-Alfocea, F.,160, I77 Peuke, A. D., 161, 186 Pfankoch, E., 64, 73 Pick, U., 79, 109 Pigott, C. D., 2, 29 Pimentel, D., 115, 186 Pineros, M., 5 , 29 Piston, D. W., 170, 187 Pitman, M. G., 80, 106, 128, 131, 134, 135, 136, 137, 140, 148, 151, 158, 167, 183, 186, I88 Pleijel, H., 34, 41, 51 Poe, M. P., 35.51 Poethig, R. S., 125, 186 Polito, V. S., 101, 108 Poljakoff-Mayber, A., 126, 182 Pollard, A., 77, 109, 112 Ponce de Le6n, I., 60, 61, 73 Powell, M. J., 123, 186 Preiss, E., 115, 186 Prins, H. B. A., 88, 111 Provart, N., 84, 85, 98, 99, 109 Prudhomme, M. P., 160, 186 Puthota, V., 6, 29

Q

Quatrano. R. S., 63, 70 Qureshi, R. H., 115, 171, 172, 186 Ragjpthama, K. G., 64,73 Rains, D. W., 76, 82, 106, 109 Rajasekhar, V. K., 65, 73 Ramadan, A. B., 37, 38, 50 Ramseyer, G. O., 170, 186 Rana, R. S., 172, 186 Randall, P. J., 8, 9, 22, 26, 30 Rao, D. N., 44, 45, 49, 51 Raschke, K., 84, 85. 86, 87, 89, 98, 99, 100, 110, 112 Ratcliffe, R. G . . 25, 28 Rathert, G., 1 1 7, 186 Rawer, W. E., 126, I87 Raven, J. A., 22, 29 Ravina, I., 100, 112 Rawson, H. M., 121, 124, 126, 135, 149, 185, 187 Read, D. J., 20, 29

Read, N. D., 55, 73 Redmann, R. E.,,128, 131, 158, 179 Regnier, F. E., 64, 73 Rehder, H., 17, 18, 27 Reid, D. A., 8, 29 Reid, J. D., 84, 85, 99, 105 Reid, R. J., 81, 100, 106, 111, 120, 178 Reimann, C., 120, 187 Reiners, W. A., 20, 26 Reinhold, L., 79, 100, 105 Reinold, S., 59, 71 Rengel, Z., 97, 109, 118, 187 Reuveni, M., 100, 110 Rhodes, P. R., 126, 187 Riazi, A., 121, 184 Richardson, A. E., 20, 29 Ridout, M., 83, 87, 89, 112 Riego, D. C., 6, 8, 26 Ringoet, A,, 155, 187 Ritchie, G. S. P.,5, 26, 29 Rizk, H. F. S., 37, 49 Roadknight, C., 36, 49 Roberts, D. M., 14,29 Roberts, S. K., 85, 86, 87, 89, 90, 91, 92, 94, 95, 98, 99, 100, 110 Robins, M. F., 118, 123, 135, 136, 139, 140, 141, 145, 159, 161, 180, 189 Robinson, D., 16, 29 Robinson, S. P., 117. 119, 187 Robson, A. D., 5 , 26 Rocholl, M., 56, 71 Rodriguez-Navarro. A,, 78, 107 Rolfe, B. G., 20, 29 Roman, G., 68, 69, 72 Romheld, V., 12, 29. 162, 189 Rook, F., 57, 59, 73 Rorison, I. H., 2, 5, 16, 17, 18, 27, 29 Ross, D. S., 5, 27 Rothenberg, M., 68, 69, 72 Rozema, J., 115, 162, 185, 187 Rozema-Dijst, E., 115, 187 Rubio, E, 81, 107, 110 Rufty, T. W., 161, 168, 169, 183 Ruiz, L. P., 16, 17, 22, 26, 29 Runge, M., 17, 29 Rush, D. W., 120, 172, 187 Rusnak, F., 99, 108 Ryan, P. C., 8, 24, 26 Ryan, P. R., 5, 8, 9, 22, 26, 28, 30 S

Sachs, T., 125, 187

200

AUTHOR INDEX

Sacks, W. R., 59, 71 Sairz, J. F., 120, 183 Sakman, B., 82, 107 Salmon, J. M., 84, 110 Sanders, D., 79, 81, 82, 83, 84, 86, 87, 88, 90, 91, 92, 95, 97, 98, 99, 102, 103, 104. 107, 108, 109, 111. 112 Sanders, G. E., 34, 51 Sanders-Mills, G., 36, 49 Sandison, D. R., 169, 170, 177, 187 Sarwar, G., 152, 154, 182 Satoh, S., 79, 111 Satter, R. L., 86, 109 Sauer, G., 155, 187 Scacchi, A., 97, 105 Schachtman, D.P., 82, 85, 86, 88, 103, 110, 119, 120, 187 Schafer, C., 57, 58, 72 Schlfer, E., 54, 55, 56, 58, 59, 60, 70, 71, 73 Schatzler, H. P., 146, 150, 156, 188 Schauf, C. L., 83, 86, 90, 110 Scheel, D., 59, 60, 65, 71, 72 Scheible, W.-R., 24, 30 Schlesinger, W. H., 47, 50 Schmelzer, E., 59, 71 Schmidhalter, U., 137, 138, 139, 161, 181 Schmidt, C., 116, 117, 118, 121, 122. 116, 130, 132, 178 Schmidt, R., 58, 73 Schnoor, J. L., 47, 50 Schnyder, H., 164, 187 Scholz, G., 156, 188 Schonhurst, M. H., 172, 179 Schreck, J., 115, 186 Schreiber. S. L., 99, 108 Schroeder, J. I., 5, 27, 81, 82, 83, 84, 85, 86, 88, 98, 99, 105, 106, 107, 108, 109, 110, 111, 112 Schubert, S., 77,96, 110 Schulze, E.-D., 24, 30 Schumaker, S., 100, 104 Sedbrook, J. C., 84, 109 Seemann, J. R., 167, 187 Sillden, G., 34, 41, 51 Sellers, E. K., 17, 18, 28 Sentenac, H., 83, 84, 85, 98, 99, 105, 106, 109, 110, 111 Serrano, R., 76, 77, 110 Setter, T. L., 117, 124, 133, 134, 136, 139, 140, 158, 176 Shacklock, P. S., 55, 73

Shah, D., 34.49 Shamsi, S. R. A., 39, 40, 42, 43, 51, 52 Shannon, M. C., 128, 159, 187 Sharkey, P. J., 155, 186 Shaw, E. J., 128, 181 Shaw, M.J., 56, 68, 69, 71 Sheen, J., 57, 73 Sheen, J.-C., 58, 71 Shevyakova, N. I., 174, 182 Shibata, J. K., 40, 41, 51 Shirasu, K., 65, 73 Shirzadegan, M., 174, 189 Sigworth, F. J., 82, I07 Silk, W.K.,124, 137, 138, 139, 140, 145, 147, 150, 158, 163, 164, 165, 177, I87 Simpson, R. J., 20, 29 Sims, A. P., 16, 27 Singh, M., 44, 45, 51 Singh, N. K., 64, 73 Skarby, L., 34, 36, 41, 49, 51 Skerrett, I. M., 100, 111 Skerrett, M., 84, 87, 89, 90, 91, 92, 95, 98, 99, 100, 103, 106, Ill Slayman, C. L., 85, 86, 87, 99. 102, 105, 110

Smalle, J., 68, 71 Smeekens, S., 57, 59, 73 Smith, C. A.. 169,177 Smith, D., 172, 179 Smith, E A., 22, 29, 81, 82, 100, 106, 108, 110. 111, 120, 178 Smith, J. A. C., 79, 104 Smith, S. M., 55, 68, 72 Snaydon, R. W., 5, 22, 26 Snowden, R. E. D.,10, 30 Sod, E. W., 168, 177 Sonnewald, U., 58, 73 Spalding, E. P., 86, 87, 102, 110 Speer, M., 91, 111, 160, 187, 188 Sprung, D.,37, 38, 50 Spurr, A. R., 129, 130, 131, 158, 178 Stapleton, A. E., 54, 59, 73 Stein W. D., 80, Ill Steizer, R., 120, 130, 135, 136, 140, 148, 167, 168, 188

Steveninck, M. E., 120, 130, 188 Steveninck, R. F. M., 135, 139, 159, 188 Stevens, C. S., 37. 38, 51 Stewart, G. R., 16, 17, 18, 21, 24, 27, 28 Stitt, M., 24, 30, 57, 58, 72, 73 Stoeckel, H., 87, 111

20 1

AUTHOR INDEX

Stolo, A. L. H., 168. 177 Stone, J. E.. 172, 179 Storey, R., 135, 136. 140, 148, 167, 188 Stout, P. R., 150, 188 Strayer, C. A., 68, 72 Strijm, L.,, 14, 15, 22, 30 Stuitje, A. R., 59, 60, 73 Stumm, W., 5 , 30 Suggs, C., 168, 169, 183 Sunderland. N.. 125. I88 Sussex, 1. M., 125, I86 Sussnian, M. R., 82, 84, 108, 109 Szabolcs, I., 76, 111

Tonneijck, A. E. G., 32, 36, 51 Tonnet, M. L.. 135. 149, 152, 153, 157, 158, 185, 189 Toroksalvy, E., 117, 120, 188 Treeby, M. T., 135, 139, 159, IN8 Trewavas, A. J., 5 5 , 68, 72, 73, 101, 108 Troke, P. F., 76, 106, 119, 120, 127, 179 Tschope, M., 65, 71 Tyemian, S. D., 84, 86, 87, 88, 89, 90, 91. 92, 95, 98, 99. 100, 103, 106. 110. 111 Tyler, G., 9, 13, 14, 15, 22, 25, 27, 30

U T Takagi, S., 12, 30 Takeda, K., 87, I l l Takemoro, T., 12, 30 Taleisnik, E., I 15, 188 Take-Messerer, C., 58, 71 Tansley. A . G., 2, 30 Tariche, S., 115, 186 Tawfik, F. S., 38, 50 Tax, E, 82, I08 Taylor, G., 6, 7, 30 Taylor, J. E., 14, 30 Taylor, 0. C., 32. 35, 50 Taylor, S. E., 167, 168. 179 Tazawa, M., 78, 97, 99, 107, I O N Tejeda. T. H., 37, 38, 51 Tel-Or, E., 117, 183 Tenhaken, R., 65, 72 Teomy, S., 79, 107 Termaat, A., 115. 118, 125, 146, 185, 188 Terry, B. R., 86, 88, 103, 110, If1 Terry, N., 119, 124, 176, 186 Terzaghi. W. B., 54. 73 Tester, M. A,, 5, 29, 82, 85, 86, 87, 89, 90, 91, 92, 94, 95, 98, 99, 100, 110, 111, 112 Thibaud, J.-B., 83, 84, 85, 98, 109. I l l Thiel, G . , 98, 99, 100, 102, I l l , 123, 134, 137, 138. 141, 145, 158. 184 Thomas, D. A., 134, 137, 151, 180 Thomashow, M. F., 55, 71 Thompson, C . R.. 35, 41, 50, 51 Thompson, R. K., 172, 179 Tibbitts, T. W., 150. 156, 186. 188 Tingey, D. T.. 32, 35, 50 Tomos, A., 164, 184 Tomos, A. D.. 117, 118. 165, 175, I80 Tomos, D., 164

Udo, W. S., 156. 188 UK FQRG 32, 51 Ullrich-Eberius, C. I.. 81, 108, Ilf Unsworth, M., 32, 35. SO Uozumi, N., 84, 85, 111 Urbach, S., 84, 106 Urwin, N. A R., 58, 59, 73

V Vaddia, Y.,126, I76 Valon, C., 68. 71 van der Eerden, L. J.. 32. 36. 51 van der Meer, I. M., 59, 60, 73 Van Duijn, B , 86, 111 van Leuken, P., 41, 51 van Steveninck, R. F. M., 120, 130, 188 van Veen, J. A,, 9, 26 Van Volkenburgh, E., 83, 87, 90, 98, 106. 118, 188 Vanbel, A. J. E., 81, 111 Venables, A. V., 115, 188 Verlin, D., 81, 82, I O N VCry, A. A.. 83, 84, 85, 98, 111 Vidal, S., 60, 61, 73 Vogelzang, S. A,, 88, 111 Vogt, K. A,, 20, 21, 28 Volenec, J. J., 164, 188 Volk, R. J., 149, 170, 171, 183 Volpe, C., 37, 38, 50 von Schaewen, A., 58, 73 von Tiedemann, A., 34, 51

W Wada, M., 79. 111 Wahid, A., 39, 40, 42, 43, 51, 52 Waisel, Y., 169, 188 Walker, N. A,, 81, 82, 108. 110, 111, 112 Walker, R. R., 117. 120, 188

202

AUTHOR INDEX

Wallihan, E. F., 12, 30 Wallin, G., 34, 41, 51 Ward, J. M,, 88, 98, 110, 112 Ward, M. R., 160, 182 Wasserman, R. H., 169, 177 Watkin, E., 117, 124, 133, 134, 136, 139, 140, 158, 176 Wayne, R., 0. 17, 27 Webb, A. A. R. 14.30 Webb, W. W. 169, 170, 177, 187 Wegner, L. H. 86, 87, 88, 89, 98, 99, 100, 106, 112 Weigel, H. J. 34, 49, 51 Weigl, J. 170-1, 184 Weiland-Heifecker, U. 69, 72 Weiss, D. 55, 59, 60, 73 Weisshaar, B. 56, 71 Weppner, J. 37, 38, 50 West, D. W. 172, 185 West, K. R. 80, 106 Wheeler, B. D. 10, 30 White, M.C . 24, 27 White, 0. 115, 186 White, P. J. 82, 83, 87, 89, 90, 92, 93, 94, 98, 100, 112 WHOLJNEP 36,52 Wickens, L. 116, 188 Wickens, L. K. 116, 120, 178 Wiebe, H. J. 146, 150, 156, 188 Wieland, E. 5, 30 Wienecke, J. 119, 120, 135, 136, 137-8, 141, 144, 145, 146, 152, 156, 182, 188

Wijnands, J. H. M. 32, 36, 51 Wilkins, D. A. 115, 188 Willis, A. J. 16, 27 Willmitzer, L. 84, 85, 98, 99, 109 Wilmitzer, L. 58, 73 Wilson, J. R. 118, 123, 124, 135, 136, 139, 140, 141, 145, 159, 161, 189 Wilson, K. J. 83, 86, 90, I10 Winicov, I. 174, 189 Witt, J. 117, 186 Wittwer, S. H. 155, 162, 177

Wolf, J. W. 41, 50 Wolf, 0. 124, 133, 134, 135, 136. 137, 138, 139, 140, 141, 152, 153, 154, 157, 158, 159, 161, 181, 182, 189 Wollenweber, B. 17, 18, 27 Wong, M. 54, 72 Woolhouse, H. W. 7, I I , 13, 22, 28, 30 Wu, L. 119, 176 WU, S.-J. 81, 112 WU,W.-H. 99, 102, 112 Wu, Y. 147, 185 Wyn Jones, R. G. 77, 89, 104, 109, 112

x Xu, Y. 64, 73 Y Yamagata, H. 61, 62, 63, 68, 70 Yeo, A. R. 76,91,106, 115, 117, 119, 120, 125, 127, 130, 135, 137, 151, 152, 156, 163, 165, 167, 178, 179, 181. 189 Yermiyahu, U. 8 , 3 0 Yienger, J. 47, 49 Yoshihashi, M. 86, 88, 103, 109 Young, J. C . 82, 108 Young, P. G. 78,107 Ypey, D. L. 86, 111

2 Zambrzuski, S. I1 9, 176 Zamski, E. 117, 183 Zayed, A. 119, 176 Zengshou, Y. 20, 21, 28 Zhang, C. 162, 189 Zhang, J. 118, 189 Zhou, L. 58, 71 Zhu, J. K. 118, 184 Zhu, J.-K. 81, 82, 106, 112 Zidan, I. 100, 112 Zierold. K. 167, 189 Zimmermann, S. 86, 88, 106 Zingarelli, L. 79, 104

SUBJECT INDEX

A abscisic acid-dependenthndependent pathway 35 abscisic acid signal transduction 68 Acetubuluriu 80, 97, 101 Agmpyron 132 Agropyoti elongututii 159 Agropvroti intermecliuni 129, 130 Agrosris cupilluris 7 aluminium and acidity in soil, 4-10 effects on plasma membrane and cytosolic processes 2 2 4 stimulation of malate synthesis 22. 24 aluminium tolerance 610. 22 aluminium toxicity 5-6, 18-20 Aly.viu rubricucrlis 1 I ammonium assimilation 22 extractable, in soil 2, 4 Anthoxunthuni odorarum 5 , 22 Arubidopsis 56, 65, 67, 68, 8 I , 84, 100, 102 Arrhenutherum elutius 10, 11 atmospheric vapour pressure, ozone uptake and 33 ATP, cation influx across plasma membrane and 102 Atrip1e.x 79, 80, 144 Arriplex uninicolu 124, 136, 139, I40 Atriplex spotigiosu 136, 140, 148 Avenu IS5 Azospirillum 9

B Befa wlguris 124, 132 beta-glucuronidase(GUS) reporter gene 61 bicarbonate in soil 4, 22 toxicity 11-13

BIOLOG plate technique 25 blue light signal transduction pathway 65-7 boron in soil 4, 20-1 Brrrssicu F1trpU.S 58 Brussicu olerucea I59 Bmniopsis heneketiii 9 BroninpsiA erecfu 18 Bronius erectci

C CAB genes 5 8 , genes 61-3 C&nus cujun 45 calcium disturbed, in young tissues 137-8 effects on plasma membrane and cytosolic processes 2 2 4 influx across plasma membrane 97-1 01 in interactions between signalling pathways 68 recirculation in the shoot 155-6 salinization, transport and 157-8 in soil 2, 4, 14-17 translocation to the shoot 129-3 I transport in growing shoot tissues 144 calcium ion permeable channels 5 Crirnelliu sinensis 8 Cupsicum antiuuni 130, 152 Carex pilulijeru 13 Curthumnus tinctorius 152 cation influx across plasma membrane 96- 103 ATP 102 cytosolic calcium and pH 101 external and cytosolic sodium 101-2 external calcium and pH 97-1 0 I voltage 97 Centururea scubinsu I6 Chuetnmorphu 80 chalcone synthase (CHS) genes 55, 56, 60 Charu 81, 101

204

SUBJECT lNDEX

Chara injlata 97 Chenopodium 57, 58 chlorosis, lime-induced 12 CHS promoter activity 61-3 Cicer arietinum 45 Citrus 161 Citrus reticulata 117 cobalt in soil 4, 20 Commelina communis 16 compartmental analysis by efflux 154-5, 163 Crassulacean Acid Metabolism (CAM) 22

D Ductylis glomerata 130, 131 Daucus carota 159 defence genes, regulation of 64-5 defence responses 59-61 Deschampsia flexuosa 10, 11, 18 dose-response relationship 56

E EDU 34,41-2 electron probe X-ray microanalysis (EPXMA) 163, 167-8 elemental deposition rates 163-6 Elodea 81 EPXMA (electron probe X-ray microanalysis) 163, 167-8 Ericaceae 18, 24 Erwinia carotovora 60-1 ethylene signal transduction 68 European Crop Loss Assessment Network 35-6 exposure-response studies to ozone 34-6

F Fesruca rubra 130, 131 FHC (frozen hydrated cryofractured) EPXMA 167-8 flavonoid biosynthetic pathway 59-61 fluorescence 170 frozen hydrated cryofractured (FHC) EPXMA 167-8

G Galium saxatile 2, 13 Galium sterner; 2 gene expression responses 54-5, 57-9 control by metabolites 57-9 genotypic sensitivity to salinization 131-2 Glycine 139, 140, 161

Gl.ycine max 42, 120, 126, 136, 137, 14I , 152, 159 Glycine tomenrella 136, I59 Glycine wightii 136, 161 Goldman-Hodgkin-Katz (GHK) equation 83, 92 Gossypium hirsutum 129, 139, 140, 159 growth dilution 164 growth kinetics analysis (GKA) 163-6 Glycine wightii 159

H Helianthus annuus 126 hexokinase, sugar signalling pathway and 58-9 Hibiscus 144, 145 Hibiscus cannabinus 45, 124, 136, 138, 140, 141 Holcus lanatus 18, 130, 131 Hordeurn 121, 144, 145 Hordeum vulgare 123, 124, 125, 129, 130, 131, 132, 137, 138, 141, 152, 153, 156, 161, I62 hypersensitive response (HR) 65

I inward-rectifying channels (IRCs) 84-8 ion excess hypothesis 119, 133, 173-4 iron acquisition 22 extractable, in soil 4 iron deficiency 11-13 iron toxicity 10-11 jasmonic acid (JA) 55 Juncus squarmsus 16

J

K Kerr Gapon equation 128 kinematic growth analysis 163-6 Koeleria mncranthu 11

L Luctuca 144, 145 Luctuca saliva 120, 124, 125, 129, 130, 131, 136, 139, 140, 141-3, 154, 159 Lens culinaris 45, 1 18 Leantodon hispidus 16 Lepidium sativunz 130 Leptochloa fusca 152, 154 lime-induced chlorosis 12

205

SUBJECT INDEX

linear variable differential transformers (LVDTs) 116 Lolium perennr 130, 13 I long-term recirculation of nutrients 151 4 Lophopyrum cdonguturn 1 3 1 , 132 Lupinus 144, 145 Lupinus ulbus 131, 136, 138, 139, 140, 152, 153, 154, 156, 157, 161 Lupinus Iicteus 16, 130, 139, 159

M magnesium extractable, in soil 2 salinization and transport of 13940, 158 translocation to the shoot 130-1 transport in growing shoot tissues 144 malate, effect of aluminium on 33 malatekitrate aluminium detoxification mechanisms 25 manganese toxicity 1&11 Maytenus biireaviann 11 Mrdicago sativu 45. 129, 17 1 Mesetnbrycmthernurn ctystallinurn 79 methyl jasmonate (MeJA) signalling pathway 63, 64 microautoradiography 170-1 microdissection 166 microelements salinization, transport and 162 in soil 20-1 molybdenum deficiency 2&1

N National Crop Loss Assessment Network (NCLAN) (US) 34-5 Nicotiancr tahacum 126 Nitrlla 81 Nitrllopsis 97, 101 nitrate assimilation 22 in soil 4 utilization 17-18 nitric oxide (NO) 42 nitrogen salinization, transport and 160-3 in soil 17-20 nitrogen dioxide as air pollutant 39, 41. 45, 46 nitrogen oxide emissions 32, 46, 47-8 nutrient status and transport, microscale Study 162-71

0 Origanirm vitlgare I6 Otyza 144 Uryza sativa 117, 124, 130, 151 osmoregulatoryhrgor hypothesis of salt tolerance 1 17-1 8, 173 Osrnotin 64 outward-rectifying channels (ORCs) 88 ozone adverse effects on crops 3 9 4 2 exposure-response studies 34-6 field chamber studies 34, 3 9 4 1 formation reactions 32 future concentrations 46-7 impacts on agricultural crops 33-6 ozone protection chemicals studies 34, 41-2 response of tropical crops and cultivars 43-6 experimental studies 43-5 field studies 45-6 rural levels in developing countries 36-9

P patch clamp technique 24-5 pathogenesis-related (PR) proteins 60 Persea americana 130, 13I , 132 Phaseofus I55 Phaseolus vulgaris 7, 36. 42, 58, 124, 125, 130, 131, 151 phenylalanine ammonia-lyase (PAL) gene 55.60 phenylpropanoid biosynthetic pathway 59-61 phloem transport under salinity 150-7 adding tracer to mature shoot tissue 152 calcium recirculation in the shoot 155-6 net fluxes and contents of xylem and phloem 1 5 2 4 pulsed labelling of roots 151-2 remobilization of nutrients from ageing shoot tissues 1 5 1 4 xyledphloem transfer 154-5 phosphate in soil 4, 13-14, 25 phospholipase C activity, inhibition of 5-6 phosphorus deficiency 25 extractable, in soil 4 salinization and transport of 13940, 158-60 short-term recirculation of 154-5

206

SUBJECT INDEX

phosphorus (cont.) transport in growing shoot tissues 144 uptake from soil 22, 25 phytochrome signal transduction 55, 56, 61-3 Phytophthora megasperma 60 Pinus muricuta 20, 21 Pisum 101 Pisum sativum 126 Poaceae 123, 136, 137, 149-50 polyphenols, effect on nitrification 20 potassium salinization, transport and 157 shoot accumulation 129 in soil 4 translocation to the shoot 129-30 transport in growing shoot tissues 144 transport in young tissue 133-7 potassium ion channels 5 primary response to salinization 115 Pseudomonus syringae pv. Glycinea 65

R Rhizobiuni 20 Ricinus communis 155, 159, 161 root signal hypothesis 118-1 19

S Saccharomyces cerevisiae 78 salicylic acid (SA) 55, 61 salinity in situ elemental and isotopic analysis 174-5 model systems 173-4 nutrient transport to growing shoot tissue 132-46 phloem transport and 150-7 reassessment of current status 171-3 shoot growth inhibition 115-21 shoot meristems 146-50 salinity tolerance 76-7 salinity toxicity 76-7 salinization cell division in leaves 125-6 cell extension 123 in dicots and monocots 121-6 nutrient transport disruptions 126-32 primordium formation and leaf emergence 123-5 timing of growth inhibition 121-3

whole shoot nutrient accumulation 128-32 salt-sensitive genotypeskell lines 103 salt tolerance 103, 117-18, 171-4 Scabiosa columbaria 5 Schizosaccharomyces pombe 78 secondary ion mass spectrometry (SIMS) 163, 167, 168-70 selectivity hypothesis 119 Shaker class of ion channel 84 short-term recirculation of nutrients 151-4 signal transduction networks ‘appropriate’ response 56-7 gene expression control by metabolites 57-9 gene expression responses and 54-5 interactions within 57-67 mechanisms in interactions between 67-9 negative regulation 57-63 networks vs pathways 55-6 phytochrome signal transduction pathways 61-3 plant defence responses 59-61 synergism 63-7 sodium carrier-mediated entry 80-2 channel-mediated entry 82-9 1 control levels of ions 127 co-residency of different channel types 89-9 1 effect of different channel types on rate of uptake 95-6 electrochemical potential differences 78-80 exclusion and genotypic tolerance 120-1 exclusion, uptake and sequestration of 77 influx across plasma membrane 101-2 ion selectivity of ion channels 82-4 semiquantitative dissection of fluxes 91-5 shoot accumulation 120 transport in young tissue 133-7 transport in growing shoot tissues 144 sodium absorption ratios (SARs) 128 sodium : calcium ratios 127-8 sodium chloride inhibition of shoot growth by 116-19, 173-4 disturbed photosynthesis I17 nutritional effect on shoot growth 119-21 soil water stress, ozone uptake and 33

207

SUBJECT INDEX

soil(s) acidic 4 calcareous 4 extractable elements 2 4 Solunum ruberosurn 41 Sorghum 144, 145 Sorxhurn bicolor 124, 132, 137, 138. 139, 140, 141, 149. 164 Spergrrlaria 80 stimulus-response pathways 56-7 Suuedu marititnu 152 sulphur dioxide emissions 32, 41, 48 synergism 63-7

T Trifoliuin alexundrinirm 124 TrijXurn pratense 124, 130, 131 Triricurn I 1 7, 132 Triticurn uesfivum 8, 13 I , 137, 139, 161 Triricum X Lophopyrum 120

U ultraviolet signal transduction pathway 65-7

V Vallisneria 81 Veronica qficina1i.s 13 Viciufubu 45 Vigna mungo 45 Vigna rudiata 45 Vignu unguiculutu 45 voltage-independent channels (VICs) 88-9

X xylem/phloem transfer 1.545

Z Zea 144, 145 Zru mays 117, 120, 121, 127, 130, 132, 138, 139, 140, 141, 159, 161


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