Desiccation and survival in plants, drying without dying

Desiccation and Survival in Plants

Drying Without Dying

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Desiccation and Survival in Plants

Drying Without Dying

Edited by

M. Black

King’s CollegeUniversity of London

UK

and

H.W. Pritchard

Royal Botanic Gardens, KewWakehurst Place

UK

CABI Publishing

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CABI Publishing is a division of CAB InternationalCABI Publishing CABI PublishingCAB International 10 E 40th StreetWallingford Suite 3203Oxon OX10 8DE New York, NY 10016UK USA

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© CAB International 2002. All rights reserved. No part of this publication may bereproduced in any form or by any means, electronically, mechanically, by photocopying,recording or otherwise, without prior permission of the copyright owners.

A catalogue record for this book is available from the British Library, London, UK.

Library of Congress Cataloging-in-Publication DataDesiccation and survival in plants : drying without dying / edited by M. Black and H.W. Pritchard.

p. cm.Includes bibliographical references (p. ).ISBN 0-85199-534-9 (alk. paper)

1. Plants--Drying. 2. Plant-water relationships. 3. Plants--Adaptation. I. Black, Michael. II. Pritchard, H. W.

QK870 .D57 2002581.4--dc21

2001043835ISBN 0 85199 534 9

Typeset in Melior by Columns Design Ltd, ReadingPrinted and bound in the UK by Biddles Ltd, Guildford and King’s Lynn

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Contents

Contributors vii

Preface ix

PART I. INTRODUCTION 1

1 Drying Without Dying 3Peter Alpert and Melvin J. Oliver

PART II. METHODOLOGY 45

2 Methods for the Study of Water Relations Under Desiccation Stress 47Wendell Q. Sun

3 Experimental Aspects of Drying and Recovery 93Norman W. Pammenter, Patricia Berjak, James Wesley-Smith and Clare Vander Willigen

4 Biochemical and Biophysical Methods for Quantifying Desiccation Phenomena in Seeds and Vegetative Tissues 111Olivier Leprince and Elena A. Golovina

PART III. BIOLOGY OF DEHYDRATION 147

5 Desiccation Sensitivity in Orthodox and Recalcitrant Seeds in Relation toDevelopment 149Allison R. Kermode and Bill E. Finch-Savage

6 Pollen and Spores: Desiccation Tolerance in Pollen and the Spores of Lower Plants and Fungi 185Folkert A. Hoekstra

7 Vegetative Tissues: Bryophytes, Vascular Resurrection Plants and Vegetative Propagules 207Michael C.F. Proctor and Valerie C. Pence

8 Systematic and Evolutionary Aspects of Desiccation Tolerance in Seeds 239John B. Dickie and Hugh W. Pritchard

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PART IV. MECHANISMS OF DAMAGE AND TOLERANCE 261

9 Desiccation Stress and Damage 263Christina Walters, Jill M. Farrant, Norman W. Pammenter and Patricia Berjak

10 Biochemistry and Biophysics of Tolerance Systems 293Julia Buitink, Folkert A. Hoekstra and Olivier Leprince

11 Molecular Genetics of Desiccation and Tolerant Systems 319Jonathan R. Phillips, Melvin J. Oliver and Dorothea Bartels

12 Rehydration of Dried Systems: Membranes and the Nuclear Genome 343Daphne J. Osborne, Ivan Boubriak and Olivier Leprince

PART V. RETROSPECT AND PROSPECT 365

13 Damage and Tolerance in Retrospect and Prospect 367Michael Black, Ralph L. Obendorf and Hugh W. Pritchard

Glossary 373

Taxonomic Index 383

Subject Index 401

vi Contents

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Contributors

Peter Alpert, Biology Department, University of Massachusetts, Amherst, Massachusetts01003-5810, USA. [email protected]

Dorothea Bartels, Institute of Botany, University of Bonn, Kirschallee 1, D-53115 Bonn,Germany. [email protected]

Patricia Berjak, School of Life and Environmental Sciences, University of Natal, Durban4041, South Africa. [email protected]

Michael Black, Division of Life Sciences, King’s College, Franklin Wilkins Building, 150Stamford Street, London SE1 6NN, UK. [email protected]

Ivan Boubriak, The Oxford Research Unit, Open University, Foxcombe Hall, Boars HillOX1 5HR, UK. [email protected]

Julia Buitink, UMR Physiologie Moléculaire des Semences, Institut Nationald’Horticulture, 16 Bd Lavoisier, F49045 Angers, France. [email protected]

John B. Dickie, Seed Conservation Department, Royal Botanic Gardens Kew, WakehurstPlace, Ardingly, West Sussex RH17 6TN, UK. [email protected]

Jill M. Farrant, Department of Molecular and Cellular Biology, University of Cape Town,7700, South Africa. [email protected]

Bill E. Finch-Savage, Horticulture Research International, Wellesbourne, Warwick CV359EF, UK. [email protected]

Elena A. Golovina, Laboratory of Plant Physiology, Department of Plant Sciences,University of Wageningen, Arboretumlaan 4, 6703 BD Wageningen, The Netherlandsand Timiryazev Institute of Plant Physiology, Botanicheskaya 35, Moscow 127276,Russia. [email protected]

Folkert A. Hoekstra, Laboratory of Plant Physiology, Department of Plant Sciences,University of Wageningen, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands.Folkert. [email protected]

Allison R. Kermode, Department of Biological Sciences, Simon Fraser University,Burnaby, BC, V5A 1S6, Canada. [email protected]

Olivier Leprince, UMR Physiologie Moléculaire des Semences, Institut Nationald’Horticulture, 16 Bd Lavoisier, F49045 Angers, France. [email protected]

Ralph L. Obendorf, Seed Biology, Department of Crop and Soil Sciences, CornellUniversity, Ithaca, New York, USA. [email protected]

Melvin J. Oliver, USDA-ARS Plant Stress and Germplasm Development Unit, 3810 4thStreet, Lubbock, Texas 79415, USA. [email protected]

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Daphne J. Osborne, The Oxford Research Unit, Open University, Foxcombe Hall, BoarsHill OX1 5HR, UK. [email protected]

Norman W. Pammenter, School of Life and Environmental Sciences, University of Natal,Durban 4041, South Africa. [email protected]

Valerie C. Pence, CREW, Cincinnati Zoo and Botanical Garden, 3400 Vine Street,Cincinnati, OH 45220, USA. [email protected]

Jonathan R. Phillips, Max-Planck-Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, D-550829 Köln, Germany. [email protected]

Hugh W. Pritchard, Seed Conservation Department, Royal Botanic Gardens Kew,Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK. [email protected]

Michael C.F. Proctor, School of Biological Sciences, University of Exeter, WashingtonSinger Laboratories, Perry Road, Exeter EX4 4QG, UK. [email protected]

Wendell Q. Sun, Department of Biological Sciences, National University of Singapore,Kent Ridge Crescent, Singapore 119260. [email protected]

Clare Vander Willigen, Department of Botany, University of Capetown, Private Bag,Rondebosch 7701, South Africa. [email protected]

Christina Walters, USDA-ARS National Seed Storage Laboratory, 1111 South MasonStreet, Fort Collins, CO 80521, USA. [email protected]

James Wesley-Smith, School of Life and Environmental Sciences, University of Natal,Durban 4041, South Africa. [email protected]

viii Contributors

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Preface

Plant survival of desiccation as sporophytic and gametophytic tissues was lastreviewed in detail in two books published in 1980. The first, by J. Levitt, onResponses of Plants to Environmental Stresses, Volume II, Water, Radiation, Saltand Other Stresses is a classic. The topic of plant water stress consumes about200 pages and is set mainly at the introductory level but is still of sufficientdetail to stimulate post-graduate researchers. Interestingly, there is no mentionin Levitt’s book of desiccation sensitivity in seeds. However, this latter topic wasspecifically covered in another book by H.F. Chin and E.H. Roberts (RecalcitrantSeeds). At that time recalcitrant seed behaviour was something of a novelty andthe book deals mainly with descriptions of germination, a listing of species withsuch seeds and an indication of how best to store the material in the short term.Aspects of plant desiccation have been considered in other publications dealingwith the biology and biophysics of dehydration and in contributions to generalworks on seeds but a comprehensive treatment of desiccation and plant survivalis not yet available.

Since 1980 there has been a revolution in plant science as new methods ofcell and molecular biology and biophysics have been applied to environmentalstress, particularly in relation to desiccation tolerance. The basic level of under-standing of how plant cells cope with extreme water stress has increasedtremendously and considerable effort has been made in the last 10 years todevelop diagnostic markers for desiccation tolerance. At the physiological level,studies have often focused on seed material and on the responses of resurrectionplants. At a more mechanistic level, model membrane systems have been usedextensively, and exploration of the molecular genetics of desiccation tolerancehas begun on developmental mutants, especially of seeds of crop species.

These progressive but fundamental changes in approach to investigating thebasis of survival of plant tissues under desiccation since the 1980s have meantthat our perceptions of this subject have altered significantly. It seems particularappropriate now to take stock of these recent developments, to assess criticallythe importance of the experimental systems available for investigation and to

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consider possible foci for future research work. This book sets out to addressthese issues. The Introduction surveys the topic of desiccation, and the remain-der of the book is divided into four parts, dealing with: (i) the technical back-ground to desiccation tolerance studies; (ii) the frequency and levels ofdehydration stress tolerance in biological systems; (iii) mechanisms of damageand tolerance; and (iv) a brief retrospect and prospect. It will not attempt toaddress in detail plant drought stress (i.e. at relatively high water potentials).This subject has been covered in detail in the last 10 years, for example inEnvironmental Stress in Plants – Biochemical and Physiological Mechanisms(Cherry. J.H. (ed.), Springer Verlag, 1989) and Plants Under Stress (Jones, H.G.,Flowers, T.J. and Jones, M.B. (eds), SEB Seminar Series, Cambridge, 1989).However, drought stress will be referred to in several places within this text.

In dealing with the different aspects of desiccation it is inevitable that certaintopics will receive consideration in more than one chapter. But the authors andeditors have attempted, as far as is possible, to avoid repetition of detail.Extensive cross-referencing has been used, to aid the reader in identifyingwhere, within the special viewpoints of the treatments, similar subjects areconsidered.

This comprehensive presentation on desiccation and survival in plantswill be of value to all researchers in the field, both beginners and the moreexperienced, and to those with interests in basic and applied plant sciences –physiology, ecology, conservation biology, agriculture and horticulture.

M. BlackH.W. Pritchard

x Preface

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Part I

Introduction

01 Desiccation -Chap 1 18/3/02 1:53 pm Page 1


1 Drying Without Dying

Peter Alpert1 and Melvin J. Oliver21Biology Department, University of Massachusetts, Amherst, Massachusetts

01003-5810, USA; 2Plant Stress and Water Conservation Laboratory, Agricultural Research Service, US Department of Agriculture, 3810 4th Street,

Lubbock, Texas 79415, USA

1.1. Introduction 41.2. Defining and Measuring Desiccation Tolerance 4

1.2.1. Operational and conceptual definitions 41.2.2. Measuring tolerance 6

1.3. A Brief History of Research on Desiccation Tolerance 61.3.1. Early work (1702–1860) on the question of whether life can

stand still 61.3.2. The next step: establishing records 7

1.4. The Occurrence of Desiccation Tolerance in Plants: Rarity and Ubiquity 81.4.1. Seeds, pollen and spores 81.4.2. Vegetative tissues 9

1.5. The Ecology of Desiccation Tolerance in Plants: a Diversity of Cycles inMarginal Habitats 131.5.1. Habitats 171.5.2. Cycles 171.5.3. Hypotheses 19

1.6. Mechanisms of Desiccation Tolerance 201.6.1. Damage 21

1.6.1.1. Damage during desiccation 211.6.1.2. Damage during rehydration 221.6.1.3. Poikilochlorophylly 23

1.6.2. Protection 241.6.2.1. Proteins 241.6.2.2. Sugars 26

1.6.3. Repair 281.7. Future Prospects and Agricultural Significance 301.8. References 31

© CAB International 2002. Desiccation and Survival in Plants: Drying Without Dying(eds M. Black and H.W. Pritchard) 3


1.1. Introduction

Water is a universal requirement for life aswe know it. Water is the most abundantcompound in all active cells, it is essentialfor metabolism and all organisms must takein water to survive. Living things thereforeface a major problem whenever theyemerge above ground on land: the air isalmost always drier than they are and takeswater from them. This is a life and deathproblem for most organisms in most habi-tats, because the air is at least sometimesdeadly dry. For example, when the relativehumidity is about 50% and the tempera-ture 28°C, a plant cell that dries to equilib-rium will drop to a water potential of about�100 MPa (Gaff, 1997). This kills over 99%of flowering plants.

Terrestrial plants appear to haveevolved two solutions to the problem ofmaintaining an aqueous self in a witheringworld. The majority solution, at least at thepresent evolutionary time, is never to dryout – to maintain a chronic disequilibriumbetween wet cells and dry air. Some of themost universal features of plant form, suchas waxy coatings on shoots, and pores thatcan open and close on leaves, seem largelydesigned to conserve water.

The minority solution is to dry up butnot die – to desiccate during drought andrehydrate and resume growth whendrought ends. About 300 species of flower-ing plants, or perhaps 0.1% of those named,are known to tolerate desiccation(Porembski and Barthlott, 2000). Some ofthese species can lose all of the free waterin their cells or remain dry for up to 5 yearsand still recover (Gaff, 1977). These prodi-gious abilities raise the first and fundamen-tal question about desiccation tolerance:How do plants survive desiccation? Mostrecent research on desiccation tolerance hasfocused on discovering the mechanisms ofdesiccation tolerance, partly in the hopes ofsome day engineering tolerance in econom-ically important species and banishing thespectre of famine from drought.

However, the ability to survive desicca-tion may not always increase the ability ofplants to survive in natural systems.

Though some desiccation-tolerant plantscan survive droughts more intense and pro-longed than any that occur almost any-where on earth, tolerant plants are in theminority. Desiccation-sensitive plants dom-inate the world’s vegetation. The rarity ofthe apparently excellent ability to toleratedesiccation raises a second, cautionaryquestion about desiccation tolerance: Howdoes surviving desiccation affect plant sur-vival? These two questions, one largelygenetic and biochemical and the othermainly physiological and ecological, framethe topic of desiccation and plant survival.

The purpose of this introductory chap-ter is to summarize some of the currentanswers to these questions and lead intothe more detailed reviews of questions andanswers about desiccation and plant sur-vival in the chapters that follow. We beginwith some terms and techniques that pro-vide concepts and methods for research ondesiccation tolerance in plants, and a briefsummary of the surprisingly lively historyof research on desiccation tolerance. Wethen give an overview of the range andecology of desiccation tolerance in plants,subjects that bear on how surviving desic-cation affects plant survival. Last, we dis-cuss mechanisms of desiccation tolerancein plants, the keys to understanding howplants survive desiccation, and considerthe potential for breeding crops that candry without dying. We will sometimesabbreviate desiccation tolerance to ‘toler-ance’, and we will call plants that cannottolerate desiccation ‘desiccation-sensitive’or ‘sensitive’. We will consider desiccationtolerance in plants and in some organismsthat are not in the plant kingdom, mainlycyanobacteria, algae and fungi.

1.2. Defining and MeasuringDesiccation Tolerance

1.2.1. Operational and conceptual definitions

Desiccation tolerance can be operationallydefined as the ability to dry to equilibriumwith moderately dry air and then resumenormal function when rehydrated, where

4 P. Alpert and M.J. Oliver


‘moderately dry’ means 50–70% relativehumidity at 20–30°C . This definition isworkable because there seems to be a widegap between the maximum tolerance ofsensitive plants and the minimum toler-ance of tolerant ones (Gaff, 1997). Almostall species known to recover from completedrying at 80% relative humidity alsorecover from drying at 50% (but seeBochicchio et al., 1998).

The reason why a gap exists between theranges of tolerance of drying in desiccation-sensitive and desiccation-tolerant plantsmay be that desiccation tolerance dependson the ability to reversibly cease metab-olism as cells dry out. There may not bevery many marginally desiccation-tolerantplants because, once metabolism hasstopped, it cannot be stopped further. Clegg(1973) argued on biochemical grounds thatmetabolism, defined as ‘systematically con-trolled pathways of enzymatic reactions’(Clegg, 2001), cannot take place at a cellwater content of less than 0.1 g H2O g�1

dry mass because not enough waterremains to hydrate intracellular proteins.Organisms this dry do show chemicalactivity. For instance, dried pollen canincorporate water vapour into organic com-pounds (Wilson et al., 1979). However,chemical reactions, even some characteris-tic of living things such as oxygen uptake,do not necessarily require metabolism: ironrusts (Clegg, 1986). We propose that desic-cation tolerance can be conceptuallydefined as reversible cessation of metab-olism in response to water loss.

This suggests that the mechanisms ofdesiccation tolerance must involve at leasttwo key elements (Section 1.6). First, theremust be an orderly shutdown of metabo-lism during desiccation. Different meta-bolic pathways must slow at compatiblerates to avoid fatal accumulations of inter-mediates and generation of free radicals.Oxidation is a major hazard of desiccation(e.g. Smirnoff, 1993), and the advantages ofminimizing photo-oxidation may explainwhy some desiccation-tolerant plants ceasephotosynthesis at relatively high watercontents during drying (e.g. Sherwin andFarrant, 1998; Tuba et al., 1998; Farrant,

2000). Some tolerant species are more dam-aged by being held at intermediate watercontents than at full hydration or completedesiccation (Gaff, 1997), and one advantageof rapid desiccation may be to minimizetime spent at intermediate levels of hydra-tion (Kappen and Valladares, 1999; Proctor,2000; Chapters 3 and 5). Second, cells mustpreserve enough cellular organization andfunctional enzymes so that metabolism canresume after rewetting. Preserving a skele-tal machinery for metabolism must involveboth protection and repair (Section 1.6).Enzymes and membranes must be pro-tected from loss of configuration and orga-nization, and the damage that accumulatesfrom degradative non-metabolic reactionswhile plants are inactive must be repaired.

Differences in effectiveness of protec-tion may explain much of why desiccation-tolerant plants do differ in the intensity(minimum water content or water poten-tial) of desiccation that they can stand. Forinstance, tolerant angiosperms tend to sur-vive equilibration with lower relativehumidities than do tolerant pteridophytesin South Africa (Gaff, 1977). Species with agreater degree of protection of molecularconfiguration and cellular organizationmay survive with smaller fractions ofwater. Differences in effectiveness of repairmay help explain why species also differ inthe duration of desiccation (length of timein the dried state) that they can stand (e.g.Sagot and Rochefort, 1996). Those withmore effective repair mechanisms may bebetter able to undo non-metabolic degrada-tion suffered while dry.

There has been some confusion aboutthe difference between ‘desiccation toler-ance’ and ‘drought tolerance’. We wouldlike to propose that desiccation tolerance isone form of drought tolerance. Drought may be defined as any level of water avail-ability that is low enough to reduce plantperformance. ‘Drought tolerance’ is mostoften used to refer to tolerance of wateravailabilities that are suboptimal but notlow enough to cause complete drying toequilibrium with the air, i.e. desiccation.Mechanisms of drought tolerance includeways of maintaining cell water content,

Drying Without Dying 5


such as osmotic regulation and stomatalclosure, whereas desiccation tolerance con-sists of ways of surviving the nearly com-plete loss of water. Some papers onintertidal algae maintain the confusion byusing ‘desiccation’ to refer to any amount ofwater loss (e.g. Leuschner et al., 1998; Bjorket al., 1999). They are probably wrong,since the Oxford English Dictionary (1989)defines ‘to desiccate’ as ‘to make quite dry;to deprive thoroughly of moisture’.

1.2.2. Measuring tolerance

Techniques for quantifying the degree ofdesiccation tolerance in different speciesare reviewed in Chapters 2–4 and 7.Chapter 2 discusses the advantages andlimitations of different measures of watercontent and techniques for distinguishingwater properties in plant cells. Chapter 3notes how the survival and recovery ratesof seeds and vegetative tissues vary withrate of drying, light conditions during dry-ing, storage conditions and length of timein the dehydrated state. In general, highlydesiccation-tolerant bryophytes can sur-vive rapid drying but tolerant angiospermscannot; this seems to be related to differ-ences in their mechanisms of tolerance(Section 1.6). A few species appear insensi-tive to rate of drying, but most probablyhave an optimal rate or optimal range ofdrying rates. For instance, desiccation inless than 6 hours or over more than 7 dayskills the otherwise highly tolerant pterido-phyte Selaginella lepidophylla (Eickmeier,1983). Rates and final levels of recoverycan decrease with increasing intensity orduration of desiccation (e.g. Gaff, 1977;Alpert and Oechel, 1987; Davey, 1997).Quantifying desiccation tolerance thereforealso requires techniques for imposingknown rates, intensities and durations ofdesiccation, and for measuring rates andfinal levels of recovery (Chapter 3). Rate ofdrying is particularly hard to standardizeacross species.

Investigating the mechanisms of desic-cation requires techniques for measuringprocesses and states in cells as they

undergo cycles of drying and wetting.Chapter 4 reviews the rapidly expandingrange of non-invasive techniques availableto study the diffusion of water, the configu-rations and interactions of macromole-cules, metabolism, thermal eventsassociated with membrane phase transi-tions, ultrastructure, oxidative stress, fer-mentation and the physical properties ofmembranes, cytoplasm and protein com-plexes during desiccation and rehydration.Chapter 7 summarizes some of the techni-cal developments in infrared gas analysisand fluoroscopy that have improved ourcapacity to quantify responses to desicca-tion on the physiological level.

1.3. A Brief History of Research onDesiccation Tolerance

The 300-year history of the science of desic-cation tolerance began with a lengthyperiod of discovery and doubt. In thecourse of discovering desiccation tolerance,scientists confronted the nature of life. Thenext step was to enumerate the organismsthat tolerate desiccation and test the limitsof their tolerance. In the 1960s, researchersstarted to investigate the physiological ecol-ogy of desiccation tolerance in plants, espe-cially the cycles of wetting and drying andtheir effects on carbon uptake in bryophytesand lichens. Since the 1980s, emphasis hasshifted to the biochemistry and molecularbiology of desiccation tolerance. We nowknow more about how plants survive desic-cation than about how tolerating desicca-tion affects plant survival.

1.3.1. Early work (1702–1860) and thequestion of whether life can stand still

It took scientists one and a half centuries toestablish that desiccation tolerance exists(Keilin, 1959). At the end of an often ran-corous debate, the nature of life had beencalled into question: Can life stop, be con-tained in a static array of molecules andrestart? Anthony von Leeuwenhoek wasapparently the first to glimpse desiccation



tolerance, soon after he invented the micro-scope. In 1702, he wrote to a friend (seeSchierbeek, 1959):

The following day the sky was very hot anddry and, about nine in the morning, I tooksome of the sediment which has been in theleaden gutter … and poured on it a smallquantity of rain-water taken out of my stonecistern … so that if there were still any livinganimalcules in it they might issue forth;though I confess I never thought that therecould be any living creatures in a substanceso dried as this was.

I was, however, mistaken; for scarce anhour had elapsed, when I saw at least ahundred of the animalcules before described.

These animals were rotifers. By the mid-1800s, others had seen desiccation toler-ance in two more phyla of animals,nematodes and tardigrades (Keilin, 1959;Alpert, 2000). However, others still flatlydenied it was possible to survive dryingout. A French biologist, Felix Pouchet(1859), wrote that: ‘Dry and completelymummified animals cannot be resuscitatedby hydration. Rational beliefs, observation,and experiment unite to demonstrate it.’The Société de Biologie in Paris conveneda special commission and conducted itsown tests on rotifers. Its report effectivelysettled the matter: ‘[organisms] reachingthe most complete degree of desiccationthat can be realized … may yet retain theability to revive in water’ (Broca, 1860).

However, the commission was silent onthe deeper question of whether this meansthat life can stop and restart. As phrased byPouchet’s main scientific opponent(Doyère, 1842), ‘Has there been a mereslowing down of the vital phenomena, …or truly an absolute destruction that onecould compare to death itself?’

The modern consensus on this questionseems to be that some organisms can slowtheir metabolism at least to the point atwhich it cannot be detected against thebackground of physical chemical reactionsand then resume normal metabolism(Keilin, 1959; Hinton, 1968; Clegg, 2001).The most convincing evidence may be thatsome desiccated tardigrades, rotifers,seeds, spores, algae, lichens and mosses

can be cooled to within 0.5°C of absolutezero, absolute zero being the temperature atwhich all molecular motion is believed tostop, and then revive when rewarmed andrehydrated (Becquerel, 1951). The discov-ery of desiccation tolerance has shown usthat living things can come to exist in threestates: alive, dead and still (Clegg, 2001).

1.3.2. The next step: establishing records

The scientific battle over whether livingthings could dry without dying was foughtover animals. Starting in the 1960s, toler-ance was identified in the larvae of at leastone insect and of some other arthropods(Hinton, 1968; Crowe et al., 1992) but hasnever been found in any life stage of anyvertebrate or in the adults of any animalsexcept microscopic rotifers, nematodes andtardigrades. In contrast, tolerance wasfound to be widespread in plants. Tolerantbryophytes were reported by 1886, ferngametophytes by 1914, fern sporophytes by1931 and angiosperms by 1921 (tables andcitations in Kappen and Valladares, 1999;Alpert, 2000; Chapter 7).

The intensity and duration of desicca-tion that plants and plant-like organismscould survive were shown to be remark-able. Like tardigrades (Doyère, 1842), cer-tain lichens, bryophytes, pteridophytes andangiosperms survived equilibration withair of nearly 0% relative humidity, inclosed volumes over concentrated H2SO4or P2O5 (e.g. Lange, 1953; Hosokawa andKubota, 1957; Gaff, 1977). The liverwortRiccia macrocarpa produced new apicalcells after 23 years of air-dryness (Breuil-Sée, 1993); the moss Grimmia laevigatagrew when rehydrated after 10 years in aherbarium (Keever, 1957); lichens survived10 years of being desiccated and frozen(Larson, 1988); and leaves of ferns andflowering plants took up neutral red dye orexcluded Evans blue dye after 5 years ofair-dryness (Gaff, 1977). Some desiccation-tolerant plants clearly survive longer andmore intense drought than ever occurswhere they grow, raising the question ofwhat has selected for such tolerance.



Dessicated plants were also shown totolerate other extreme stresses. Various taxasurvived extreme cold (Becquerel, 1951;Pence, 2000), and some mosses survivedheating to 100°C (Glime and Carr, 1974;Norr, 1974). Eickmeier (1986) found thatmore desiccation-tolerant populations ofSelaginella were also more heat-tolerant.The fungus Schizophyllum commune pro-duced hyphae after 34 years in a vacuum ofless than 0.01 mm Hg (Bisby, 1945), show-ing long-term tolerance of both desiccationand lack of oxygen. Takács et al. (1999) cor-related desiccation and UV-B tolerance in aset of bryophyte species.

The correlation between tolerance of des-iccation and tolerance of cold, heat andanoxia has suggested that there may besome basic properties or mechanisms thatconfer ‘broad-spectrum’ tolerance. Sincefreezing often dehydrates cells, cold anddesiccation stress have an obvious func-tional link. Another parallel between desic-cation and cold tolerance is that both can be‘softened’ by periods of low stress and‘hardened’ by ones of moderate stress.Plants may lose some of their desiccationtolerance after prolonged periods of fullhydration (e.g. Gaff, 1977; Schonbeck andBewley, 1981; Kappen and Valladares,1999). Desiccation tolerance can vary sea-sonally (Dilks and Proctor, 1976; Gaff, 1980)and increase in winter (Kappen, 1964).However, the correlation between toleranceof desiccation and other stresses is notabsolute. Wood and Gaff (1989) saw no cor-relation between desiccation and salinitytolerance in species of the grass Sporobolus.

As records of desiccation toleranceaccumulated, pictures emerged of the taxo-nomic and geographic ranges of desicca-tion tolerance in plants. These picturesremain somewhat haphazard because therehave been few systematic surveys for desic-cation tolerance within taxa or habitats.Relatively extensive lists exist for seeds(Chapter 8). A survey of all the soil algae atone site was published by Evans (1959).The lists of tolerant vascular plants fromdifferent regions published by Gaff and co-workers (e.g. Gaff, 1977, 1986; Gaff andLatz, 1978) and from rock outcrops by

Porembski and co-workers (e.g. Porembskiand Barthlott, 2000) are probably the clos-est approaches to surveys for whole plants.The overall pattern one sees is taxonomicand geographic breadth contrasted withecological narrowness.

1.4. The Occurrence of DesiccationTolerance in Plants: Rarity and Ubiquity

Part of the puzzle of desiccation tolerance inplants is that it is both very uncommon andnearly universal (Alpert, 2000). The relativebiomass of desiccation-tolerant plants in allbut the most arid or frigid habitats is verylow, and fewer than one in a thousandspecies of flowering plants is known to toler-ate desiccation. At the same time, desicca-tion-tolerant species are found on allcontinents, in all major plant groups exceptgymnosperms, and among species of allgrowth forms except trees; and the greatmajority of flowering plants and also gym-nosperms have desiccation-tolerant seeds orpollen or both. Desiccation tolerance appearsto be a universal evolutionary potential ofplant cells that has been little selected forexcept in resting stages of the life cycle andin organisms that have not evolved effectiveways of avoiding desiccation.

Detailed reviews of the occurrence ofdesiccation tolerance in seeds, pollen andother spores, and vegetative tissues aregiven in Chapters 5, 6, 7 and 8. Other recentreviews of the occurrence of tolerance inadult plants and non-plant non-animalsinclude those of Kappen and Valladares(1999), Alpert (2000), and Porembski andBarthlott (2000). In this section, some of themain points in these reviews will be dis-cussed, a few of the examples they give willbe mentioned and some additional exam-ples and points will be presented.

1.4.1. Seeds, pollen and spores

As in adult plants, the desiccation toler-ance of seeds can vary greatly betweenspecies within genera, between individualswithin species and between tissues within



individuals; and there is a continuum ofdegree of tolerance across species(Chapters 5 and 8). However, whereas des-iccation tolerance is rare in adult floweringplants, it is so much the rule in their seedsthat tolerant seeds are traditionally knownas ‘orthodox’ and desiccation-sensitiveseeds as ‘recalcitrant’. Desiccation sensitiv-ity may be a derived character in seeds,evolved through neoteny, and is probablyassociated with large seeds and trees(Chapter 8). Another difference betweendesiccation tolerance in adult plants andseeds is that tolerance and desiccation areenvironmentally induced in adults but maybe developmentally programmed in seeds.Seeds become tolerant as part of develop-ment and dry because the parent withholdsor withdraws water from them. Once theygerminate, the seedlings of desiccation-sen-sitive species with desiccation-tolerantseeds lose their tolerance within hours.The obvious ecological advantage of ortho-doxy is that seeds can survive periods ofdrought and disperse the offspring of aplant more widely in space and time,although orthodoxy is not a prerequisite fordormancy (Chapter 5). Two advantages ofbeing recalcitrant are that seeds need neverstop growing and may germinate morerapidly – as in whole plants, there may bea trade-off between desiccation toleranceand productivity in seeds.

Desiccation tolerance is probably alsothe rule rather than the exception in pollenand spores, and tolerance and desiccationare developmentally programmed in sporesas in seeds (Chapter 6). However, there areat least three differences between tolerancein seeds and in spores. Tolerant pollen hasno dormancy, it survives no more than afew months of dry storage at room tempera-ture, and spores of some pteridophytes cansurvive cycles of drying and wetting.Desiccation-sensitive pollen is relativelycommon in species of Poaceae,Cucurbitaceae and Araceae (Chapter 6),and may be associated with hot, humidhabitats. The prevalence of desiccation tol-erance in seeds and spores is one reason tobelieve that the genetic potential to toleratedesiccation exists in all plants.

1.4.2. Vegetative tissues

Desiccation tolerance appears commonthough not universal in bryophytes (e.g.Richardson, 1981; Proctor, 1990), commonin lichens (Kappen and Valladares, 1999),uncommon in pteridophytes and rare inangiosperms (Chapter 7). No gymnospermsare known to tolerate desiccation (Gaff,1980; Chapter 7), even though gym-nosperms may have desiccation-tolerantseeds or pollen (Chapters 5 and 6).Desiccation tolerance occurs in non-lich-enized fungi, cyanobacteria and algae (Ried,1960; Mazur, 1968; Bertsch, 1970;Schonbeck and Norton, 1978; Potts, 1994,1999; Dodds et al., 1995) but little is knownabout its extent. It must be very common infree-living algae and bacteria that grow onthe surface of plants or soil, where they arevery probably subject to desiccation.

Different vegetative parts of a plantmay have different degrees of tolerance.There seem to be two main patterns. First,in some species only the perennatingstructures survive desiccation, such ascorms in Limosella grandiflora (Gaff andGiess, 1986) or special dry-season organsin the small shrub Satureja gilliesii(Montenegro et al., 1979). As in plantsthat are desiccation-sensitive but havedesiccation-tolerant seeds, tolerance inthese species is confined to relativelyinactive plant parts. Second, leaves maybe more desiccation-tolerant whenyounger. Younger leaves are more tolerantthan older ones in Chamaegigas intre-pidus (Gaff and Giess, 1986) and somespecies of Borya (Gaff, 1989). In the leavesof some grasses, only the basal meristem-atic zone tolerates drying (Gaff andSutaryono, 1991). This suggests that sometissues may lose tolerance as they differ-entiate or age; the processes involvedcould conceivably parallel those thatcause loss of tolerance after germinationof seeds. In all these examples of differen-tial tolerance in leaves, there are con-geners whose leaves remain tolerant asthey mature, offering inviting systems forcomparative studies of the ecology andmechanisms of desiccation tolerance.



No one appears to have assessed the rel-ative prevalence of desiccation tolerance indifferent taxa of bacteria, cyanobacteria,fungi and algae. Acinetobacter radioresis-tans survives 150 days at 31% relativehumidity, which helps make it a persistentsource of infection in hospitals (Jawad etal., 1998). At least 400 species of algae andcyanobacteria tolerate desiccation (e.g.Davis, 1972; Potts, 1994, 1999; Trainor andGladych, 1995). Evans (1959) found thatmany but not all of the freshwater algae inpond mud survived desiccation in thefield; at least two species survived 69 daysof desiccation in the laboratory withoutforming resting stages. Two interesting phe-nomena that have been reported from somegreen algae but apparently not from othergroups are dependence of tolerance onnutrient availability (McLean, 1967, citedin Chandler and Bartels, 1999) and loss ofcapacity to reproduce after desiccation(Hsu and Hsu, 1998). We know of fewreports of desiccation tolerance in non-lichenized fungi (Bisby, 1945;Zimmermann and Butin, 1973), but there isan extensive literature on tolerance inlichens, at least 50 species of which havebeen shown to tolerate desiccation(Kappen and Valledares, 1999).

Desiccation tolerance is broadly butunevenly distributed among taxa in plants.Most of the 25,000–30,000 species ofbryophytes probably tolerate at least briefdesiccation of low intensity (Chapter 7);the proportion of desiccation-tolerantspecies appears to differ between orders ofmosses and to be higher in mosses than inliverworts. There are also desiccation-toler-ant hornworts (Oliver et al., 2000).

Porembski and Barthlott (2000) estimatedthat there are 275–325 desiccation-tolerantspecies of vascular plants. At least nine fam-ilies of pteridophytes and seven families ofangiosperms contain desiccation-tolerantsporophytes (Chapter 7). Some fern gameto-phytes also tolerate desiccation (e.g. Pence,2000). Groups of ferns and allies that seemto be relatively rich in desiccation-tolerantspecies include the family Pteridaceae andthe genera Cheilanthes and Selaginella(Gaff, 1977; Gaff and Latz, 1978; Kappen

and Valladares, 1999; Porembski andBarthlott, 2000). Desiccation-tolerant mono-cotyledons outnumber tolerant dicotyle-dons. The monocotyledonous familyVelloziaceae may have over 200 tolerantspecies (Kubitzki, 1998). At least 39 speciesof Poaceae tolerate desiccation (Gaff, 1997).One very small family of angiosperms, theMyrothamnaceae, is entirely desiccation-tolerant (Porembski and Barthlott, 2000). Atthe other extreme, some species, such asBorya nitida (Liliaceae), contain both toler-ant and sensitive individuals (Gaff, 1981).Phylogenetic analysis suggests that desicca-tion tolerance in active phases of the lifecycle has evolved at least eight separatetimes in vascular plants (Oliver et al., 2000).

Desiccation-tolerant angiosperms arealso widely but unevenly geographicallydistributed. They occur on all continentsexcept Antarctica, but very few species areknown from Europe or North America. TheEuropean species are all in two genera fromone family (Ramondia and Haberlea in theGesneriaceae) (Muller et al., 1997; Drazic et al., 1999). The North American speciesinclude three grasses (Iturriaga et al., 2000).The greatest concentrations of known desic-cation-tolerant angiosperms are in southernAfrica, western Australia and eastern SouthAmerica (Figs 1.1 and 1.2; Gaff, 1977, 1987;Gaff and Latz, 1978; Porembski andBarthlott, 2000). Different taxa predominatein each of these three areas.

Desiccation-tolerant plants have a widerange of morphological and physiologicalcharacteristics (Porembski and Barthlott,2000). There are desiccation-tolerant annualsand perennials, graminoids and forbs, andherbs, shrubs and arborescent rosette plants.Tolerant species may be caespitose, stolonif-erous or rhizomatous. Some species are xero-morphic, such as B. nitida (Gaff andChurchill, 1976); others are not, such as Boeahygroscopica (Gaff, 1981). A few desiccation-tolerant species, like C. intrepidus, have mor-phological features typical of aquatic plants(Gaff and Giess, 1986), and at least onespecies is succulent (Barthlott andPorembski, 1996). Desiccation-tolerantangiosperms can have crassulacean acidmetabolism (Barthlott and Porembski, 1996;




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Markovska et al., 1997) and probably C4 pho-tosynthesis (Lazarides, 1992). However, noplants more than 3 m tall and hence no treesare known to tolerate desiccation, possiblybecause they cannot re-establish upwardmovement of water once the xylem cavitatesduring desiccation (e.g. Sherwin et al., 1998).

The wide distribution of desiccation tol-erance in plants has suggested to someauthors that the basic mechanism of toler-ance must be simple (Chandler and Bartels,1999). According to the ‘water replacementhypothesis’ of Crowe et al. (1998a), theevolution of desiccation tolerance in all


Fig. 1.2. The large, isolated, granitic or gneissic outcrops known as inselbergs are a major habitat for desiccation-tolerant vascular plants in Australia, Brazil and Africa. (a) An inselberg in the Mata Atlantica of Brazil; (b) a mat ofthe pteridophyte Selaginella sellowii on a Brazilian inselberg; (c) an arborescent Brazilian monocot (Velloziaceae);(d) a species of Borya (Boryaceae, shown desiccated) in Australia; (e) Afrotrilepis pilosa (Cyperaceae, showndesiccated), a dominant, mat-forming species on inselbergs in West Africa. (Photos by S. Porembski.)

(b) (c)

(d) (e)

(a)


organisms depends on the selection andsynthesis of sufficient concentrations ofmolecular substitutes for water (Clegg,2001). Under certain circumstances, tre-halose may even induce desiccation toler-ance in human cells (Guo et al., 2000).However, tolerance in plants also involvesother mechanisms (Section 1.6), and theecology of desiccation-tolerant plants sug-gests that the evolution of tolerance inplants is constrained by its consequencesfor growth and competition.

1.5. The Ecology of DesiccationTolerance in Plants: a Diversity of Cycles

in Marginal Habitats

Desiccation-tolerant plants grow mainly inthe interstices and on the margins of theworld’s vegetation, in microhabitats and

habitats where desiccation-sensitive plantsdo not live (Fig. 1.3). In habitats wherewater availability and temperature aremoderate and sensitive plants are abun-dant, desiccation-tolerant vascular plantsgrow mostly on outcrops of bare rock(Porembski and Barthlott, 2000). In thedriest and coldest habitats, especiallywhere dew and fog are major water sources,desiccation-tolerant bryophytes, lichens,algae or cyanobacteria may form the onlyvegetation (e.g. Thompson and Iltis, 1968;Friedmann and Galun, 1974; Davey, 1997).Despite the ability of some of these speciesto tolerate a drought that is longer andmore intense than occurs in these habitats,the most xeric microsites are often stillbare (e.g. Alpert, 1985). On rocks and soilin the desert, small differences in exposureto the sun may determine whether a patchof soil or stone is colonized or not.


Fig. 1.3. (a) Exposed surfaces of granitic boulders in the western foothills of the Cuyamaca Mountains insouthern California are colonized mainly by an assemblage of desiccation-tolerant lichens and bryophytes.(b) Two of the most common mosses are Grimmia laevigata (left) and Grimmia apocarpa (right), shownhydrated. Crevices support desiccation-tolerant pteridophytes such as Pentagramma triangularis (gold-backfern), shown (c) desiccated and (d) hydrated. (Photos by P. Alpert.)

(a) (b)

(c) (d)


These patterns appear tied to the differ-ent sources of water that different desicca-tion-tolerant plants can use to rehydrate,the rates at which they rewet and dry out,and their ability to recover after desicca-tion and achieve a cumulative net gain ofresources. Lichens and bryophytes mayrewet from dew, recover in minutes and

dry out again in hours. Desiccation-tolerantvascular plants are only known to rewetfrom rain; they recover in hours to daysand dry out in days to weeks. The cumula-tive effect of repeated cycles of desiccationon net photosynthesis and growth mayexplain why desiccation-tolerant plants failto survive in the most exposed microsites.


Fig. 1.4. Desiccation-tolerant bryophytes are common even in cool, moist climates. The mosses (a)Orthotrichum anomalum, (b) Anomodon viticulosus, and (c) Tortula latifolia all occur in the UK. Each isshown in its desiccated and its rehydrated state. (Photos by M.C.F. Proctor.)

(c)

(a) (b)



Fig. 1.5. The most intensively studied desiccation-tolerant bryophyte is (a) Tortula ruralis, shown in the fullyhydrated state (top), after slow drying (lower right), and after rehydration for 2 min (lower left). T. ruralis iscommon in dry habitats in North America, as shown on rocks at Mesa Verde National Park, USA (b). One of themost studied desiccation-tolerant angiosperms is the grass Sporobolus stapfianus (c), shown after drying in a potfor 14 days (left) and after subsequent immersion in water for 24 h (right). (Photos by M.J. Oliver and B. Mishler.)

(c)

(a)

(b)



Fig. 1.6. Effects of desiccation and rehydration on ultrastructure in leaves of the moss Tortula ruralis. Thetransmission electron micrographs (after Bewley and Pacey, 1978) show papillose cells in (a) the fullyhydrated state (note chloroplast (C) with grana stacks (g), starch grains (s), and plastoglobuli (p, labelled in(b)); a vesicle (V) and rough (RER) and smooth endoplasmic reticulum (SER); a mitochondrion (m) withprominent internal membranes or cristae; and electron-dense bodies (E); and (b) after desiccated plants hadbeen rehydrated for 5 min (note that the chloroplasts are swollen but the nucleus (N) is not). Thefreeze–fracture micrograph (c) (from Platt et al., 1994) shows a portion of a cell from a slowly dried leaf(note the large, tightly appressed grana stacks (G) in the portion of the chloroplast visible and themitochondrion (M) outside the chloroplast.)

(a) (b)

(c)


Trade-offs between tolerance and growthand competition with sensitive plants mayexplain why desiccation-tolerant plants,though they can rise again from ‘apparentdeath’ (Doyère, 1842), have not dominatedthe earth.

1.5.1. Habitats

In contrast to the wide taxonomic and geographical ranges and the broad mor-phological diversity of desiccation-tolerantvascular plants, their ecological range isnarrowly confined to chronically or sea-sonally dry habitats or microhabitatswhere desiccation-sensitive plants aresparse or absent. Porembski and Barthlott(2000) estimated that 90% of desiccation-tolerant vascular plants are associatedwith rock outcrops, mainly in tropical tolower temperate latitudes. Some speciesgrow on exposed rock surfaces, while oth-ers are associated with crevices (Nobel,1978; Gildner and Larson, 1992).Ephemeral pools on rock outcrops inAfrica harbour a set of aquatic, desicca-tion-tolerant vascular plants (e.g. Volk,1984; Gaff and Giess, 1986). Tolerantangiosperms and pteridophytes also growin semiarid or desert grasslands, especially(Eickmeier, 1983; Gaff, 1987; Kappen andValladares, 1999) though not invariably(Gaff and Sutaryono, 1991), on shallowsoils. There are exceptions to this narrow-ness of ecological range. A few tolerantvascular species, such as Boeah hygro-scopica (Gaff, 1981) and Pentagrammatriangularis (P. Alpert, personal obser-vation), occur in forest understoreys.

Desiccation-tolerant bryophytes, lichensand algae occupy a much wider ecologicalrange than do tolerant vascular plants,including both less and more arid sites(Fig. 1.4). For example, tolerant bryophytesand lichens are common on rocks, trunksand soil in moderately moist forests. Theymay be common in tundra, although theSphagnum species characteristic of tundrado not necessarily recover net photosyn-thesis after losing more than about 10%of the water content they hold at the com-

pensation point for photosynthesis(Schipperges and Rydin, 1998). In warmand cold deserts, tolerant algae and lichensgrow inside or on the underside of translu-cent rocks (Friedmann and Galun, 1974;Kappen, 1993; Nienow and Friedmann,1993). Species of Nostoc, Anacystis andother cyanobacteria form desiccation-tolerant crusts on bare walls and rocksfrom the tropics to the boreal zone (Potts,1994; Lüttge, 1997). Algae, lichens andbryophytes join cyanobacteria to formcrusts on desert soils, which are importantin nitrogen cycling (e.g. Nash and Moser,1982; Lange et al., 1994, 1997). Sincelichens and bryophytes dry so rapidly, theymay be active mostly when conditions areeffectively mesic and function as ‘shadeplants’, even in exposed, xeric habitats(Green and Lange, 1994; Proctor, 2000).

Degree of desiccation tolerance seems toexplain some of the relative ability of toler-ant species to occupy xeric microsites orhabitats (e.g. Hernandez-Garcia et al., 1999;Franks and Bergstrom, 2000). For instance,ability to tolerate desiccation (Mitchell etal., 1999), to maintain photosynthesis dur-ing desiccation (Robinson et al., 2000) andto recover photosynthesis after repeatedcycles of desiccation (Davey, 1997) areassociated with occurrence of bryophytesin relatively dry sites in Antarctica. Theability to tolerate prolonged desiccationand to recover quickly upon rehydrationappeared necessary but not sufficient toallow mosses to colonize highly insolatedsurfaces on boulders in chaparral inCalifornia (Alpert, 1985; Alpert andOechel, 1987). A species of Selaginellafrom dry habitats recovered net photosyn-thesis faster than one from moister habitats(Eickmeier, 1980). Shirazi et al. (1996)reported differences in desiccation toler-ance between populations of lichens fromdifferent habitats.

1.5.2. Cycles

Desiccation-tolerant plants vary greatly inthe rates at which they dry out, rehydrateand recover upon rehydration and there-



fore in the rhythms of desiccation andgrowth they experience in nature (Tuba etal., 1998; Kappen and Valladares, 1999;Chapter 7). In general, bryophytes andlichens dry out in hours in the sun,whereas ferns and angiosperms take a dayor more. Minimum times to net photosyn-thesis after rehydration range from minutesin lichens and mosses rewetted with liquidwater, to hours in lichens and mosses rehy-drated with water vapour and in some vas-cular plants rewetted with liquid, to daysin other vascular plants (Fig. 1.5; Lange,1969; Lange and Kilian, 1985; Gaff andGiess, 1986; Reynolds and Bewley, 1993a;Scott and Oliver, 1994; Scheidegger et al.,1997; Tuba et al., 1998).

Desiccated lichens can resume net pho-tosynthesis by taking up water vapour(Hahn et al., 1993; Schroeter et al., 1994),but only if the phycobiont is a green algarather than a cyanobacterium (Kappen andValladares, 1999). A few mosses canrecover at least very slow rates of net pho-tosynthesis by taking up water vapour afterdesiccation (Lange, 1969; Rundel andLange, 1980). Both bryophytes and lichenscan rehydrate with dew (Lange et al., 1994;Csintalan et al., 2000). Despite the lack of acuticle, differences in thallus, leaf andshoot morphology and packing produceseveral-fold differences in drying ratesbetween different species of bryophytesand lichens (e.g. Gimingham and Smith,1971; Proctor, 1982; Scott, 1982;Valladares, 1994); differences in morpho-logical control of water loss may helpexplain differences in ability to colonizexeric microhabitats.

Desiccation-tolerant angiosperms areknown to rehydrate in nature only afterrain. Woody angiosperms may take longerto desiccate and rehydrate than herbaceousones (Sherwin and Farrant, 1996; Farrant etal., 1999). The slowest to recover from des-iccation are the poikilochlorophyllous des-iccation-tolerant plants, monocots thatdismantle their photosynthetic machinerywhen they dry and reassemble it againwhen they rehydrate (Sherwin and Farrant,1996; Tuba et al., 1998). In one desiccationstudy, the poikilochlorophyllous species

Xerophyta scabrida began to respire within20 min after rehydration, reached full ratesof respiration within 6 h, began to synthe-size chlorophyll after 12 h and did notcomplete synthesis until 36 h (Tuba et al.,1994). Poikilochlorophylly appears to be aprogrammed rather than a pathologicalresponse to desiccation. For instance, it is anecessary component of tolerance in theleaves of some species: when these leavesare detached before drying, they stay greenas they dry but they die (Gaff, 1981).

Natural cycles of wetting and dryinghave been followed for a number ofmosses and lichens (e.g. Kappen et al.,1979; Lange et al., 1994; Sancho et al.,1997) but very few desiccation-tolerantvascular plants (Nobel, 1978; Gaff andGiess, 1986). During a year in the NegevDesert, thalli of the lichen Ramalina maci-formis underwent a cycle of wetting anddrying almost daily, mostly from dew(Kappen et al., 1979). Some bryophytes insemiarid grasslands can likewise experi-ence diurnal desiccation cycles driven bydew during dry seasons (Csintalan et al.,2000). At the other extreme, someangiosperms may undergo a single periodof desiccation per year, with a cycle ofactivity almost like that of an annual plant(e.g. Gratani et al., 1998).

Three factors that determine how cyclesof desiccation translate into growth arelight damage, nutrient relations and carbonbalance. Photodamage can occur as plantsdry or while they are desiccated, due atleast in part to light absorption withoutenergy transfer to photosynthesis (e.g. Seelet al., 1992; Gauslaa and Solhaug, 1996).Desiccation-tolerant plants show a varietyof mechanisms likely to reduce photodam-age, including leaf curling, accumulation ofanthocyanin and carotenoids, and xantho-phyll metabolism (e.g. Muslin andHomann, 1992; Eickmeier et al., 1993;Lebkeucher and Eickmeier, 1993;Calatayud et al., 1997; Deltoro et al., 1998;Beckett et al., 2000; Farrant, 2000).Antarctic mosses, which could be subjectto photodamage during freezing, showreversible photoinhibition and zeaxanthinactivity (Lovelock et al., 1995).



Little is known about the interactionbetween desiccation tolerance and nutrientrelations. Uptake and metabolism of min-eral nutrients must be interrupted fromsome point during drying to some pointduring rehydration and recovery. Inmosses, leakage of solutes during rehydra-tion could also reduce net nutrient uptake.Increased frequency of desiccation cyclesdecreases potassium content but not cumu-lative phosphorus uptake in Tortula ruralis(Badacsonyi et al., 2000). Activity of nitratereductase decreases rapidly during desicca-tion in T. ruralis (Mahan et al., 1998;Badacsonyi et al., 2000); activity canrecover in less than 8 h after rehydration ifthe moss has dried slowly, but may take 24 h after rapid drying (Mahan et al.,1998), and may decrease during the firsthour of rehydration (Marschall, 1998).However, Bates (1997) found that weekly,24 h desiccation did not decrease uptake ofN, P or K in two mosses compared touptake during continuous hydration, andBadacsonyi et al. (2000) saw no differencebetween the effect of low water potentialon nitrate reductase activity in desiccation-tolerant and sensitive mosses.

Cycles of desiccation tend to reduce netcarbon gain by favouring respiration overphotosynthesis and by decreasing theamount of time that plants are active.Desiccation increases the ratio of respira-tion to photosynthesis because: (i) photo-synthesis ceases before respiration duringdrying and resumes after respiration duringrehydration; (ii) respiration in some speciesincreases above normal levels during recov-ery from desiccation; and (iii) plants tend tostay hydrated at night when they cannotphotosynthesize but do respire, and to des-iccate most rapidly when light levels arehigh (e.g. Alpert, 1979; Proctor, 1982; Langeet al., 1994; Tuba et al., 1998). Tuba et al.(1999) examined the hypothesis that anincrease in atmospheric CO2 might improvecarbon balance during cycles of desicca-tion. Elevated CO2 does prolong photosyn-thesis during drying in X. scabrida, but theauthors concluded that this aspect of globalchange was unlikely to favour desiccation-tolerant over sensitive species.

These factors are most important in des-iccation-tolerant plants that tend to haveshort periods of hydration or frequentcycles of desiccation, such as lichens andmosses in arid habitats, and are probablyone reason why these species grow soslowly (Stark, 1997; Kappen andValladares, 1999; Badacsonyi et al., 2000).Since brief periods of hydration can resultin net carbon loss (Lange et al., 1994;Csintalan et al., 2000), it is possible thatsome desiccation-tolerant plants may havebeen selected for traits that help preventrewetting by small amounts of water. Waterrepellence in epilithic lichens (Bertsch,1966) and hair points on some epilithicmosses (P. Alpert, unpublished data) couldbe examples.

1.5.3. Hypotheses

The rarity of desiccation-tolerant vascularplants in habitats where other vascularplants are abundant suggests that survivingdesiccation may have negative as well aspositive effects on survival overall. Onehypothesis is that there is a trade-offbetween tolerance and growth, and that tol-erant plants are out-competed by sensitiveones where the latter can survive, becausethe latter grow faster and larger. Thisshould cause selection against tolerance inhabitats where plants can acquire and con-serve enough water to avoid desiccation.An alternative possibility is that toleranceis merely lost in plants that are notexposed to desiccation, due to lack ofselection pressure to maintain tolerance.

Does desiccation tolerance entail areduction in growth rate or maximumsize? There are a number of reasons tosuppose this, but little direct evidence.Kappen and Valladares (1999) proposedthat some morphological features that pro-mote tolerance also conflict with produc-tivity. In angiosperms, hairs or scales thatreduce water loss and can thus prolongperiods of net photosynthesis also inhibitrehydration (Kappen and Valladares,1999). In lichens and bryophytes, havinghigh maximum water content tends to



prolong hydration but inhibits photosyn-thesis, since much of the water is typi-cally held externally or in upper layers ofthe lichen thallus and so slows gas diffu-sion (Green and Lange, 1994; Valladares,1994; Lange et al., 1996; Tuba et al.,1996a; but see Sojo et al., 1997).Populations of Ramalina capitata in dry,bright sites tend to have greater capacityto store water and slower gas diffusionthan populations in more shaded sites(Pintado et al., 1997), suggesting thatselection favours water storage more whenlight is less limiting. Proctor (2000) pro-posed that bryophytes have been selectedfor rapid desiccation to minimize timespent at intermediate water contents,which most dispose plants to damage.Rapid desiccation would also reduce timeavailable for photosynthesis and growth.Cellular mechanisms of tolerance such assugar and protein synthesis (Section 1.6)seem likely to impose metabolic costs andthus reduce growth. If cavitation duringdesiccation precludes desiccation-tolerantplants from exceeding 3 m in height(Sherwin and Farrant, 1998), then theywill be overtopped wherever trees cangrow. Some comparative studies onmosses (Bates, 1997; Arscott et al., 2000)and anecdotal reports on grasses (Gaff,1989) have found that more productivespecies are less desiccation-tolerant. Thelong-standing hypothesis (Grime, 1979)that stress tolerance conflicts with pro-ductivity is intuitively appealing butmechanically elusive. Further compara-tive studies on desiccation-tolerant plantscould help reveal mechanisms that dictatetrade-offs between tolerance and growth.

The absence of desiccation-tolerantplants in some highly xeric habitats whereno other plants occur suggests that surviv-ing desiccation may not assure survival,even where competition is not a factor. Onehypothesis to explain why tolerant plantsare not more abundant in barren habitats isthat the plants cannot maintain a cumula-tive positive carbon balance under certainregimes of water availability (Ried, 1960).Does carbon balance limit the survival ofdesiccation-tolerant plants in xeric habi-

tats? This hypothesis has been partiallytested in bryophytes and lichens (e.g.Alpert, 1990; Pintado et al., 1997; Williamsand Flanagan, 1998; Kappen andValladares, 1999). For example, during amorning after nocturnal rain or dew,bryophytes and lichens growing on slopedsurfaces in north temperate latitudes tendto dry more rapidly if they are on surfacesthat face south or east than if they are onsurfaces that face north or west (Kappen etal., 1980; Alpert and Oechel, 1985). Thoseon north- and west-facing surfaces aremore likely to recoup the respiratory lossesincurred during the night before desicca-tion arrests photosynthesis in the morning.This probably at least partly explains whymosses are ‘more common on the north sideof the tree’. There appear to be no studieson the effect of microsite on carbon balancein desiccation-tolerant vascular plants.

Oliver et al. (2000) hypothesized thatdesiccation tolerance was once the major-ity solution to the problem of living in dryair. They suggested that tolerance is a prim-itive characteristic in green plants thatallowed them to colonize the land. Onceplants evolved vascular tissues and effi-cient internal water transport, they losttheir tolerance of desiccation except inparts that had to be cut off from watertransport – their spores, seeds and pollen.Tolerance in adult plants then re-evolvedseveral times in different lineages.Porembski and Barthlott (2000) proposedthat this re-evolution occurred asangiosperms colonized the bare rock out-crops where their desiccation-tolerantspecies are now most diverse. If this sce-nario is correct, then desiccation tolerancein plants has evolved not just as a way ofsurviving in marginal habitats, but as a wayof colonizing frontiers, first from water onto land and then from soil on to stone.

1.6. Mechanisms of DesiccationTolerance

Until the mid-1970s, it was generallybelieved that the mechanisms of desicca-tion tolerance in plants were mechanical



(see reviews by Bewley, 1979; Oliver andBewley, 1984). Structural features such asflexible cell walls, small vacuoles andlack of plasmodesmata were suggested askey elements in tolerance (Gaff, 1980;Bewley and Krochko, 1982; Oliver andBewley, 1984). In a landmark paper,Bewley (1979) articulated the alternativeview that desiccation tolerance is primar-ily protoplasmic in nature. This theoryargues that certain plants and plant tis-sues achieve desiccation tolerance as aresult of the inherent properties of theircellular contents (protoplasm). Most evi-dence now supports this view, thoughstructural features are clearly importantin desiccation tolerance in some cases(Sherwin and Farrant, 1996; Farrant etal., 1999).

Bewley (1979) further defined threecritical features of desiccation tolerancebased on the observation that many desic-cation-tolerant plants exhibit cellularchanges, some of which can be describedas extensive damage, during and follow-ing desiccation. The plant or tissue must:(i) limit damage to a repairable level; (ii)maintain its physiological integrity in thedried state (perhaps for extended periodsof time); and (iii) mobilize mechanismsupon rehydration that repair damage suf-fered during desiccation and rehydration.These criteria laid the experimental foun-dation for the field from the 1980sonwards and continue to influence theway we think about how plants survivedesiccation. In particular, it is nowwidely accepted that the cells of desicca-tion-tolerant plants employ mechanismsthat protect them from the rigours ofextensive water loss and also mecha-nisms, at least in the case of vegetativecells, that repair damage suffered duringdesiccation or rehydration (Bewley andOliver, 1992). This introductory overviewof the mechanisms of desiccation toler-ance will therefore concentrate on cellu-lar features (the so-called ‘inherentproperties’ of desiccation-tolerant cells)that have been suggested to play a majorrole in protection and repair. Details arecovered in subsequent chapters.

1.6.1. Damage

Before discussing what is known of thecellular protection and repair mecha-nisms of desiccation tolerance, it is worthreviewing the effects of desiccation andrehydration on cellular integrity in desic-cation-tolerant plants. The critical ques-tion, when deciding what type ofmechanisms desiccation-tolerant plantsemploy, is when might damage occur? Isit during the drying process or upon rehy-dration? For instance, if damage does notactually occur during desiccation, thenthere is good reason to believe that pro-tective mechanisms are in place. If dam-age occurs upon rehydration, and the cellsubsequently recovers, repair mecha-nisms are probably operative. In addition,the amount of damage and the rate atwhich cells return to a normal statusmeasure the effectiveness of protectiveand repair processes and the overall levelof desiccation tolerance.

1.6.1.1. Damage during desiccation

The timing of damage is still controversial,but a consensus is building that little dam-age occurs during drying in desiccation-tolerant tissues. Much of the work in thisarea has focused on the plasma membrane.All desiccation-tolerant tissues leaksolutes during rehydration (Simon, 1978;Bewley, 1979; Bewley and Krochko, 1982),indicating that the cell membrane has beencompromised. Early electron microscopyof seeds (Webster and Leopold, 1977;Morrison-Baird et al., 1979) and bryophytetissues (reviewed by Oliver and Bewley,1984) suggested that membranes in driedplant cells were completely disorganized.With the advent of more sophisticatedtechnologies, these observations weredetermined to be artefacts of samplepreparation and chemical fixation (Bewley,1979; Thompson, 1979; Bewley andKrochko, 1982; Oliver and Bewley, 1984).The use of non-aqueous fixatives elimi-nated some of these artefacts but the heavyuse of chemical treatments still madeinterpretation difficult (Thompson, 1979;



Öpik 1980, 1985; Tiwari et al., 1990;Smith, 1991). Freeze–fracture electronmicroscopy, however, has yielded the mostreliable data. Dried tissues are eminentlysuited for freeze–fracture preparationbecause their low water content virtuallyeliminates the formation of ice crystals,which make high-quality replicas difficultto obtain. Freeze–fracture studies clearlydemonstrated that the membranes of seeds(Thompson and Platt-Aloia, 1982; Bliss etal., 1984) and pollen (Platt-Aloia et al.,1986) could retain normal bilayer organi-zation and dispersal patterns of intramem-branous particles at water contents as lowas 0.08 g H2O g�1 dry mass. The plasmaand organelle membranes of vegetativecells of the desiccation-tolerant pterido-phyte Selaginella lepidophylla and themoss Tortula ruralis also retain normalorganization and dispersal patterns in thedried state (Platt et al., 1994; Fig. 1.6).

The effects of desiccation on cellularcomponents that cannot be observed byfreeze–fracture microscopy are more diffi-cult to evaluate, largely due to the likeli-hood of partial rehydration and theproduction of artefacts during chemicalfixation. In seeds, the uncertainty is com-pounded by the fact that the tissues are part of a developing system.Nevertheless, observations tend to sug-gest that desiccation of tolerant plantsgenerates an ordered ‘collapse’ of the cellular milieu that results in little ultra-structural damage (Oliver and Bewley,1984; Gaff, 1989; Goldsworthy andDrennan, 1991; Sherwin and Farrant,1996; Farrant et al., 1999). If desiccation-tolerant plants successfully avoid damageduring the dehydration process, as itappears they do, is there any consequenceat all of desiccation in these plants? Theanswer appears to be yes. All desiccation-tolerant plants and plant tissues showsigns of cellular damage when the driedtissue is rehydrated. It is, however, debat-able whether or not the damage occursduring the drying process (but is notobservable at an ultrastructural level) oras the result of the inrush of water intothe cells during rehydration.

1.6.1.2. Damage during rehydration

As noted above, all plant tissues leaksolutes when rehydrated following a dry-ing event. In desiccation-tolerant tissues,however, this is a transient event (Simon,1978; Bewley, 1979; Bewley and Krochko,1982). Several hypotheses have beenoffered to explain imbibitional (or rehydra-tive) leakage (Simon, 1974; Senaratna andMcKersie, 1983a,b; Crowe et al., 1989,1992; Hoekstra et al., 1992). The prevailinghypothesis is that imbibitional leakage isthe result of lipid-phase transitions occur-ring in the plasma membrane as a result ofdehydration and rehydration (Crowe et al.,1992). During drying, membranes passfrom the liquid crystalline to the gel phase,and they return to the liquid crystallinephase during rehydration. In artificialmembranes, this transition can lead to atransient leakage event (Hammoudah et al.,1981), and, since phase transitions havebeen demonstrated in drying and rehydrat-ing desiccation-tolerant cells (Crowe et al.,1989; Hoekstra et al., 1992), it has beengenerally accepted that phase transition isthe basis of imbibitional leakage in mostdesiccation-tolerant tissues. In seeds, how-ever, it is thought that membrane-phasechanges do not occur because of the pres-ence of a seed coat, which impedes thepassage of water to the dried cells.Hoekstra et al. (1999) suggested that theslow rate of penetration of water may setup a ‘pre-hydration’ state where the mem-branes are in a liquid crystalline statebefore liquid water surrounds the rehydrat-ing cells. Since leakage does occur duringthe rehydration of these tissues (Hoekstraet al., 1992; Tetteroo et al., 1996), it hasbeen concluded that leakage must occurthrough an intact lipid bilayer, as suggestedby Senaratna and McKersie (1983b).

Recently, a new hypothesis has emergedfrom some exciting new studies on dehy-drating and rehydrating pollen (Hoekstra etal., 1997, 1999; Golovina et al., 1998;Buitink et al., 2000; Chapter 10). This body ofwork using amphiphilic spin probes demon-strates that during dehydration endogenousamphiphilic substances partition from the



aqueous cytoplasm into pollen membranes.Using data obtained from liposome-based ex-periments, Golovina et al. (1998) suggestedthat it is the presence of these amphiphilesin the membrane that causes imbibitionalleakage and that as the pollen rehydrates theamphiphilic substances move out of themembranes and leakage stops. This hypo-thesis could explain how transient leakagecan occur through an intact membrane. In amore recent study, Buitink et al. (2000)demonstrated that the movement ofamphiphilic compounds into membranesalso occurs in imbibing radicles of peas andcucumbers. This study, using electron para-magnetic resonance (EPR) spectroscopy andinserted nitroxide spin probes, demon-strated a difference in partitioning behav-iour between desiccation-tolerant andsensitive tissues. Spin probes partitionedinto the membranes at higher water contentin desiccation-sensitive tissues than in toler-ant tissues. These authors suggest, from invitro portioning experiments, that it is themicroviscosity of the cytoplasm that con-trols portioning of amphiphilic compoundsinto the plasma membrane. What remains tobe determined is the role of the nativeamphiphilic compounds in membrane dam-age and, if they are important in desiccationtolerance, the role they play in the long-termstability of membranes in the dried state.

Golovina et al. (1998) speculated thatamphiphiles may have antioxidant proper-ties that protect membranes from damageby free radicals generated during desicca-tion and rehydration. If so, imbibitionalleakage may be a necessary trade-off forprotection. Much work will be requiredbefore the importance of nativeamphiphilic compounds in desiccation tol-erance can be determined, but amphiphilesare an intriguing new development in ourunderstanding of desiccation tolerance.

Rehydration-induced damage other thanleakage is difficult to distinguish from nor-mal development in seeds and pollen butis clearly evident in the tissues of mostdesiccation-tolerant vegetative tissues,especially in organelles (reviewed by Bewleyand Krochko, 1982; Oliver and Bewley,1984; Gaff, 1989; Oliver and Wood, 1997).

Within minutes after rehydration, thechloroplasts of the green gametophytic tis-sues of desiccation-tolerant bryophytesappear swollen and globular. Their outermembranes are folded and separated fromthe thylakoids, which are no longer com-pacted (Oliver and Bewley, 1984). Theextent of thylakoid disruption increaseswith the rate of prior desiccation. Thechloroplasts of desiccation-tolerantangiosperms tend to be more resistant todisruption than those of bryophytes,although vesicularization within thechloroplast internal membranes is common(Gaff and Hallam, 1974; Gaff et al., 1976;Sherwin and Farrant, 1996). In all desicca-tion-tolerant plants, mitochondria swelland exhibit disruption of the cristae(reviewed by Bewley and Krochko, 1982).Swelling and disruption of mitochondriaare not affected by rate of desiccation. Inall cases, organelles regain normal struc-ture within 24 h.

1.6.1.3. Poikilochlorophylly

At least eight genera of desiccation-tolerantmonocots are ‘poikilochlorophyllous’, i.e.they reversibly lose their chlorophyll anddismantle their chloroplasts during desic-cation (Gaff, 1989; Tuba et al., 1998). Thethylakoid system within desiccated chloro-plasts is completely replaced by smallgroups of plastoglobuli and by osmophilic,stretched lipid material, which appears tooccupy the positions previously occupiedby the thylakoids (Hallam and Luff, 1980;Tuba et al., 1993a,b; Sherwin and Farrant,1996). After 10–12 h rehydration, whenfull turgor and maximum leaf water con-tent are reached, synthesis of chlorophyllsand carotenoids and the reassembly of thy-lakoids begin. Early in reassembly, sets oftwo primary thylakoids stack to formgrana. Within 72 h the chloroplasts appearnormal and full photosynthetic capacity isrestored (Tuba et al., 1993b, 1994). Fromthese studies and later physiological in-vestigations (Tuba et al., 1997), it appearsthat these changes can be classified asgenetically programmed responses to des-iccation rather than damage.



1.6.2. Protection

Much of what we know of the cellular pro-tection mechanisms involved in desiccationtolerance in plants comes from studies oforthodox seeds (Bewley and Black, 1994;Chapter 5) and, to a slightly lesser extent,pollen (Crowe et al., 1992; Hoekstra et al.,1992). The ability of seeds to withstanddesiccation is acquired during their devel-opment. This acquisition is usually sub-stantially earlier than the culmination ofthe drying event itself, which is the termi-nal event in orthodox seed maturation.Seeds of some species can withstand pre-mature desiccation well before the mid-point of their development (Bewley andBlack, 1994; Chapter 5). Among the meta-bolic changes that take place just prior to orduring drying is the synthesis of proteinsand sugars, which have long been postu-lated to form the basis of a series of overlap-ping protective mechanisms that limitdamage to cellular constituents (Bewley,1979; Leprince et al., 1993; Oliver andBewley, 1997). These two components havesince been widely implicated as being criti-cal for desiccation tolerance in all plantcells including vegetative cells (Ingram andBartels, 1996; Oliver and Bewley, 1997;Scott, 2000). Over the years it has alsobecome clear that the synthesis of antioxi-dants and enzymes involved in oxidativemetabolism also play a critical role in cellu-lar protection and desiccation tolerance(Chapter 10). However, this aspect of pro-tection will not be addressed here.

1.6.2.1. Proteins

Only one subset of proteins that accumu-late at the time of the acquisition of desic-cation tolerance has been extensivelyinvestigated, the late embryogenesis abun-dant (LEA) proteins, first described in cot-ton (Galau and Hughes, 1987; Galau et al.,1987, 1991; Chapter 5). The genes thatencode LEA proteins in developing cotton-seeds are comprised of two distinct classeswhose regulation is coordinated. One classcontains six different lea transcripts, whichappear relatively early in development and

reach a maximum about three days beforethe seed begins to desiccate (Galau andHughes, 1987; Galau et al., 1987). Theother class contains 12 transcripts, whichappear late in maturation and achieve max-imum expression just before and duringdesiccation. LEA proteins make up 30% ofthe non-storage protein and 2% of the totalsoluble protein in the mature cottonembryos and are uniformly localizedthroughout the cytoplasm (Roberts et al.,1993). LEA proteins and the acquisition ofdesiccation tolerance during seed matura-tion have been linked in other dicots (e.g.soybean: Blackman et al., 1995) and inmonocots (e.g. maize: Mao et al., 1995;Wolkers et al., 1998).

A set of LEA proteins arises in develop-ing barley and maize embryos at the timethat tolerance of desiccation is acquired. Asmall subset of these proteins is inducedwhen barley embryos at the intolerant stageare cultured in abscisic acid (ABA) (Bartelset al., 1988; Bochicchio et al., 1991), and acausal relationship between ABA and leagene expression has been suggested.Evidence for, and against, this relationshipexists in the literature. In cotton embryos,high expression of the first class of leagenes occurs as ABA content increases.High expression of the second set of leagenes, however, occurs at the start of, andduring, maturation drying, when theendogenous ABA content is low. There areexplanations for this lack of correlation,e.g. there is an early-regulated, ABA-con-trolled mechanism, which operates onlylater when drying commences. On theother hand, an ABA-independent pathwaymay be involved in the synthesis of thesecond group of LEA proteins.

LEA proteins have been identified in thevegetative tissues of all desiccation-tolerantplants studied so far (Ingram and Bartels,1996; Oliver and Bewley, 1997; Blomstedtet al., 1998) and proteins related to some ofthe LEA proteins, e.g. dehydrins (seebelow), have been associated with theresponse of non-tolerant plants to waterstress (Skriver and Mundy, 1990; Bray,1997). In nearly all instances, the inductionof LEA protein synthesis in vegetative tis-



sues can be elicited by exogenous ABAapplication (Ingram and Bartels, 1996;Campalans et al., 1999).

LEA proteins fall into five groups byvirtue of sequence similarities (Dure et al.,1989; Ingram and Bartels, 1996; Cuming,1999). All are highly hydrophilic and all arevery stable, as evidenced by their resistanceto the denaturing effects of boiling (with theexception of Group 5 LEA proteins). Group1 LEA proteins are characterized by a 20-amino acid motif and are represented by thewheat Em protein, the first LEA proteinidentified (Cuming and Lane, 1979). Group2 LEA proteins are characterized by a 15-amino acid motif, the K-segment, a stretchof serine residues and a conserved motifnear the N-terminus of the protein (Close,1997). This group of proteins is also calledthe dehydrins and these are the most wide-spread and most studied of the LEA pro-teins. Group 3 LEA proteins share acharacteristic 11-amino acid repeat motif(Dure et al., 1989), which is predicted toform an amphipathic �-helix. These amphi-pathic helices are postulated to form intra-and intermolecular interactions that mayhave important consequences for their func-tion (Baker et al., 1988; Dure, 1993a). Theleast studied of the LEA proteins are those inGroups 4 and 5, which are somewhatatypical (Dure, 1993b; Galau et al., 1993).Group 5 LEA proteins are more hydrophobicthan other LEA proteins and are not resistantto high temperature. Most of the LEA proteingroups have been identified in many differ-ent plants. All groups are thought to play arole in desiccation tolerance, and the evi-dence for this viewpoint is growing.

The evidence for the involvement ofLEA proteins in desiccation tolerance iscircumstantial but compelling. LEA proteinsynthesis in seeds, as mentioned above, isassociated with both the acquisition of des-iccation tolerance and the final stage ofseed maturation just prior to desiccation.In addition, ABA-deficient (aba) and ABA-insensitive (abi3) double-mutants ofArabidopsis seeds do not dry on the parentplant, do not tolerate desiccation and lackseveral LEA proteins (Koorneef et al., 1989;Meurs et al., 1992).

LEA protein synthesis is also highlyinduced in the vegetative tissues of desic-cation-tolerant angiosperms during drying(Bartels et al., 1993; Blomstedt et al., 1998;Bartels, 1999). Callus derived from vegeta-tive tissue of the desiccation-tolerant plantCraterostigma plantagineum is not inher-ently tolerant but can be made so by theapplication of ABA (Bartels et al., 1990).The application of ABA to this tissueresults in the synthesis of novel proteins,some of which are LEA proteins includingthe Group 2 LEA proteins, the dehydrins(Bartels et al., 1993). The desiccation-toler-ant moss T. ruralis utilizes a more primi-tive mechanism of desiccation tolerance(Oliver et al., 2000), which involves a con-stitutive cellular protection strategy, and inthis plant, unlike others, dehydrins are notinduced by dehydration or by ABA but areconstitutively expressed (Bewley et al.,1993). Dessication-sensitive speciesexposed to sub-lethal dehydration stressalso respond by synthesizing LEA proteinsand LEA-like proteins, in particular dehy-drins (Close, 1997). These examples andmany more all point to the importance ofLEA proteins in dehydration responses anddesiccation tolerance.

The most convincing pieces of evidenceto suggest that LEA proteins have animportant role in cellular protection comefrom transgenic studies using a barleyGroup 3 lea gene, HVA1. This gene, whenexpressed in a constitutive fashion intransgenic rice, increased its tolerance towater and salt stress (Xu et al., 1996).HVA1 overexpression in wheat, driven by amaize ubiquitin promoter, resulted intransgenic lines that performed in a supe-rior fashion under soil-water deficits(Sivamani et al., 2000).

There are a variety of suggested mecha-nisms by which LEA proteins might pro-tect cellular components. Many LEAproteins have extensive regions of randomcoiling, which has been postulated to pro-mote the binding of water, helping to main-tain a minimum water requirement (Ingramand Bartels, 1996). For instance, the Emprotein of wheat is considerably morehydrated than most common proteins, and



over 70% of the Em protein is configuredas random coils (McCubbin et al., 1985).Baker et al. (1988) suggested that the ran-dom coil nature of some LEA proteins mayallow them to conform to the shape of cel-lular constituents and thus, by virtue oftheir hydroxyl groups, help to maintaintheir solvation state when water isremoved. These authors also suggested thatthe Group 2 LEA proteins (dehydrins), byvirtue of their amphipathic helical repeats,provide surfaces when bundled togetherthat would sequester ions. This may becrucial as the increasing ionic strength dur-ing drying could cause irreversible damageto cellular proteins and structural compo-nents. Recently, Velten and Oliver (2001)described an LEA-like protein from T.ruralis that contains 15 15-amino-acidrepeats predicted to form amphipathichelices. This protein appears to be synthe-sized during the rehydration event andmay serve to trap valuable ions that wouldotherwise be lost. Studies using individualLEA proteins in in vitro assays also add tothe possible mechanisms by which theseproteins exert protection of cellular compo-nents. Wolkers (1998) suggested from dataobtained from the study of a pollen Group3 LEA protein and its effect on sucroseglass formation that LEA proteins may actas anchors in a structural network that sta-bilizes cytoplasmic components duringdrying and in the dried state.

At this point it seems likely that eachindividual group of LEA proteins may havedifferent, complementary effects. Most des-iccation-tolerant tissues contain a represen-tative of most, if not all, of the differentgroups of LEA proteins, and it is also likelythat all are needed to achieve the highestdegree of desiccation tolerance.

There is mounting evidence that anotherclass of proteins, the small heat-shock pro-teins (HSPs), may play a role in cellularprotection during desiccation. Small HSPsaccumulate in maturing seeds of manyplant species (Vierling, 1991; Wehmeyer etal., 1996) prior to desiccation. Alamillo etal. (1995) reported that small HSPs areexpressed constitutively in the vegetativetissues of C. plantagineum and increased

in accumulation during desiccation.Constitutive expression of HSPs is unusualin vegetative tissues and resembles theexpression pattern of these proteins inseeds. In addition, exogenous ABAinduced both the expression of HSPs andthe acquisition of desiccation tolerance inC. plantagineum callus tissues (Alamillo etal., 1995). Finally, a LEA-like HSP, HSP-12,from yeast was shown to be capable of pro-tecting liposomal membranes from thedamaging effects of desiccation in a waysimilar to that seen with the sugar tre-halose (Sales et al., 2000). Thus it appearsthat small HSPs may also play a role in cel-lular protection during desiccation: per-haps this capability is related to theirchaperonin-like activities, which may helpmaintain protein structure under denatur-ing conditions. Other proteins whose tran-scripts accumulate during the dehydrationphases of vegetative desiccation-tolerantangiosperms have been identified but littlehas been done to confirm their roles in des-iccation tolerance (Kuang et al., 1995;Ingram and Bartels, 1996; Blomstedt et al.,1998; Bockel et al., 1998; Neale et al.,2000). See Chapters 5 and 11 for furtherdiscussion of all these proteins.

1.6.2.2. Sugars

The accumulation of soluble sugars is alsostrongly correlated to the acquisition ofdesiccation tolerance in plants and otherorganisms (for reviews see Crowe et al.,1992; Leprince et al., 1993; Vertucci andFarrant, 1995; Chapters 5 and 10). Solublesugars, especially sucrose, accumulate inseeds (Leprince et al., 1993), pollen(Hoekstra et al., 1992) and in desiccation-tolerant vegetative tissues (Bewley andKrochko, 1982; Ingram and Bartels, 1996;Oliver and Bewley, 1997). In Craterostigmaplantagineum, 2-octulose stored in thehydrated leaves is converted to sucroseduring drying to such an extent that in thedried state it comprises about 40% of thedry weight (Bianchi et al., 1991).

Sucrose is the only free sugar availablefor cellular protection in desiccation-toler-ant mosses, including Tortula ruraliformis



and T. ruralis (Bewley et al., 1978;Smirnoff, 1992). The amount of this sugarin gametophytic cells of T. ruralis isapproximately 10% of dry mass, which issufficient to offer membrane protectionduring drying, at least in vitro (Strauss andHauser, 1986). Moreover, neither dryingnor rehydration in the dark or light resultsin a change in sucrose concentration, sug-gesting that it is important for cells tomaintain sufficient amounts of this sugar(Bewley et al., 1978). The lack of anincrease in soluble sugars during dryingappears to be a common feature of desicca-tion-tolerant mosses (Smirnoff, 1992).

It is thought that sugars protect the cellsduring desiccation by two mechanisms.First, the hydroxyl groups of sugars maysubstitute for water to maintainhydrophilic interactions in membranes andproteins during dehydration (Crowe et al.,1992). This has so far only been demon-strated in vivo, using liposomes and iso-lated proteins (Crowe et al., 1992).Secondly, sugars are a major contributingfactor to vitrification, the formation of abiological glass, of the cytoplasm of drycells (Leopold et al., 1994; Chapter 10).This mechanism has been the subject ofintense research over the last 15 years.

Vertucci and Leopold (1986) suggestedthat desiccation tolerance in seeds had tobe associated with some feature or solutecombination that would avoid crystalliza-tion of the cytoplasm as dehydration pro-gressed. Burke (1986) proposed that highconcentrations of sugars lead to vitrificationof the cytoplasm during desiccation andthus prevent crystallization. Glass forma-tion has since been demonstrated in seeds(Williams and Leopold, 1989; Leopold etal., 1994; Leprince and Walters-Vertucci,1995), pollen (Buitink et al., 1996) and inleaf tissues of C. plantagineum (Wolkers etal., 1998). Walters (1998) went as far as tosay that glass formation is an intrinsic prop-erty of any complex system that can survivedesiccation. However, glass formation maynot be sufficient to confer desiccation toler-ance since desiccation-sensitive tissues arecapable of forming cytoplasmic glasses(Sun et al., 1994; Buitink et al., 1996).

Cytoplasmic glass formation has alsobeen postulated to maintain the structuraland functional integrity of macromolecules(Sun and Leopold, 1997; Crowe et al.,1998b), which has been well demonstratedwith in vitro models (Roos, 1995).Intracellular glasses, by virtue of their highviscosity, drastically reduce molecularmovement and impede diffusion of reac-tive compounds in the cell. It is by thisproperty that glasses are thought to prolongthe longevity of desiccated tissues by slow-ing down degradative processes duringstorage. Buitink et al. (1998) recentlydemonstrated a strong relationshipbetween molecular mobility and storagelongevity in both pollen and pea seeds.Thus, although glass formation may not beimportant in the initial acquisition of des-iccation tolerance, it may be crucial for sur-vival of the dried state (as suggested byBuitink, 2000; Chapter 10).

Other carbohydrates besides sucroseaccumulate in desiccation-tolerant tissues,the principal ones being the oligosaccha-rides stachyose and raffinose (Horbowiczand Obendorf, 1994), and have been postu-lated to play a part in desiccation toler-ance. The presence of these compoundshas also been correlated with seedlongevity (Hoekstra et al., 1994;Horbowicz and Obendorf, 1994), whichhas linked them to a possible role in thestabilization of intracellular glasses(Leopold et al., 1994; Bernal-Lugo andLeopold, 1995; Sun, 1997). However,Buitink et al. (2000) demonstrated that thereduction in oligosaccharides in primedseeds did not alter Tg (the glass-to-liquidtransition temperature) or viscosity andthus they contended that oligosaccharidesdo not affect the stability of intracellularglasses. These results support the earlierstudies of Black et al. (1999), which hadshown a lack of a temporal correlationbetween the induction of desiccation toler-ance by a mild dehydration treatment andthe appearance of raffinose in wheatembryos. These studies cast doubt on therole of oligosaccharides in the acquisitionof tolerance and the maintenance of viabil-ity in the dried state.



1.6.3. Repair

The repair processes associated with des-iccation tolerance have been difficult todetail and characterize. In seeds, repairmechanisms are difficult to separate fromevents that are associated with germina-tion and early seedling growth, but evi-dence for repair does exist. In vegetativeangiosperms, the major emphasis appearsto be on effective cellular protection andmuch of the research in angiosperms hasfocused on this component. The mostpromising models for investigating a directrole for cellular repair in desiccation toler-ance appear to be the highly desiccation-tolerant bryophytes.

In seeds, most of the evidence for cellu-lar repair derives from investigations intothe causal relationship between cellulardamage and loss of viability during storage(Bewley and Black, 1994). Consequently,one has to keep in mind that the repairprocesses that have been identified maynot play a major role in desiccation toler-ance per se but rather in the ability to sur-vive long term in the dried state. There aretwo reports, however, that indicate therepair of cellular components, proteins andDNA, which may directly affect desicca-tion tolerance as well as storage longevity.Mudgett et al. (1997) demonstrated thatproteins containing abnormal L-isoaspartylresidues could be repaired in aged barleyseeds by the activity of the enzyme L-isoas-partyl methyltransferase. These authorsargue that this type of repair is particularlyimportant during dehydration where pro-tein turnover rates are slow. Boubriak et al.(1997) demonstrated that one of the earliestactivities seen in imbibing cereal grains isthe repair of damage to genomic DNAincurred whilst the seeds were dry and instorage. If the repair processes wereblocked during imbibition then DNAdegradation became severe. If universal,DNA repair would certainly qualify as akey process in the mechanism of desicca-tion tolerance in seeds (Chapter 12).

The identification of repair processes invegetative tissues of desiccation-toleranttracheophytes has received little attention.

In the desiccation-tolerant fernPolypodium virginianum rehydrating tis-sues do accumulate novel transcripts buttheir identities have not been investigated(Reynolds and Bewley, 1993b). Recentwork with the desiccation-tolerant grassSporobolus stapfianus has identified twotranscripts that accumulate during theearly phases of dehydration but also accu-mulate during rehydration. The first ofthese is a transcript coding for a plantRab2, a small GTP-binding protein that inother systems is an important protein inthe targeting of membrane vesicles invesicular trafficking pathways and a path-way directly involved in membrane con-struction (O’Mahony and Oliver, 1999a).The second transcript encoded a polyubi-quitin, a protein involved in proteinturnover (O’Mahony and Oliver, 1999b). InC. plantagineum very few transcripts wereidentified as being specific for the rehydra-tion process. Those that were appeared tobe involved in the metabolism of sugarsthat is required to re-establish the pools ofoctulose required for the generation ofsucrose during dehydration (Bernacchia etal., 1996).

Cellular repair, as a component of desic-cation tolerance mechanisms, is more easilydefined in desiccation-tolerant bryophytes.Desiccation-tolerant bryophytes are thoughtto employ a mechanism for desiccation tol-erance that represents the most primitiveform expressed in land plants (Oliver et al.,2000). Unlike the acquisition of desiccationtolerance in seeds, which may be develop-mentally programmed, and in desiccation-tolerant angiosperms, which isenvironmentally induced by drying, desic-cation tolerance in most bryophytesappears to be constitutive (Oliver andBewley, 1997). The difference in mecha-nisms of tolerance in these systems isreflected in their biology. Desiccation-toler-ant angiosperms have morphological andphysiological adaptations in place that canretard the loss of water. The mechanism ofdesiccation tolerance that has evolved inthese plants takes advantage of these adap-tations by being inducible. As the rate ofwater loss is relatively slow, there is time



to establish the protective measuresrequired, and the plant can thus survive adrying event. If water loss is too rapid,these plants succumb to the damagingeffect of water loss and die (Gaff, 1989;Bewley and Oliver, 1992; Oliver andBewley, 1997). Bryophytes, on the otherhand, have little in the way of adaptationsto retain water within the plant and, as aresult, the internal water content of theseplants rapidly equilibrates to the waterpotential of the environment (Proctor et al.,1998). A consequence of this is that manybryophytes experience drying rates that areextreme and therefore have insufficienttime to induce and set in place protectivemeasures. Thus, it appears that bryophyteshave evolved a constitutive mechanism fordesiccation tolerance, one that has protec-tive measures that are always in place. Thisconclusion is supported by the observa-tions that both sucrose and LEA proteinsare maintained at constant levels in desic-cation-tolerant bryophytes during dryingand rehydration (see above). The constitu-tive protection mechanism appears to beparticularly effective in preventing damageto the photosynthetic apparatus, as evi-denced by the very rapid recovery of pho-tosystem II activity (Tuba et al., 1996b;Csintalan et al., 1999; Proctor andSmirnoff, 2001).

How does this relate to cellular repairand the uniqueness of bryophytes forstudying this aspect of desiccation toler-ance? It appears that the level of protec-tion that bryophytes are capable ofmaintaining is not sufficient to completelyprevent damage, especially to membranes,during rehydration. To achieve desicca-tion tolerance, bryophytes thus rely heav-ily on repair mechanisms induced duringthe initial phases of hydration followingrewetting (Oliver and Bewley, 1997;Oliver et al., 1998).

Most work on repair in mosses has cen-tred on the proteins whose synthesis isinduced immediately upon rehydration ofdesiccated gametophytic tissue. Earlywork (see Bewley, 1979, for review) estab-lished the ability of T. ruralis and othermosses to rapidly recover synthetic meta-

bolism when rehydrated. During the first 2h following rehydration of dried T. ruralis,the synthesis of 25 proteins (termedhydrins) is terminated or substantiallydecreased, and the synthesis of 74 proteins(termed rehydrins) is initiated or substan-tially increased (Oliver, 1991). Controlsover changes in synthesis of these twogroups of proteins are not mechanisticallylinked. It takes a certain amount of priorwater loss to fully activate the synthesis ofrehydrins upon rehydration. Perhaps thisis a strategy that has evolved to link theamount of energy expended in repair tothe amount of damage potentiated by dif-fering degrees of drying.

In T. ruralis, there also appears thecapability of preparing the cell for a rapidrecovery if drying rates are sufficientlyslow (4–6 h). Using cDNA clones corre-sponding to T. ruralis transcripts that arepreferentially translated during rehydra-tion (Scott and Oliver, 1994), it was deter-mined that several ‘recovery’ transcriptsaccumulate during slow drying (Oliverand Wood, 1997; Wood and Oliver, 1999).Recent studies clearly demonstrate thatthese transcripts are sequestered in thedried gametophytes in messenger ribo-nucleoprotein (mRNP) particles (Woodand Oliver, 1999). Of 18 rehydrin cDNAsisolated (Scott and Oliver, 1994) andsequenced (Oliver et al., 1997; Wood etal., 1999) in T. ruralis, only three exhibitsignificant sequence homology to knowngenes in the Genbank databases. Tr155 hasa strong sequence similarity to an alkylhydroperoxidase linked to seed dormancyin barley (Aalen et al., 1994) andArabidopsis embryos (Haslekas et al.,1998), and in rehydrated but dormantBromus secalinas seeds (Goldmark et al.,1992). Tr213 exhibits a high degree of sim-ilarity to polyubiquitins from severalplant sources, suggesting that proteinturnover may be an important part ofrepair in mosses as well as inangiosperms. Tr288 encodes an LEA-likeprotein (see above), which suggests thatLEA proteins may have a protective roleduring rehydration and a role in cellularrepair (Velten and Oliver, 2001).



1.7. Future Prospects and AgriculturalSignificance

Our present agricultural system is almosttotally dependent upon the ability of ortho-dox seeds to tolerate desiccation. The iden-tification and functional analysis of genesinvolved in the developmentally pro-grammed desiccation tolerance stage ofseeds is a vital step in understanding thiscomplex trait. The knowledge gained fromsuch studies will impact on many diverseareas of agricultural concerns such asgermplasm preservation, seed productionand seedling establishment. Apart fromgenetic considerations, the ongoing studieson the role of biological glasses in desicca-tion tolerance and the determination ofwhich proteins and sugars are important intheir stability will affect our ability to pre-serve viable germplasm for longer periodsof time, preserving genetic diversity forfuture breeding needs. This goal will alsobe benefited by our continued progress inunderstanding of how desiccation-toleranttissues deal with oxidative stress.

Over 35% of the world’s land surface isconsidered to be arid or semiarid, experi-encing precipitation that is inadequate formost agricultural uses. Ramanathan (1988)has argued, based on predictions of globalenvironmental changes, that developingcrops that are more tolerant to waterdeficits while maintaining productivitywill become a critical requirement in theearly part of this century. Understandinghow plant cells tolerate water loss is a vitalprerequisite for developing strategies thatcan influence agricultural and horticulturalcrop productivity and survival under theseconditions of decreasing water availability.Much has been accomplished in the dis-covery and characterization of those genesthat are expressed during the response ofcrop and model plants to water deficit orsalt stress. From this work, our knowledgeof stress tolerance has improvedimmensely and some success has beenachieved, but these traits are very complexand breeding progress has been slow. Thisapproach is also restricted in that mostcrops have a limited capacity for drought

tolerance and thus the genetic informationnecessary for expanding their drought tol-erance may not be exploitable or indeedpresent. In contrast, more may be gained byunderstanding how stress-tolerant plants orplant structures accomplish tolerance, andfrom such sources genes that contributedirectly to tolerance can be identified. Aspointed out by Bartels and Nelson (1994),the limiting factor for the improvement ofabiotic stress tolerance in crops is ‘theavailability of structural genes and regula-tory elements which positively contributeto stress tolerance improvement’. If ourefforts to utilize our understanding of theunderlying mechanisms of desiccation tol-erance are to bear fruit, we must discoverand identify the genes that are central tothis trait.

Cushman and Bohnert (2000) describe astrategy for cataloguing genes central toparticular traits in their discussion ongenomic approaches to plant stress. Theinitial phase of gene discovery is the large-scale sequencing of randomly selectedcDNA clones, termed Expressed SequenceTags (ESTs), which are synthesized frommRNA pools representing a specific devel-opmental stage or response state. EST col-lections that will enhance gene discoveryin desiccation tolerance have been started,the most extensive of which is that fordeveloping seeds of Arabidopsis thaliana(Girke et al., 2000). Smaller EST collec-tions have been made for C. plantagineumleaves that had been dried for an hour orfully dried (Bockel et al., 1998) and S. stap-fianus leaves during dehydration(Blomstedt et al., 1998; Neale et al., 2000).Wood et al. (1999) have established a lim-ited sample of ESTs (152) from a cDNAlibrary developed from the mRNP fractionof slowly-dried T. ruralis gametophytes. Inthe EST collections from vegetative desic-cation-tolerant plant tissues, many of thesequenced clones were of unknown iden-tity (71% for Tortula) and/or not previ-ously associated with water stress.Cushman and Bohnert (2000) suggestedthat this may indicate, as suggested above,that these plants may possess ‘unique genecomplements or regulatory processes that



contribute to desiccation stress’ and, byinference, novel genes that may prove use-ful in breeding for drought stress tolerance.

The cataloguing of gene products that areexpressed during the acquisition and estab-lishment of desiccation tolerance is only thefirst step. The ultimate goal is to determinewhich genes are central to desiccation toler-ance and what functions they perform. Themajor approach used to gain a functionalunderstanding of individual gene productshas been the overexpression of genes intransgenic plants. We have already men-tioned the studies of Xu et al. (1996) andSivamani et al. (2000) concerning the over-expression of HVA1, a Group 3 lea gene, andtheir success in improving drought toler-ance in rice and wheat. Two other groupshave attempted to modify sugar metabolismto improve tolerance by engineering tre-halose 6-phosphate synthetase genes fromnon-plant sources, from yeast (Holmström etal., 1996) and from bacteria (Pilon-Smits etal., 1998) into tobacco. The aim was to pro-mote trehalose accumulation in leaf cells,and both groups were successful andachieved greater tolerance to water deficitsin tobacco. The exciting conclusion fromthese studies, and those with the Group 3LEA protein, is that the engineering of a sin-gle gene can achieve results that affect sucha complex trait as drought tolerance. It willbe interesting to see how these results willtranslate into an advancement in our under-standing of how these gene products func-tion to achieve greater tolerance and if theseplants will have an impact on drought-tolerance breeding efforts.

The next step in the search for insightinto gene function and regulatory controlsin desiccation tolerance will come from theuse of expression profiling with cDNAmicroarrays. Such work is under way butlittle has been reported as yet. The firstinformation is likely to come from theanalysis of Arabidopsis EST microarrays inextensions of the studies reported by Girkeet al. (2000), which may pinpoint tran-scripts to the exact time of the acquisitionof desiccation tolerance in developingseeds. Direct approaches to elucidate func-tionality of individual genes or gene fami-lies will be slower to develop. The vastarray of genetic tools, such as transgeniccapability, mutant generation and screen-ing tools, T-DNA and transposon-taggedknockouts, and map-based cloning tech-nologies, will make Arabidopsis and seeddesiccation tolerance the initial foci offunctional studies. Nevertheless, tools arebecoming available for vegetative desicca-tion-tolerant model plants and these willplay an important role in evaluating targetgenes. An example of this has been theexciting use of activation tagging, by trans-genic random insertion of a highly activeforeign promoter to ‘activate’ native genes,by Furini et al. (1997) to isolate a gene(cDT-1) involved in regulation of theresponse of C. plantagineum callus to des-iccation. Advances in the fields of molecu-lar biology, genetics, genomics andbiophysics have put us on the threshold ofa new era in our quest to understand one ofthe most complex and important traits inplant biology: desiccation tolerance.


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Part II

Methodology

02 Dessication - Chap 2 18/3/02 1:53 pm Page 45


2 Methods for the Study of Water RelationsUnder Desiccation Stress

Wendell Q. SunDepartment of Biological Sciences, National University of Singapore,

Kent Ridge Crescent, Singapore 119260

2.1. Introduction 482.2. Expression of Water Status 48

2.2.1. Mass-based measures for tissue hydration 482.2.1.1. Water content 482.2.1.2. Relative water content 49

2.2.2. Thermodynamic measures for tissue hydration 502.2.2.1. Water activity 502.2.2.2. Chemical potential of water and water potential 51

2.3. Measurement of Tissue Water Potential 532.3.1. Psychrometric and hydrometric methods 532.3.2. Osmometric or cryoscopic method 542.3.3. Isothermal equilibrium method 55

2.4. Water Relations – the Thermodynamic Approach 552.4.1. The Höfler diagram and pressure–volume curve 55

2.4.1.1. Change of cell turgor pressure during desiccation 552.4.1.2. Change of osmotic potential during desiccation 572.4.1.3. The volume of water in symplast, apoplast and

intercellular spaces 572.4.1.4. Volumetric elasticity of the cell wall 59

2.4.2. Analysis of water sorption isotherms 602.4.2.1. Theoretical models 602.4.2.2. Temperature dependency of water sorption 622.4.2.3. Monolayer hydration and water-clustering function 652.4.2.4. Occupancy of water-binding sites 66

2.5. Measurement of Drying Rate and Desiccation Stress 682.5.1. Driving force for water loss and expression of drying rate 682.5.2. Quantification of desiccation stress 68

2.6. Water Relations – the Kinetic and Functional Approach 702.6.1. General considerations 72



2.6.1.1. Time scale 722.6.1.2. Structural complexity and dynamics of

molecular ordering 722.6.1.3. The model-dependent interpretation: the pitfalls 73

2.6.2. Biophysical techniques 742.6.2.1. Differential scanning calorimetry 742.6.2.2. Thermally stimulated current (TSC) method 752.6.2.3. Nuclear magnetic resonance (NMR) 762.6.2.4. Electron spin resonance 77

2.7. Concluding Remarks 782.8. References 79

Appendix 84

48 W.Q. Sun

2.1. Introduction

In hydrated plant cells, water is the mainconstituent. The organization of cellularstructures (both supramolecular assembliesand micromolecular structures) and the over-all biochemistry (the thermodynamics of bio-logical processes and their rate parameters) ofan organism depend on water. Water is a sol-vent and a medium in which diffusion ofsolutes and biochemical reactions take placein plant cells. It is often a participant and/ora product of various biochemical reactions.In low-moisture systems such as naturallydried pollen grains and plant seeds, cellularwater also plays an important role as a plasti-cizer, influencing the translational or rota-tional motions of entire molecules, orsegments of macromolecules and intramolec-ular motions. Water is involved in virtuallyevery dynamic process in a living cell.

The loss of water from plant cells is animportant environmental stress. Changes inthe aqueous environment influence thecomplex thermodynamics and kinetics ofstructural stability and all aspects of biologi-cal functions. The accurate measurement ofthe status of cellular water is essential forthe study of both desiccation stress in plantsand the mechanisms of plant desiccationtolerance. The method of quantification andinterpretation must be applicable not just tothe narrowly defined desiccation condi-tions, but also to all other types of physio-logical stresses with a dehydrationcomponent, such as freezing and salinity. Inthis chapter, several fundamental principles

of water relations that are directly related todesiccation tolerance of plant tissues will beintroduced. The strengths and limitations ofvarious methods or techniques of measure-ment of water relations during desiccationwill be discussed. An effort will be made togive a basic understanding of terms andconcepts concerning cellular water statusand the expression of dehydration stress.

2.2. Expression of Water Status

The most important quantity that has to bemeasured in all studies of desiccation toler-ance is the degree of dehydration stress. Sofar, there is no agreed parameter of dehydra-tion stress measurement. The change inwater content of plant tissues and organs isoften used as an indicator of dehydration.However, insufficient attention has beenpaid to problems commonly associated withthe use of water content as an indicator ofdehydration stress. For example, differentconcepts and approaches are currently usedby research groups working on biologicalsystems, ranging from bacteria and fungalspores to microscopic animals, pollengrains, large seeds and resurrection plants.

2.2.1. Mass-based measures for tissuehydration

2.2.1.1. Water content

Water content on a wet-weight basis (WC, %w.b.) is widely used in the literature of desic-


cation studies, and is adopted by theInternational Seed Testing Association(ISTA, 1993) for the expression of seed watercontent. WC (% w.b.) is the percentage massfraction of water of the total tissue mass:

WC (% w.b.) = (fresh weight � dryweight)/fresh weight � 100 (1)

WC (% w.b.) is not a linear expression ofwater content in tissues, because freshweight appears in both the numerator termand the denominator term in Equation (1).When WC (% w.b.) is used to monitor theloss of water during desiccation, thedecrease of WC (% w.b.) does not necessar-ily reflect the exact extent of dehydrationstress. The change of WC (% w.b.) duringdrying is, in fact, related to the change ofthe reciprocal of tissue fresh weight. Forexample, when the tissue of 80% WC (%w.b.) is dried to 70% and 60% water con-tent, the tissue actually loses 41.7% and62.5% of its initial water quantity, respec-tively, not just 12.5% and 25% reduction asimplied by the values of WC (% w.b.). Thequantity of water lost during dehydrationfrom 80% to 70% WC (% w.b.) is twice asmuch as water loss during dehydrationfrom 70% to 60% WC (% w.b.).

Water content on a dry-weight basis mea-sures the mass ratio between water and thedry mass in tissues, and is often expressedby g water per g dry weight (i.e. g g�1 dw):

WC (g g�1 dw) = (fresh weight � dryweight)/dry weight (2)

WC (g g�1 dw) is a linear expression ofwater content, and the change of WC (g g�1

dw) during drying is proportional to theloss of water in a tissue. On the mass basis,a tissue with a WC of 0.20 g g�1 dw ishydrated exactly twice as much as the tis-sue with a WC of 0.10 g g�1 dw, and four-fold as much as the sample with a WC of0.05 g g�1 dw. For this reason, someresearchers have argued that WC (g g�1 dw)is a more sensible expression than WC (%w.b.) for the measurement of dehydration.At WC < 15%, the difference between WC(% w.b.) and WC (g g�1 dw) is fairly small.WC (% w.b.) is converted to WC (g g�1 dw)using the following equation:

WC (g g�1 dw) = WC (% w.b.)/[100 – WC (% w.b.)] (3)

2.2.1.2. Relative water content

Relative water content (RWC) is anothermass-based parameter. RWC is widely usedin the pressure–volume analysis of planttissue water stress. RWC is a simple anduseful measure of the extent to which a tis-sue is in water deficit. It is related to tissuewater content at full turgor (WCF). During adehydration experiment, RWC is calcu-lated by dividing water content at a giventime by water content at full turgor, andexpressed as a fraction value or as a per-centage. If water content in the tissue isdetermined as WC (g g�1 dw), the calcula-tion of RWC is straightforward, beingWC/WCF. But, if water content is deter-mined as WC (% w.b.), RWC is calculatedby the equation:

RWC = [WC (100 – WCF)]/[WCF(100 – WC)] � 100 (4)

RWC is a linear expression of moisturecondition. The change in RWC over timeserves as a good indicator for the rate ofdehydration. Physiological responses ofplant water deficit are highly correlatedwith RWC (Sinclair and Ludlow, 1985). Theuse of RWC is particularly advantageous forcomparative studies, in which initial watercontent at full turgor or full hydrationvaries considerably among differentspecies, different tissues of the samespecies or the same tissue at differentdevelopmental stages, such as seeds. In cer-tain cases, it may be even preferred overwater potential, because RWC also accountsfor the effect of osmotic adjustment inaffecting plant water status. For example,two plants with the same leaf water poten-tial can have different RWCs if they differin their ability for osmotic adjustment.

In many desiccation studies on higherplants, water content of a tissue at full tur-gor was not specifically determined, andinstead water content after full hydration inwater was used. Typically, leaf samples (e.g.discs or sections) of higher plant speciesare taken and weighed immediately. The

Methods for Studying Water Relations Under Stress 49


samples are then hydrated in distilledwater for 4–6 h, after which they areweighed again and their water contents aredetermined by drying the samples in anoven. In higher plants, the amount of inter-cellular water is small or non-existent(Oertli, 1989). However, it should be notedthat some plant tissues do contain intercel-lular water, which is held in spacesbetween cells of a tissue at relatively highwater potential (near zero). Therefore, watercontent at full turgor has different physio-logical meaning from water content of thetissue at full hydration when the tissue con-tains intercellular water. If intercellularwater is present, water content of the tissueat full turgor has to be estimated from theplot of water potential on water content (gg�1 dw). To determine the water content ofthe tissue at full turgor, two linear regres-sion lines can be fitted, respectively, withdata points where water potential remainsalmost unchanged during initial water lossand the next few points where water poten-tial starts to fall. The intercept of these tworegression lines gives the water content atfull turgor. Beckett (1997) reported that theamount of intercellular water varied greatlyamong species of bryophytes. If intercellu-lar water exists in a tissue, correction needsto be made to the raw RWC readings, whichare calculated according to the water con-tent at full hydration. The method used tocorrect the raw RWC readings wasdescribed in detail by Beckett (1997).

There are shortcomings in using mass-based parameters for the expression ofwater content. Plant tissues are heteroge-neous, complex biological systems, inwhich carbohydrates, proteins and lipidsand other components have different hydra-tion properties. As a consequence, whenplant tissues of various species are equili-brated under given conditions of tempera-ture and relative humidity, equilibriumwater content varies considerably amongspecies. For example, seeds with large lipidreserves equilibrate to lower water contentsthan starchy seeds, even though the chemi-cal potential of water molecules is the samefor all tissues when equilibrium isachieved. The disadvantage of using mass-

based parameters for the expression ofdehydration stress has been shown by anumber of studies. The critical onset waterpotential of Quercus rubra (Pritchard, 1991)and Quercus robur (Pritchard and Manger,1998) is about –3 MPa, but the correspond-ing mass water contents vary substantiallydue to different seed oil content. Sun andGouk (1999) studied the water relationresponses of three recalcitrant (desiccation-sensitive) seeds (Aesculus hippocastanum,Andira inermis, Q. rubra) during controlleddehydration. The critical water potentialsfor seeds are quite similar for all threespecies (�7 to �8 MPa), but their corre-sponding critical water contents are 0.45,1.10 and 0.35 g g�1 dw for A. hippocas-tanum, A. inermis and Q. rubra, respec-tively. If the critical water content wereused to express the relative desiccation tol-erance, one would conclude incorrectlythat seeds of A. hippocastanum and Q.rubra are much more desiccation-tolerantthan seeds of A. inermis. Therefore, a mass-based parameter of water loss may not be areliable indicator for the degree of desicca-tion stress in plant tissues.

2.2.2. Thermodynamic measures for tissuehydration

The response of plant tissues to desiccationis related to the thermodynamic andkinetic status of tissue water, rather than toactual water content. The water status ofplant tissues can be expressed in terms ofenergy status of water molecules, i.e. thepartial molar Gibbs’ free energy or waterpotential. This thermodynamic approach ispreferred to the mass-based expression oftissue hydration, because thermodynamicparameters (i.e. energy status) are relateddirectly to the numerous biophysical andphysiological events that contribute to des-iccation stresses and desiccation toleranceof plant tissues.

2.2.2.1. Water activity

Water activity (aw) is used to describewater status in the studies of desiccation

50 W.Q. Sun


tolerance and storage survival for spores,pollen, seeds and resurrection plants (Elliset al., 1990, 1991; Berjak and Pammenter,1994; Vertucci et al., 1994, 1995; Walters,1998a). Water activity is measured as theratio of the vapour pressure of water in asystem to the vapour pressure of purewater at the same temperature. It is relatedto the equilibrium relative humidity (RH)of the air surrounding the system (i.e. RH =aw � 100). Water activity can be viewed asthe ‘effective’ water content, which is ther-modynamically available to various physi-ological processes in cells. For the survivalof organisms under water stress, the ‘effec-tive’ water is more important than the totalamount of water present in the tissue.

Water activity of fresh plant tissues mayvary only between 0.980 and 0.996. Withinthis narrow range, it is not useful for theexpression of dehydration stress or tissuewater status. However, for the studies ofsevere water stress and extreme desicca-tion, water activity has several advantagesover water content, including its concep-tual simplicity, measurability, easy experi-mental manipulation, and its applicabilityto both simple and complex systems. Anumber of physiological processes that arerelevant to desiccation tolerance or damagehave been shown to occur at specific wateractivities, and some of those are presentedin Fig. 2.1.

Water activity in plant tissues can bedetermined using the hygrometric methodand the isothermal equilibrium sorptionmethod. The hygrometric instrumentmethod directly measures the equilibriumRH of plant tissues in a closed chamber.With the equilibrium sorption method,samples of plant tissues are equilibrated toa series of known water activities at a spec-ified temperature. The relationshipbetween water content and water activityupon equilibrium (i.e. the sorptionisotherm curve) is then used to calculatewater activity of plant tissues at differentwater contents. Water activity is defined atequilibrium. However, plant tissues at lowand intermediate moisture levels may notbe in a true state of equilibrium at all, butin an amorphous metastable state instead.

In such cases, the measured vapour pres-sure of water may not be the equilibriumvapour pressure, but the vapour pressure ofa ‘stationary’ state that is time-dependent.However, studies in food sciences havesuggested that water activities measuredare likely to be close to equilibrium and thedifferences should be within the uncer-tainty associated with the experimentaldetermination (Chirife and Buera, 1996).The usefulness of the water activity con-cept in seed storage stability has been dis-cussed by Walters (1998b).

2.2.2.2. Chemical potential of water andwater potential

The quantity of free energy of a component(µj) in a system is measured by its chemicalpotential. The chemical potential of water(µw) in a system is defined by:

µw = µ*w + RT ln aw + V–

wP + zwFE + mwgh (5)

where µ*w is the chemical potential of purewater at ideal reference conditions. Thesecond term RT ln aw is for water activity. Ris the gas constant (8.314 � 10�3 kJ mol�1

K�1), T is the absolute temperature (K, inkelvin), and aw is water activity (RH/100).V–

w is the partial molal volume of water (i.e.the differential increase or decrease in vol-ume when a differential amount of water isadded or removed). V

–w is influenced by the

presence of solutes and is also tempera-ture-dependent (1.805 � 10�5 m3 mol�1 at20°C). P is the hydrostatic pressure onwater in excess of atmospheric pressure(MPa, 1 MPa = 103 kJ m�3). The term V

–wP

represents the effect of pressure on thechemical potential of water and isexpressed in energy per mole (kJ mol�1).zwFE is the electrochemical potential ofwater, which equals zero because water isuncharged (zw = 0). The last term mwgh isthe gravitational term, representing thework needed to move 1 mole of water to agiven height. Practically, mwgh will remainconstant in most circumstances of desicca-tion studies.

The water potential is proportional tothe chemical potential of water (µw � µ*w) ina system as described in Equation (5).



Therefore, water potential is actually thepotential energy of water per unit mass.While water content tells how much wateris in a sample, water potential tells youhow available that water is. By convention,water potential is defined as follows:

� = �P + �π + �h (6)

where �p = P and is hydrostatic pressureon water as defined in Equation (5), �π is

osmotic potential and �h is the gravita-tional potential. The total water potential isthe sum of hydrostatic, osmotic and gravita-tional components. The gravitational term(�h) depends on the position of water in agravitational field and is not relevant tomost desiccation studies. Osmotic potentialdepends on the concentration of dissolvedsubstances in water. Osmotic potential isrelated to water activity by the equation:

52 W.Q. Sun

Wat

er p

oten

tial o

f wat

er v

apou

r (M

Pa)

–250

–200

–150

–100

–50

0

20 40 60 80 100

Ort

hodo

xse

eds

typi

cally

sur

vive

at l

ower

aw

Wat

er v

apou

r ab

ove

satu

rate

d C

aCl 2

sol

utio

n

Typi

cal e

xpos

ure

of N

osto

cco

loni

es in

situ

DN

A d

isor

dere

d an

d da

mag

edat

low

er a

w

Des

icca

tion

tole

ranc

e of

em

bryo

s of

Cof

fea

spec

ies

Nuc

leic

aci

ds a

nd p

rote

ins

fully

hyd

rate

d

Wat

er v

apou

r ab

ove

satu

rate

d N

aCl s

olut

ion

Lyso

zym

e ac

tivity

sto

ps

Min

imum

req

uire

d fo

r ph

otos

ynth

esis

Cel

l res

pira

tion

star

ts to

cea

se

Mea

n of

min

imum

val

ue fo

r ba

cter

ial g

row

th

Relative humidity (%)

Fig. 2.1. Water activities (relative humidities) that limit physiological activities and cell growth. Physicalparameters and physiological processes are drawn with data from Wolfe and Leopold (1986), Potts (1994)and Sun and Gouk (1999). The relationship between relative humidity and water potential is calculatedaccording to Equation (7) at 25°C. A similar diagram that is specific to a plant tissue can be established.Such a diagram would serve as a valuable reference for experimental design and data interpretation, since itgives a clear concept about the possible sequence of potential physiological and biochemical events andtheir interactions as the tissue loses water.


V–

w �π = RT ln aw (7)

During dehydration, the water content inseed tissues is reduced, resulting in anincrease in the concentration of solutes andthus a decrease in water activity andosmotic potential (i.e. �π becomes morenegative). A similar situation occurs duringfreezing. The formation of ice leads to thedehydration of the protoplast and the con-centration of solutes.

The interactions of water with biologicalsurfaces and interfaces are of great impor-tance to desiccation tolerance of plant tis-sues, especially at low moisture levels. Theinfluence of such interactions on waterpotential in a tissue is commonly called‘matric’ potential. Rapid water uptake bydry seeds during the early stage of germina-tion is mainly attributed to large matricpotentials. Another example is the reducedrate of water loss as the tissue is dried tolower water content. Matric potentialdepends on the adsorptive forces that bindwater to a matrix. The amount of matrix-bound water in recalcitrant Q. roburembryonic axes is as high as 0.25–0.30 gg�1 dw (Pritchard and Manger, 1998).However, the forces of such water–matrixinteractions are adequately represented bytheir contributions to hydrostatic pressure(P) and osmotic potential (�π). For exam-ple, the presence of aqueous interfaces incells lowers water activity through interfa-cial attractions and binding of water neartheir surfaces, which has already beenincluded in the osmotic component inEquation (7). Therefore, matric potentialdoes not represent additional new forces.

2.3. Measurement of Tissue WaterPotential

A pressure chamber (pressure bomb) is com-monly used to measure directly leaf waterpotential of higher plants. The detached leafis sealed in a steel chamber with the cutpetiole protruding out. Pressure that isapplied to the chamber is taken as thexylem (leaf water) potential when the sapmeniscus appears at the petiole xylem sur-

face. This technique, however, is unsuitablefor many desiccation-tolerant plant tissues,e.g. lichens, bryophytes, spores, pollen grainand seeds. This is because the pressurechamber method measures the xylem ten-sion, which is broadly equal to the leafwater potential. Water potential of plantparts that do not have vascular systems can-not be measured with the pressure chambermethod. However, water potential of planttissues can be measured by a number ofother techniques. These techniques useeither the relationship of the sample waterpotential to the equilibrium vapour pressureimmediately around the sample or the prin-ciple of the freezing-point depression in theliquid solution.

2.3.1. Psychrometric and hydrometricmethods

Both methods are widely used for the mea-surement of tissue water potential. The mea-surement of water potential by apsychrometer and a hydrometer is calledthe wet-bulb depression method and thedew-point depression method, respectively.A psychrometer measures water potential ofsamples (placed in closed chambers)through its ability to determine the RH ofthe closed environment. The instrumentuses high-sensitivity thermometers to mea-sure temperature reduction resulting fromthe heat of vaporization of water in a samplerelative to pure water. It can measure waterpotential of solid tissue materials anddroplets of solutions. The sample is firstsealed in a small chamber containing a ther-mocouple. After an equilibration period, acooling current is applied to the thermocou-ple in order to condense water on the ther-mocouple junction. The amount ofcondensed water is proportional to thewater potential of the tissue. The water isallowed to evaporate, causing a change inthe thermocouple output, and the output iscalibrated for water potential, using stan-dard salt solutions. On the other hand, ahydrometer maintains the dew-pointdepression temperature during the measure-ment using a thermocouple. The dew-point



depression temperature is the temperatureto which the air in the sealed chamber mustbe reduced so that the air becomes saturatedwith water vapour. Psychrometric andhydrometric methods can be used to mea-sure both water potential and osmoticpotential of plant tissues. To measureosmotic potential, a sample has to undergothe freeze–thaw cycles to disrupt the cellu-lar structures before the measurement,whereas the water potential is measuredusing undisrupted tissues.

A psychrometer is very sensitive to tem-perature change because it measures verysmall temperature differences. A change inwater potential of 1.0 MPa is reflected by achange in wet-bulb temperature depressionof only approximately 0.085°C. A hydro-meter is less affected by the changes inambient temperature during the measure-ment compared with a psychrometer.Psychrometric and hydrometric methodsare suitable only for plant tissues of highwater content. At low water content, theequilibrium may take several hours toachieve. The nominal range of the Peltierthermocouple measurement is limited from0 to �6.0 MPa for these two methods. Yetmany desiccation-tolerant plant tissues cansurvive far below �6.0 MPa. Even if oneuses the Richards thermocouple, it extendsonly to –25 MPa and the accuracydecreases to –0.1 MPa at –10 MPa(Decagon Devices Inc., Pullman,Washington, USA), corresponding to an RHof ~84% at 25°C.

2.3.2. Osmometric or cryoscopic method

A freezing-point osmometer measures theosmotic concentration of a biological liquidusing the principle of the freezing-pointdepression. The freezing-point depressionis one of the four colligative properties of asolution. The freezing point is the uniquetemperature at which the ice phase and theliquid phase can coexist in equilibrium atstandard pressure. When a solute is dis-solved in the water, the freezing point ofthe water is lowered in proportion to theosmotic potential of the solution. For a 1.0

osmolal aqueous solution, the osmoticpotential at 0°C is ideally �2.271 MPa, andthe freezing-point depression is 1.86°C.(An osmole is the mass of a substance thatwhen dissolved in 1 kg water generates anosmotic pressure equivalent to that pro-duced by 1 mole of an ideal solute dis-solved in 1 kg water. After dissolving, anideal solute gives 6.023 � 1023 osmoticallyactive particles.) Theoretically, the osmoticpotential of an unknown sample can beestimated from the depression of its freez-ing point by the following relationship:

where �T is the depression of the freezingpoint. The effect of osmotic potential onfreezing-point depression also holds fornon-ideal solutions such as plant saps.However, freezing-point depression is non-linear with concentration changes duringdehydration. Water potential (MPa at 0°C)can be derived by the empirical equation(Crafts et al., 1949):

Ψ = �1.206�T + 0.0021�T2 (9)

With the osmometric method, a sampleis usually supercooled a few degrees belowits freezing point to induce immediatecrystallization. As the heat of fusion isreleased, the sample temperature rises toits freezing point, and its equilibrium tem-perature is measured. Alternatively, thetemperature at which ice crystals startmelting can be measured and taken as theequilibrium freezing temperature (i.e.Ramsay’s method). The applicability ofEquations (8) and (9) to the measurementof water potential or osmotic potential inplant tissues was examined by Sun andGouk (1999), using seed tissues that werepre-equilibrated with saturated salt solu-tions (from �3 to �35 MPa). The freezing-point depression was determined with adifferential scanning calorimeter, using theonset temperature for the exotherm oncooling. Calculated water potentials werefound to be very close to the pre-freezingwater potentials of seed tissues, withEquation (9) fitting the data slightly betterthan Equation (8).

Ψ ∆ ∆π =−

°= −

2 2711 86

1 221.

..

MPaC

(8)T T

54 W.Q. Sun


2.3.3. Isothermal equilibration method

It is more difficult to measure directlywater potential of low-moisture systems.Perhaps the convenient, yet accurate andreliable method is first to establish theempirical relationship between water con-tent and water potential for a particularplant tissue. Samples of plant tissues areequilibrated over different salt solutionsthat would maintain a series of constantwater vapour pressures (i.e. RH) in closedcontainers. Upon equilibrium, the watercontents of tissue samples are determinedgravimetrically, and their water potential atequilibrium is then the same as the waterpotential of the air in the closed containers,which in turn equals the osmotic potentialof the salt solutions used. Therefore, waterpotential of tissue samples is calculated bythe equation:

� = RT ln %RH (10)V–

w 100

where R is the gas constant, T is theabsolute temperature (kelvin), V

–w is the par-

tial molal volume of water, and %RH is thepercentage relative humidity inside thecontainers. (Note that Equation (10) isessentially the same as Equation (7).) Theempirical relationship between water con-tent and water potential can be describedby exponential and polynomial (Poulsenand Eriksen, 1992) or other functions (Sunand Gouk, 1999). The derived mathematicalexpression is then used to calculate waterpotential of plant tissues at any water con-tent within the limit of experimental range.This method is particularly useful in moni-toring the change of tissue water potentialduring desiccation. Water potential of dehy-drating tissues can be calculated immedi-ately from the data of water loss.

Constant RH can be achieved using satu-rated or non-saturated salt solutions, poly-ethylene glycol solutions and glycerolsolutions. Physico-chemical data of varioussalts and their solutions are presented in theAppendix. This technique does not need spe-cial instruments to measure water potential,and can avoid the difficulty in measuring RHaccurately. This method has been used suc-

cessfully in seed desiccation studies by anumber of workers (Pritchard, 1991; Poulsenand Eriksen, 1992; Vertucci et al., 1994; Sunet al., 1997; Tompsett and Pritchard, 1998).

2.4. Water Relations – theThermodynamic Approach

2.4.1. The Höfler diagram and thepressure–volume curve

Water relations parameters of plant tissuescan be presented by the Höfler diagram andthe pressure–volume curve (PV curve). TheHöfler diagram shows the relationshipbetween water potential and relative watercontent (Fig. 2.2a). The PV curve is a plotbetween the reciprocal of water potential(�1/�) and RWC or water loss (1 – RWC)during desiccation (Fig. 2.2b). Both theHöfler diagram and the PV curve arewidely used to characterize water relationsof plant tissues. To construct a Höfler dia-gram or a PV curve, the changes in waterpotential and RWC are monitored as thetissue is dehydrating. Several importantparameters can be obtained by analysingthe components of cell water potential,including the osmotic potentials at full tur-gor and at the partially dehydrated state,the apoplastic and symplastic water vol-ume in tissues, a plot of turgor pressure(i.e. hydrostatic water potential in Equation(6)) as a function of RWC, and the tissuebulk modulus of elasticity. Without know-ing these biophysical metrics, it would beimpossible to identify different kinds ofcellular stresses associated with the loss ofwater in the tissue and to examine the sig-nificance of an array of biochemical andphysiological responses during desicca-tion. Moreover, valid comparisons of theresponse of cell function to water stressamong different organisms cannot be madewithout such knowledge.

2.4.1.1. Change of cell turgor pressure duringdesiccation

In fully turgid cells, turgor pressure is equalto the osmotic potential (with opposite



56 W.Q. Sun

0.0

–0.2 0.0 0.2 0.4 0.6 0.8 1.0

1 – RWC

Externalwater

�p

�

��

�p

0

0.7 1.0 1.3

RWC

�p

(�p = 0)–1/��

Incipientplasmolysis–1

/� (

MP

a–1 )

(a)

(b)

2

0

–2

–4

–6

0.0 0.2 0.4 0.6 0.8 1.0 1.2

Relative water content (RWC)

Wat

er p

oten

tial (

MP

a)R

ecip

roca

l of w

ater

pot

entia

l

Fig. 2.2. Cellular water relations. (a) Höfler diagram showing the components of cell water potential.Intercellular or external water (RWC > 1.0) in many plant tissues is held at near-zero water potential and,during the initial dehydration, cell water potential (�) and turgor pressure (�p) do not change significantly(the horizontal dashed line). Maximum osmotic potential is found at the point of full turgor (RWC = 1.0),where �p = ��π. As the plant tissue loses water, turgor pressure decreases, and at the turgor-loss point(RWC = ~0.8), � = �π (the vertical dashed line). At RWC < ~0.8, the relationship between RWC and �πfollows a rectangular hyperbola (RWC = a + b/�π). Osmotic potential at RWC = ~0.8–1.0 is extrapolatedfrom the rectangular hyperbola relationship. Turgor pressure is the difference between the measured waterpotential and the extrapolated osmotic potential. (b) The pressure–volume curve showing the relationshipsbetween �, �p and �π during dehydration. The reciprocal of water potential is plotted against (1 � RWC).Beyond the turgor-loss point (incipient plasmolysis), the relationship between (1 � RWC) and 1/� (or 1/�π)is linear. The extrapolation of this linear relationship toward the y-axis intercept gives osmotic potential (thedashed line) of the tissue when the tissue is still turgid. The difference between the measured water potentialand the extrapolated osmotic potential is turgor pressure (inset).



signs). During dehydration, the PV curve ofa plant tissue initially displays a concaveregion, beyond which the curve is linear(Fig. 2.2b). The loss of turgor is marked bythe point at which the relation of 1/� to(1 � RWC) deviates away from linearity.Turgor pressure (�p) is calculated as thedifference between the extrapolated linearportion of the PV curve and the waterpotential actually measured, and is oftenplotted as a function of RWC. The relation-ship between turgor pressure and RWC canbe described sufficiently by a quadratic orcubic function.

Certain plant tissues might develop nega-tive turgor pressure before the cells collapseand �p can become zero under severe waterstress. When negative �p develops, the PVcurve would fall below the extrapolated lin-ear portion of the graph (Fig. 2.3a). If thecells are sufficiently strong, do not collapseand the plasma membrane remains firmlyattached to the cell wall, the formation of anintracellular gas bubble will increasinglybecome possible (cavitation). The develop-ment of negative turgor pressure and intra-cellular cavitation appear to play some rolesin desiccation tolerance of certain cells. Agood example of a cell surviving large nega-tive turgor pressure and cavitation is theascospore of Sordaria (Milburn, 1970). Thevolume of Sordaria ascospores changes verylittle, and the protoplast remains in contactwith the spore wall at all times. Underwater stress (by air-drying or in osmoticsolution), these cells might generate nega-tive �p as much as –4 MPa. Beyond thisnegative turgor pressure, a small bubbleappears inside the protoplasm suddenly,which increases slowly in size andapproaches the walls quite closely withoutlosing its spherical appearance. Honegger(1995) and Scheidegger et al. (1995) alsoshowed that ascomycetous lichen myco-bionts form large intracellular gas bubbleswhen desiccated. More recently, the PVanalysis by Beckett (1997) suggested theexistence of negative turgor pressure in veg-etative cells of several desiccation-tolerant(poikilohydric) plants (e.g. Dumortiera hir-suta and Myrothamnus flabellifolia). PVcurves of most plants do not show any indi-

cation of negative turgor pressure. It is con-ceivable that the development of negativeturgor pressure may reduce mechanicaldamage on cellular structures by preventingcollapse of the cells.

2.4.1.2. Change of osmotic potential duringdesiccation

When cell turgor pressure falls to zero dur-ing desiccation, the water potential of thecell is equal to the osmotic potential (seeEquation (6)). As desiccation continues,osmotic potential and cell water potentialare equal and inversely proportional to thevolume of osmotically active water. The rela-tionship between RWC and the reciprocal ofosmotic potential is a straight line. Theosmotic potential at full turgor is calculatedfrom the extrapolation of the linear portionof the PV curve to the RWC at full turgor (i.e.RWC = 1.0 in Fig. 2.2b). In the Höfler dia-gram, the relationship between osmoticpotential and RWC is represented by a rec-tangular hyperbolic function to the datapoints corresponding to the linear part of thePV curve (dashed part of the �π in Fig. 2.2a).

2.4.1.3. The volume of water in symplast,apoplast and intercellular spaces

In hydrated plant tissues, water may existin the symplast, in the apoplast (i.e. theporous spaces in the cell wall) and in theintercellular spaces (large voids) as dis-cussed before. Intercellular water, alsocalled ‘external’ cell water by some work-ers, may account for up to 35% of totalwater in certain plant tissues, such aslichens, liverworts, mosses and fern fronds(Beckett, 1997; Proctor, 1999) and develop-ing embryos of higher plants (W.Q. Sun,unpublished data). During desiccation,water potential and turgor potential do notfall with initial water loss at RWC > 1.0(Fig. 2.2a and b inset). The volume of waterthat is lost before turgor pressure starts tofall is assumed to be intercellular water.The volume of symplastic water representsthe amount of osmotically active water inthe tissue, and is obtained by subtractingthe apoplastic water volume from the water


content at full turgor. Symplastic watergenerally declines over a range of waterpotential from about �0.5 to �10 MPa, inline with that of osmotic potential.

Apoplastic or osmotically inactive wateris present in very small pores and strongwater-binding sites of biological surfaces inplant tissues. This fraction of water is held

58 W.Q. Sun

�p

0

0.6 0.8 1.0RWC

12

(a)

(b)

Rec

ipro

cal o

f wat

er p

oten

tial

–1/�

(M

Pa–

1 )

1

2

0.0

–0.2 0.0 0.2 0.4 0.6 0.8 1.0

1 – RWC

Wat

er p

oten

tial (

–MP

a)

1000

100

10

1.0

0.1

0.0 0.2 0.4 0.6 0.8 1.0

Relative water content (RWC)

Fig. 2.3. (a) The pressure–volume curves of plant tissues that develop negative turgor pressure (curve 1) andintracellular cavitation (curve 2) during desiccation. The inset in (a) shows the change of cell turgor pressure(�p) during the early stage of drying. When intracellular cavitation occurs, the �p suddenly changes to zero(curve 2, inset), and � is equal to �π (curve 2). If intracellular cavitation does not occur, the cell wall willcollapse or deform when the �p develops beyond the threshold to which the cell wall can resist (i.e. (1 �RWC) > 0.15). The collapse or deformation of the cell wall will lead to a gradual increase in � (curve 1)and �p (curve 1, inset). (b) The semi-logarithmic plot between RWC and tissue water potential. The highRWC break point corresponds to the turgor-loss point, whereas the low RWC break point corresponds to thevolume of apoplastic water. Drawn with data from Quercus rubra seeds (Sun, 1999).


by matric and molecular forces, and lostonly when plant tissues are desiccated to awater potential less than �15 MPa(Meidner and Sheriff, 1976). The loss ofapoplastic water in some species extendsto approximately �800 MPa. The amountof such matrix-bound water in plant tissuescan be as high as 0.1–0.2 RWC or up to0.25–0.35 g g�1 dw. This fraction of waterdoes not generally act as a solvent in cells,and is not readily freezable. From theHöfler diagram, the apoplastic volume isestimated from the fitted hyperbolic func-tion. From the PV curve, the volume ofapoplastic water is commonly estimated byextrapolation of the linear relationshipbetween RWC and the reciprocal ofosmotic potential to the (1 � RWC) axisafter the loss of turgor pressure. However,the simple extrapolation from the PV curveis not a reliable method of estimating theapoplastic volume, and in some cases givesnegative values (Proctor et al., 1998).

The apoplastic volume of water shouldbe derived with data from the isothermalsorption study at low water potentials(water activity), rather than the extrapola-tion method, because the linear relation-ship between RWC and the reciprocal ofosmotic potential does not hold for theapoplastic volume of water (which isosmotically inactive). Compared to theremoval of osmotically active (symplastic)water, the measured osmotic potential(including the term of matric potential)declines much more rapidly when theapoplastic water is removed. Therefore, thevolume of apoplastic water is marked bythe point at low water content at which therelationship of 1/� to (1 – RWC) againdeviates away from linearity (Fig. 2.3b).The volume of apoplastic water roughlycorresponds to the primary hydration intissues (including both strong and weakwater-binding sites).

One can expect that plant tissues wouldrespond differently to the loss of external,symplastic and apoplastic water. The loss ofsymplastic water can cause osmotic pertur-bation of physiological and biochemicalprocesses, whereas the loss of apoplasticwater may disrupt the structure and func-

tion of cellular membrane and molecularassemblies. So far, workers have paid littleattention to the location of water in planttissues. The difference in the relative vol-ume of external, symplastic, and apoplasticwater should be taken into account in thecomparative studies on mechanisms of des-iccation tolerance among cells, tissues orplants. A similar analysis of water relationswas found to be very useful in developing amechanistic understanding of the role ofdehydration in freezing tolerance in earth-worms (Holmstrup and Zachariassen, 1996).

2.4.1.4. Volumetric elasticity of the cell wall

The cell wall may undergo elastic expan-sion or contraction. Elastic (mechanical)properties of cell walls play an importantrole in cell water relations. For example,the negative turgor pressure that candevelop in a cell largely depends on themechanical properties of the cell wall. Theelasticity of the cell wall is represented bythe volumetric elasticity module �, where �depends on both �p (turgor pressure) andV (cell volume) and is defined as:

��p� = V (11)

�V

where �V is volume change caused by agiven pressure change ��p. Equation (11)indicates that a high value of � implies arigid cell wall, whereas a low value impliesa more elastic cell wall. The � value can becalculated from the relationship between�p and RWC (Steudle et al., 1977;Stadelmann, 1984). The change of � as afunction of RWC is given by the first deriv-ative of the quadratic or cubic function ofturgor pressure on RWC. The value of the�p/RWC derivative curve at RWC = 1.0 isusually taken as the bulk modulus of elas-ticity and used for purposes of comparison.

A pressure probe technique can be useddirectly to determine the turgor pressureand the � for individual plant cells. Thistechnique is useful for continuous mea-surement of cell turgor pressure, cell wallelasticity and hydraulic conductivity of thecell membrane in single cells (Hüsken etal., 1978). The intracellular hydrostatic



pressure is transmitted to the pressuretransducer via an oil-filled microcapillaryintroduced into the cell, which transformsinto a proportional voltage. This techniquepermits volume changes and turgor pres-sure changes to be determined with anaccuracy of 10�5–10�6 µl and 3–5 � 10�3

MPa, respectively.At present, very little information is

available on cell wall properties of desicca-tion-tolerant plant tissues. Proctor (1999)found that two highly desiccation-tolerantliverworts have low values of bulk elasticmodulus. He thought that extensible cellwalls might be a part of structural adapta-tion to rapid changes of cell volume intheir intermittently desiccated habitats.Ultrastructural studies on dry mesophyllcells of desiccation-tolerant Selaginellalepidophylla by Thomson and Platt (1997)showed highly folded cell walls and con-tinuous apposition of plasmalemma to thewalls. Vicre et al. (1999) studied the cellwall architecture of leaf tissues ofCraterostigma wilmsii (a resurrectionplant), and also observed extensive foldingof the cell wall during desiccation. Thefolding of the cell wall allows the plasmamembrane to remain firmly attached to thewall as the cell loses water. Biochemicalmodifications of the cell wall wereobserved during desiccation and rehydra-tion, leading to the change in its tensilestrength that may prevent the total collapseof the walls in the dry tissue and avoidrapid expansion upon rehydration. Thechange in cell wall elasticity during desic-cation can be determined easily by takingthe first derivative of the function of turgorpressure on RWC.

2.4.2. Analysis of water sorption isotherms

The water sorption isotherm is the depen-dence of water content on water activity ofthe surrounding environment at a giventemperature. There are two types of sorp-tion isotherms: desorption isotherm andadsorption isotherm (Fig. 2.4a).Conventionally, a desorption isotherm isdeveloped by drying fresh tissues over satu-

rated salt solutions in closed desiccatorsuntil constant weights are achieved,whereas an adsorption isotherm is devel-oped by rehydrating dried tissues over satu-rated salt solutions. A desorption curve canalso be developed during drying of tissuesin any atmospheric condition by measur-ing, at various points in time, the watercontent of the tissue and the equilibriumRH of its surrounding air in a closed con-tainer. Similarly, the dry tissue can be rehy-drate with a given quantity of water to raisethe water content and equilibrium RH.Sophisticated instruments such as con-trolled atmosphere microbalance anddynamic vapour sorption systems (SurfaceMeasurement Systems, London, UK) usethe latter methods. Desorption and adsorp-tion isotherms are used, respectively, tostudy the properties of dehydration andrehydration of plant tissues. Desorption andadsorption curves are rarely the same: thedesorption curve usually gives a higherwater content than the adsorption isotherm.The difference in the equilibrium watercontent between two curves is called hys-teresis. Hysteresis is evidence of the irre-versibility of the sorption process, andtherefore indicates the limited validity ofthe equilibrium thermodynamic approachto investigate the dehydration–rehydrationproperties of plant tissues. Hysteresis mightbe an important issue when consideringcritical water activities for desiccationstress during dehydration–rehydrationcycles and when investigating storage stabil-ity after manipulation of moisture content ofseeds and pollen.

2.4.2.1. Theoretical models

Plant tissues show a sigmoid sorptionisotherm (Fig. 2.4a). The inflection point ofthe isotherm is believed to indicate either achange of water-binding capacity and/orthe relative amount of ‘bound’ or ‘free’water. Water sorption data are normallyanalysed using theoretical models, fromwhich useful biophysical parameters ofwater relations are derived. Commonlyused models include the Brunauer–Emmett–Teller (BET) model, the

60 W.Q. Sun


Guggenheim–Anderson–de Boer (GAB)model and the D’Arcy–Watt model.

The BET model (Brunauer et al., 1938)is derived from statistical and thermody-namic considerations. The equation can bewritten as:

where aw is the water activity, Mw is equi-librium water content in the tissue, Mm isthe BET monolayer (water content corre-

aM a

CM

aM C

w

w w mw

m

(12)1

1 1−( ) = −

+


(a)

(b)

Wat

er c

onte

ntS

orpt

ion

enth

alpy

Water content

Water activity

III III

Free energy

Entropy

Enthalpy

Adsorption

Desorption

I II III

Fig. 2.4. The analysis of water sorption isotherms. (a) The typical shape of desorption curves and adsorptioncurves of plant tissues. The difference between these two curves shows hysteresis, which indicates theirreversibility of water sorption in the tissues during dehydration and rehydration. The sigmoid shape ofsorption curves is presumably due to the existence of three types of water-binding sites in tissues (strong (I),weak (II) and multilayer molecular sorption sites (III)). (b) Differential enthalpy (�H), free energy (�G) andentropy (�S) of hydration. Desorption curves can be used to calculate �H and �S of tissue hydration. Seetext for detailed discussion.


sponding to the monolayer hydration) andC is temperature dependence for sorptionexcess enthalpy (Brunauer et al., 1938,1940). BET equation parameters, Mm and C,can be calculated by plotting aw/[Mw (1 �aw)] against aw. The y-axis intercept of thestraight line is equal to 1/(MmC) and theslope is equal to (C � 1)/Mm. The BET isvalid only for aw < 0.5, thus data pointswithin that range are used to estimate themonolayer value (Mm). The BET model isan effective method for estimating theamount of water bound specifically topolar sites (monolayer), but cannot be usedto give a complete estimation of specifichydration parameters.

The GAB model is an extension of theBET model, taking into consideration themodified properties of the sorbing materi-als in the multilayer region and the bulkliquid properties through the introductionof a third constant, K. The GAB equation iswritten as:

Mw =MmCKaw

(1 � Kaw)(1 � Kaw + CKaw)(13)

where C and K are temperature-dependentcoefficients. Constants, Mm, C and K areestimated via the curve fitting of sorptiondata. In the field of food sciences, the GABmodel is the most widely accepted due toits accuracy and its validity over a widerange of water activities from 0.05 to 0.9(Rahman and Labuza, 1999).

The D’Arcy–Watt model was developedfor the analysis of sorption isotherms ofnon-homogeneous materials (D’Arcy andWatt, 1970). This model assumes that thereis a fixed number of water-binding siteswith different discrete binding energies. TheD’Arcy–Watt equation can be written as:

where K�, K, c, k� and k are equation coeffi-cients (adjustable parameters). The equationhas three terms, which represent theamounts of water that are strongly bound,weakly bound and sorbed in multimolecularwater clusters, respectively. For a tissue thatis in equilibrium with a given aw, the

amount of water in those three regions canbe estimated. K� is the number of strongwater-binding sites, multiplied by the mole-cular weight of water and divided byAvogadro’s number (6.023 � 1023); K is thestrength of the attraction of the strong water-binding sites for water; c is a measure (lin-ear approximation) of the affinity and thenumber of weak water-binding sites; k�relates to the number of multimolecularwater sorption sites; and k relates to theactivity of water (D’Arcy and Watt, 1970).The number of water-binding sites in tissuescan be calculated from the derived equationcoefficients. The number of strong, weakand multimolecular water-binding sites areK�N/M, cN/(M�o), and k�N/M, respectively,where N is Avogadro’s number, M is themolecular weight of water and �o is the satu-rated vapour pressure of pure water.

The D’Arcy–Watt model has been usedextensively for the analysis of desiccation-tolerant and desiccation-intolerant planttissues (Vertucci and Leopold, 1986, 1987a,b; Sun et al., 1997). Both the GAB and theD’Arcy–Watt models are valid over a widerrange of water activities for plant tissues.The GAB model has some advantages overthe D’Arcy–Watt model, which assumes thethree types of water-binding sites. The GABmodel does not have such an assumption.For biological systems it is more reasonableto assume that the number of water-bindingsites is changing continually along with thebinding energies. Moreover, the GAB modelcan be more easily applied to other thermalanalyses (e.g. water-clustering function).

2.4.2.2. Temperature dependency of watersorption

Desiccation involves the transfer of liquidwater in plant tissues into the vapourphase. Temperature influences evaporationrate through the heat supply as well asthrough its effect on the partial watervapour pressure in air and the energy sta-tus of water in plant tissues. In isothermalconditions, air acts as an osmotic mem-brane and equilibrium is often slow anddependent on temperature. An increase intemperature generally results in a decrease

MK Ka

Kaca

k kaaww

w

w

w

w

= (14)� �

1 1++ +

−κ

62 W.Q. Sun


in equilibrium water content of plant tis-sues at a given RH (i.e. water activity) or anincrease in equilibrium water activity atconstant tissue water content. The shift ofwater activity at the constant water contentby temperature is mainly due to the changein water binding, dissociation of water,physical state of water or increase in solu-bility of solute in water. Tensile strength ofwater, the pressure holding moleculestogether, increases by 81.6 mbars on aver-age for a reduction of 1°C. Temperaturedependence of isotherm shift is describedby the Clausius–Clapeyron equation:

where q is the excess heat of sorption; w isthe latent heat of vaporization for water(44.0 kJ kg�1 at 25°C); R is the gas constant;aw1 and aw2 are water activities for a givenequilibrium water content at temperatureT1 and T2, respectively. The plot of ln awagainst 1/T at any given tissue water con-tent is a straight line and its slope gives (q� w)/R, from which the excess heat ofsorption, q, can be derived (Fig. 2.5a).

In practice, some thermodynamic quan-tities of tissue hydration can be calculatedaccording to isotherms at two differenttemperatures. The aw1 and aw2 for a givenequilibrium water content at two tempera-tures can be taken from water sorptioncurves or calculated from fitted sorptionequations (Fig. 2.5b and Fig. 2.7a).Differential enthalpy of hydration (�H,including q and w), differential freeenergy of hydration (�G) and differentialentropy of hydration (�S) are given by:

�H �RT1T2 ln

aw1

T2 � T1� aw2

� (16)

�G � RT ln (aw) (17)

�S � �H � �GT

(18)

These thermodynamic quantities are thefunctions of water content in tissues. Therelationships of �H/WC, �G/WC and�S/WC provide important information withregard to the hydration properties of tissues(Fig. 2.4b). Water sorption is an exothermic

event. A high negative �H value at lowwater content suggests the strong affinity ofadjacent water molecules toward ionic sitesand/or other polar sites of the substrate. Aswater content increases, the �H becomesless negative (Fig. 2.4b). The primary hydra-tion process (i.e. strong and weak bindingsites) is considered to be completed whenthe differential enthalpy of hydration (�H)approaches zero (Luscher-Mattli and Ruegg,1982; Rupley et al., 1983; Bruni andLeopold, 1991). The change of �S reflectsthe relative degree of order, and the �S peakis presumably associated with the saturationof all primary hydration sites. It should beclearly noted that the relationships of�H/WC, �G/WC and �S/WC describe ther-modynamic interactions between water andbiomaterials, but not necessarily the func-tions of water and biological structures inphysiological processes. A possible associa-tion between water sorption behaviours anddesiccation tolerance of plant tissues wasdiscussed in a number of studies (Vertucciand Leopold, 1987b; Farrant et al., 1988;Pritchard, 1991; Sakurai et al., 1995; Eira etal., 1999; Sun, 2000). No consistent differ-ence in water sorption characteristics hasbeen found between desiccation-sensitive(recalcitrant) and desiccation-tolerant(orthodox) seed tissues (Sun, 2000).

The van’t Hoff relationship providesanother convenient means to analyse tem-perature dependence of sorption isotherm.The van’t Hoff equation and theClausius–Clapeyron equation are essen-tially the same in theory, but different intheir mathematical treatment of experimen-tal data. The Clausius–Clapeyron equationhandles two temperature points, whereasthe van’t Hoff equation can handle a seriesof temperature points at once. The van’tHoff equation expresses the relationship ofthe equilibrium water activity (aw) for agiven water content against the tempera-ture (1/T) (Fig. 2.5b), and is written in itsdifferential mathematical form as:

∂ ln aw � ��H

∂(1/T)R

(19)

where T is absolute temperature in kelvin,and R is the gas constant. The �H is the

ln = (15)w2

w1

waa

qT T

= + −

R1 1

2 1



differential enthalpy of water sorption. It isimportant to note that the relationshipbetween ln(aw) and (1/T) is not necessarilya straight line. Within a relatively narrowrange of temperature, linear approximationmay be used to calculate �H accurately.However, there is considerable interest instudying water sorption properties of bio-logical tissues at a much wider range oftemperature. For example, long-termpreservation of genetic resources may

require the storage of desiccation-sensitiveseeds and other tissues in a refrigeratedcondition or at liquid nitrogen tempera-ture. When the extrapolation is used, thenon-linear nature of the relationshipbetween ln(aw) and (1/T) needs to be takeninto consideration. The study on tempera-ture dependence of water sorption usingthe van’t Hoff equation (Fig. 2.5a and b) isused to establish the theoretic frameworkfor the optimization of germplasm preser-

64 W.Q. Sun

(a)

(b)

0.09 g g–1 dw

0.04 g g–1 dw

0.02 g g–1 dw

0.0

–1.0

–2.0

–3.0

–4.0

3.3 3.4 3.5 3.6

1/Temperature (K, � 10–3)

ln (

a w o

r R

H/1

00)

Temperature ( C)

0 5 10 15 20 25

10% RH

50% RH

Wat

er c

onte

nt (

g g–

1 dw

)

0.14

0.12

0.10

0.08

0.06

0.04

0.02

Fig. 2.5. (a) Temperature dependence of water sorption for the same seed material at different watercontents (i.e. the van’t Hoff plot). Drawn with data from Eira et al. (1999). (b) Equilibrium water content atspecific water activities as a function of temperature for whole seeds of Coffea arabica cv. Mundo Nova.This relationship is called ‘isopleth’.


vation protocols (Vertucci et al., 1994,1995; Eira et al., 1999).

2.4.2.3. Monolayer hydration and water-clustering function

The monolayer hydration values of planttissues, the amount of water bound to spe-cific polar sites, can be easily determined,using BET or GAB isotherm models. Formost plant tissues and their major chemi-cal components, the monolayer value atambient temperature is estimated to bebetween 0.04 and 0.09 g g�1 dw using theBET or GAB model (Rahman and Labuza,1999). The BET monolayer value of manyorthodox seeds was also found to be in thisrange (Vertucci and Leopold, 1987a,b;Bruni and Leopold, 1991; Vertucci andRoos, 1993; Sun et al., 1997). The mono-layer value of Typha pollen was much lessthan that of orthodox seeds (Buitink et al.,1998b). The monolayer hydration is gener-ally complete at a water activity of0.20–0.30 (i.e. �150 to �250 MPa). It isimportant to note that the monolayer valuedecreases rapidly as temperature increases,and increases as temperature declines. Themonolayer water is of great importance forthe survival of many dry organisms (e.g.spores, pollen grains and seeds) duringstorage. In food science, the water activityat the monolayer value is defined as thecritical water activity. At a water activityabove 0.20–0.30, the rate of chemical reac-tions begins to increase significantlybecause of the greater solubility and mobil-ity of the reactants. At water contentsbelow the monolayer value, the rate oflipid oxidation and associated free radicaldamage increases. The presence of mono-layer water inhibits the undesirable inter-actions between polar groups on adjacentcarbohydrate or protein molecules, therebypreserving their rehydration ability andbiological functions (Rahman and Labuza,1999).

There is no defined monolayer parame-ter in the D’Arcy–Watt model. However,the first term of the D’Arcy–Watt equationmay be used as an approximation, as it rep-resents water that is bound strongly to

polar hydration sites. A recent study usingthe D’Arcy–Watt model suggested thatwater redistribution among different typesof hydration sites might be related to therapid loss of seed viability during storageafter osmotic priming and drying back (Sunet al., 1997).

Water clustering in binding sites isanother important hydration event that isof significance to desiccation tolerance ofplant tissues and the survival of tissue inthe dried state. Clustering formation isrelated to a number of transport phenom-ena. For example, clustering reduces theeffective mobility of water by increasingthe size of the diffusing molecular group orby increasing the tortuosity of diffusionpaths (Stannett et al., 1982). The range ofwater activity where the self-association ofwater takes place can be examined by theclustering function (Lugue et al., 1995;Dominguez and Heredia, 1999). The clus-tering function is written as:

G11/V1 � �V2[�(aw/V1)/�aw] � 1 (20)

where G11/V1 is the clustering function, V1is the volume fraction of water, V2 is thevolume fraction of biopolymers, and aw iswater activity (Zimm and Lundberg, 1956).The subscript ‘11’ in G11/V1 denotes thewater–water interaction as a function ofwater content (component 1). The cluster-ing function can be applied to an isothermsorption model such as the GAB equationwith some modifications. The GAB equa-tion needs to be rewritten in terms of vol-ume fraction instead of weight fraction.The GAB equation can be rewritten as:

Mw�V1p1/V2p2� MmCKaw

(1�Kaw)(1�Kaw�CKaw)(21)

or

aw/V1 �(1�Kaw)(1�Kaw�CKaw)

MmCKp2V2

(22)

where p1 and p2 are the density of waterand biopolymers. The density of sorbedwater is assumed to be equal to 1.0 g cm�3.Substituting aw/V1 in Equation (20) withEquation (22), the clustering function canbe expressed as:



G11/V1�(2K�CK�MmCK)�(2CK2�2K2)aw

MmCKp2

(23)

According to Equation (23), G11/V1 is pro-portionally related to aw and the reciprocalof polymer density (p2) function. G11/V1can be solved easily by the substitution ofMm, C and K constants from the GAB equa-tion. Figure 2.6 shows a plot of the water-clustering function of soybean axes. Theclustering plot is basically a straight lineagainst water activity. When G11/V1 isgreater than �1, water is expected to clus-ter (Zimm and Lundberg, 1956). The auto-association (clustering) of water in a fewdesiccation-tolerant seeds is observed tooccur at water activity ranging from 0.55 to0.60 (W.Q. Sun, unpublished data).

2.4.2.4. Occupancy of water-binding sites

The D’Arcy–Watt model can be used toexamine the occupancy of water-bindingsites as a function of water content accord-ing to Luscher-Mattli and Ruegg (1982).The occupancy represents the amount ofwater attached to certain hydration sites,

expressed as the percentage of the corre-sponding maximum value in the fullyhydrated tissues (Fig. 2.7b). Therefore, theoccupancy relationship indicates thedegree of hydration for different types ofhydration sites during desiccation. Figure2.7b shows that the occupancy for threetypes of hydration sites changes as thewater content of Q. rubra seed tissuesdecreases during desiccation. Desiccationof seed tissues to 0.30 g g�1 dw (the criticalwater content) removed about 90% of mul-tilayer molecular sorption water, but onlyabout 10% of water molecules attached tothe weak hydration sites in seed tissues.The removal of water from weak hydrationsites appears to be related to desiccationdamage in Q. rubra seeds (Sun, 1999). Thecritical water content of Q. robur axes alsocorresponds to the amount of matrix-boundwater (Pritchard and Manger, 1998).However, the question of whether thewater-binding or sorption behaviour inseed tissues is related to their desiccationtolerance remains unresolved. The loss ofviability in many recalcitrant seeds occursat a water content that is much higher than

66 W.Q. Sun

Clu

ster

ing

func

tion

8

4

0

–4

–8

–12

0.0 0.2 0.4 0.6 0.8 1.0

Water activity

Fig. 2.6. Water-clustering function showing the water�water association in soybean seed axes as a functionof equilibrium water activity. Apparent water clusters first appear at a water activity of 0.58 (arrow). Thewater-clustering function Equation (23) was solved through the study on biopolymer volumetric changeduring hydration [i.e. P2= f (V1)] by applying water sorption analysis. See text for further explanation.


that of ‘bound’ water (Pammenter et al.,1991; Berjak et al., 1992). Clearly, morecomprehensive studies are needed.

Readers who wish to know more aboutwater sorption analysis may refer to a

recently released manual by Bell andLabuza (2000). This book generally dis-cusses water activity in food materials, butthe principles are also applicable to plantdesiccation tolerance studies. Practical


0.8(a)

(b)

0.6

0.4

0.2

0.0

0.0

0.2 0.4 0.6 0.8 1.0Water activity

Wat

er c

onte

nt (

g g–

1 dw

) WC = 4.974/�

+ 0.0673/� + 0.0219/�

1 + 90.2/� 1 – 0.990/�

5 C

25 C

WC = 1.279/�

+ 0.0373/� + 0.026/�

1 + 27.4/� 1 – 0.986/�

100

80

60

40

20

0

0.1 0.2 0.3 0.4 0.5 0.6 0.7

Water content (g g–1 dw)

Sor

ptio

n si

tes

occu

pied

(%

)

Strong binding site

Weak binding site

Multilayer sorption

5 C25 C

0.0

Fig. 2.7. (a) The interpretation of desorption isotherms of Quercus rubra cotyledonary tissues, using theD’Arcy–Watt model. Equation coefficients are derived though curve-fitting of experimental data (/o = aw).See text for further explanation. (b) The occupancy for three types of hydration sites in Q. rubracotyledonary tissues at different water contents. The occupancy is based on the percentage of thecorresponding maximum values in the fully hydrated state (i.e. full turgor). The change of occupancy revealshow and when water is removed in different types of hydration sites during dehydration.


examples are provided to elucidate how tosolve many equations.

2.5. Measurement of Drying Rate andDesiccation Stress

2.5.1. Driving force for water loss andexpression of drying rate

The loss of water from tissues depends ontwo factors: the gradient in water potentialbetween tissue surface and external air orsolution, and the hydraulic conductivity ofthe tissue. The volume flow of water fromthe tissue to air can be described by:

Vw � ALp (�o � �i) (24)

where Vw is the volume flow of water perunit time (m3 s�1), A is the surface area ofthe tissue (m2) and Lp is the hydraulic con-ductivity of the tissue (m s�1 Pa�1). The �oand �i are water potentials of external airand the tissue, respectively. The differencein water potential (�o � �i) is a measure ofthe driving force for dehydration. �i is afunction of time that describes the decreasein tissue water potential during drying. Thehydraulic conductivity of the tissue (Lp) is ameasure of the diffusional resistance of thewater transport pathway within the tissue.

A good measure for the drying rate isessential for comparative studies on desic-cation tolerance. According to Equation(24), the rate of water loss from the tissue istime-dependent. Under constant RH andtemperature, the water content of the tissueis expected to decrease exponentially overtime until �i reaches �o (Fig. 2.8a). Thecurve of water loss can be described by:

WC = � exp(��t) (25)

where � is the initial water content, � is therate constant of water loss, and t is time ofdrying. This relationship was first used byTompsett and Pritchard (1998) to comparethe dehydration rate of A. hippocastanumseeds. Drying curves of other seed tissueshave been examined under a wide range ofdesiccation conditions, and they conformto Equation (25) (Li and Sun, 1999; Liangand Sun, 2000). Typically, water content

curves are biphasic. During the first dryingphase, the loss of water follows a simpleexponential function. During the secondphase, water content does not decreasemuch because the tissue is very muchcloser to achieving equilibrium with theair. Because water loss during the firstphase is described by an exponential func-tion, the rate constant (�) of water loss canbe used as an expression of drying rate.

2.5.2. Quantification of desiccation stress

The response of plant tissues to desiccationis significantly affected by dehydration con-ditions, such as drying rate (see Chapter 3).Under slow-drying conditions, plant tissuesstay longer at intermediate water contents.Fast drying is often reported to improvedesiccation tolerance of recalcitrant plantseeds (reviewed by Pammenter and Berjak,1999). There is no doubt that the level ofdesiccation stress would vary with dryingrate, and the questions are: (i) how desicca-tion stress can be quantified; and (ii) howdrying rate affects the level of desiccationstress. The change in chemical potential ofcellular water is a good measure for thedegree of desiccation stress. When thechemical potential of water is compared,�*w and mwgh in Equation (5) cancel out.The difference in chemical potential of cel-lular water between the dehydrated state(�D

w) and the hydrated state (�Hw) is:

�Hw��D

w�RT(ln aHw�ln aD

w)�V–

w(PHw�PD

w) (26)

According to Equation (26), the degree ofdesiccation stress is proportional tochanges in osmotic potential and hydrosta-tic pressure (P) in cells. Therefore, thechange of water potential, d�/dt, can beused to quantify the level of desiccationstress. Figure 2.8b shows the plots of tissuewater potential against drying time. Underthe conditions of constant temperature andrelative humidity, such plots are straightlines down to the fraction of apoplasticwater. Water potential of the tissuedecreases faster and deviates away fromthe straight line when the apoplastic wateris lost (see Fig. 2.3b, the low-RWC break

68 W.Q. Sun


point). The slope of each straight line por-tion (d�/dt) represents the degree of directphysical stress under different desiccationconditions. The relationship betweend�/dt and the rate constant (�) of waterloss (drying rate) is linear (Fig. 2.9). A

mathematical evaluation of this linear rela-tionship will not be presented here.

If the plant tissue is viewed as a viscoelas-tic system, the mechanical stress caused bywater loss can be considered as a simplestress–strain response. The physico-chemical


3.2

2.4

1.6

0.8

0.0

Wat

er c

onte

nt (

g g–

1 dw

)

(a)

(b)

� = 0.00502

� = 0.103

0 80 160 240 320 400

Drying time (h)

0 80 160 240 320 400

Drying time (h)

33% RH

88% RH

94% RH

33% RH

88% RH

94% RH

0

–5

–10

–15

–20

Wat

er p

oten

tial (

MP

a)

d�/dt = –0.689

d�/dt = –0.160

d�/dt = –0.032

� = 0.0211

Fig. 2.8. Measurement of drying rate and quantification of desiccation stress for Theobroma cacao axes. (a)Drying curves of isolated axes in three constant relative humidities (RHs). The data are fitted withexponential functions (WC = � exp(��t)), and the rate constants of water loss, �, are shown near eachcurve. (b) Plots of tissue water potential against drying time. The mechanical stress on tissue caused by thewater loss can be considered as a simple stress–strain response. The slope of �/time plot, d�/dt, is directlyrelated to the intensity of desiccation stress. Data from Liang and Sun (2000).


aspect of desiccation stress can be assessed byintegrating the function of tissue water poten-tial over time (Fig. 2.8b). Figure 2.10a and bshows an example of the quantitative analysisof desiccation stress. The cumulative waterstress during desiccation at three different RHswas plotted against drying time and watercontent, respectively. Under the slow-dryingcondition (94% RH), the mechanical stress(d�/dt) is small (Fig. 2.8b); however, thecumulative physico-chemical stress is remark-ably high because the time to dry to the samewater content increases exponentially as thedrying rate decreases (Fig. 2.10a and b). Thequantitative analysis of mechanical andphysico-chemical aspects of desiccation stresshas led to an understanding of the physiologi-cal basis of the optimal drying rate to achievethe maximum desiccation tolerance ofTheobroma cacao axes (Liang and Sun, 2000).

2.6. Water Relations – the Kinetic andFunctional Approach

The thermodynamic approach to waterrelations has its limitations, because it

treats biological systems as fully reversibleones and does not give much considera-tion to the term time, one of the mostimportant factors in any biologicalresponse. This limitation is particularlyrelevant to the study of desiccation. Theapplication of thermodynamics is gener-ally sufficient in many cases for fullyhydrated tissues. However, at intermediateor low moisture levels, the non-equilib-rium, kinetic principles play a moreimportant role. During desiccation, thebiological system basically shifts from athermodynamic state to a non-equilibriumkinetic state (Leopold et al., 1994; Sun etal., 1994; Sun, 1997, 1998). The thermo-dynamic approach does not sufficientlyaddress the kinetics of various reactionsand processes in intermediate- to low-moisture systems.

The kinetic and functional approach tocellular water relations focuses on how theinteractions between water and other cellu-lar components can influence the struc-tures and biological properties of eachother. In this chapter, principles of thekinetic approach and the interactions

70 W.Q. Sun

1.6

1.2

0.8

0.4

0.0

Deh

ydra

tion

rate

(–d

�/d

t)

0.00 0.06 0.12 0.18 0.24 0.30

Rate constant of water loss (�)

Fig. 2.9. The relationship between the rate constant of water loss (�) in Equation (25) and dehydration rate(�d�/dt) for Theobroma cacao (cocoa) axes. Isolated axes were dehydrated at 16°C under constant relativehumidities ranging from 6 to 94% to achieve different drying rates and stress conditions. Drawn using datafrom Liang and Sun (2000).



between water and many other biomole-cules will not be discussed in detail. Thesetopics will be covered in other chapters ondesiccation damage and mechanisms ofdesiccation tolerance (see Chapters 9–12).

Instead, a general account will be offered,so that readers can be confident aboutchoosing the appropriate method to quan-tify particular water properties in studieson desiccation tolerance.

(a)

(b)

0 80 160 240 320 400

Drying time (h)

33% RH

88% RH

94% RH

33% RH

88% RH

94% RH

2000

1500

1000

500

0

Cum

ulat

ive

wat

er s

tres

s (M

Pa

� h

)

2000

1500

1000

500

0

Cum

ulat

ive

wat

er s

tres

s (M

Pa

� h

)

0.0 0.6 1.2 1.8 2.4 3.0

Water content (g g–1 dw)

Fig. 2.10. (a) Cumulative water stress during desiccation as the function of drying time. The cumulative waterstress is calculated by integrating the �/time function (see text for details). (b) Cumulative water stress as thefunction of tissue water content under different desiccation conditions. Cumulative stress is much higher inslow-drying conditions because the dehydration time increases exponentially as drying rate decreases.


2.6.1. General considerations

2.6.1.1. Time scale

Any study on the state of water by a bio-physical technique involves measuringparameters of time scale directly or indi-rectly. Biophysical techniques often use thediffusional correlation time as the timescale to make comparisons of the measure-ments on water. Many of the physical prop-erties of water are theoretically related tothe diffusional correlation time (�D) ofwater, the average time between jumps inposition for water molecules in the system.Two critical numbers on this time scale (�D)are 10�5 s and 10�11 s. The �D of water in iceis 10�5 s and in pure liquid is 10�11 s, onemillion times faster. Under normal condi-tions, water in biological systems exists in astate somewhere between the solid state ofcrystalline ice and the liquid hydrogen-bonded lattice of pure water. In low-moisture systems, however, the �D of watermolecules in the intracellular glasses wouldbe much slower than 10�5 s. Readers mayrefer to a series of studies on molecularmobility in seeds and pollen by Buitink etal. (1998a, 1999, 2000a,b,c; Chapter 10 ofthis volume).

The ability of a biophysical technique toyield useful information about the physicalstate of water largely depends on how fast ameasurement can be made. Slow tech-niques which require measurement timesgreater than the diffusional correlation timeyield an average over all molecules in thepopulation with a kinetic contribution fromdiffusion, whereas fast techniques can yieldinstantaneous information about intramole-cular factors such as H–O bond lengths andhydrogen bond angles (geometric factors).To choose the appropriate technique, onehas to bear in mind that the type of prop-erty or structure that a technique can probeis related to the time scale. Generally speak-ing, fast techniques would probably pro-duce data of instantaneous structures ofwater and other biomolecules, but slowtechniques would provide more informa-tion about the interactions between watermolecules and their environment. Taking

the study on water in skeletal muscle as anexample, fast techniques (e.g. laser Ramanspectroscopy, infrared spectroscopy anddielectric relaxation) could not find anyintramolecular differences in hydrogenbond lengths, angles or strength betweenmuscle water and pure water (Beall, 1981).However, slower techniques, such asnuclear magnetic resonance (NMR) (Fungand McGaughy, 1974), electron paramag-netic resonance (EPR) (Belagyi, 1975) (seeChapter 4), fluorescence polarization(Knight and Wiggins, 1979) and freezingbehaviour (Rustgi et al., 1978), showed arestricted motion of at least a portion of cellwater. This situation is identical to pho-tographing moving objects with differentshutter speeds. If the shutter speed is veryhigh relative to the velocities of two movingobjects (e.g. 1/800 s), the photo will proba-bly not record any information as towhether one object is moving faster thanthe other. On the other hand, if the shutterspeed is too slow (e.g. 1/2 s), the images ofboth moving objects will be blurred and nomeaningful information can be obtainedfrom such a photo. Only with a propershutter speed can the photo reflect the dif-ference in the velocity between the twomoving objects.

2.6.1.2. Structural complexity and dynamicsof molecular ordering

Hydration of protein is a good example,illustrating the complexity of structuresand functions for water in biological sys-tems. When a mole of lysozyme is hydratedby 60 moles of water (~ 0.07 g g�1 dry pro-tein), water is primarily located to chargedgroups, and its mobility is reduced at leastby 100 times relative to pure water. At thislow hydration level, no structural differ-ence is observed in the protein. As hydra-tion increases to 220 moles of water permole of protein (~ 0.25 g g�1 dry protein),water begins to form clusters of varioussizes and arrangements around the chargedand polar sites of the protein. Internal pro-tein motion (H exchange) increases by 1000times to be comparable to that of solution,while the protein sample is still a solid. At

72 W.Q. Sun


a hydration level of 300 moles of water permole of protein (~ 0.38 g g�1 dry protein),enzymatic activity, mobility of bound lig-and and fast water motion become easilydetectable. Dielectric relaxation measure-ments show two water relaxation times,one of 2 � 10�11 s, close to that of bulkypure water, and the other of 10�9 s, indicat-ing the heterogeneous nature of waterbehaviours and functions (Rupley et al.,1983). At the hydration level of 0.38 g g�1

dry protein, each water molecule covers, onaverage, 20 Å2 of protein surface, which istwice the effective area of a water molecule.Yet several populations of water moleculesare observed at such low hydration.

The surfaces of membranes, proteinsand other macromolecules impose geomet-ric limitations on the possible arrange-ments of hydration water. Interfacial watermolecules, being part of the network of bio-logical interfaces, are dynamically orientedand exhibit restricted motion (i.e. are‘bound’). The ordering of molecules on var-ious biological surfaces is strictly local,and may fluctuate rapidly between possiblearrangements. Ideally, a biophysical tech-nique used to study the state of water inbiological systems should have the resolu-tion to differentiate closely related struc-tures or populations. However, as discussedearlier, kinetic measurements reflect onlyaverage or time-average properties over allmolecules in the system and, in manycases, do not provide definitive answers tothe questions of interest. To interpret thedata of kinetic measurements, the investiga-tor must impose a conceptual model, whichmay be controversial (Beall, 1983). Suchstudies are often misunderstood and misin-terpreted by readers who are less familiarwith the biophysical techniques used.

2.6.1.3. The model-dependent interpretation:the pitfalls

The selection of a theoretic model or thedevelopment of a new model is an impor-tant step in any kinetic study, whichshould be done before actual measure-ments are made. The assumptions that amodel contains, and the specific predic-

tions that a model allows, have to be takeninto consideration for experimental designand implementation. If possible, additionalexperiments should be conducted to con-firm the results and to examine whether theassumptions are satisfied. Unfortunately,biophysical models or equations have fre-quently been used to analyse the actualdata without checking their assumptions.Worse still, sometimes a model wasselected after the entire experiment wascompleted (Beall, 1983).

Conceptual models have been used forthe interpretation of the data on water inbiological systems, with many pitfalls. Forexample, the ‘two-fraction fast-exchangemodel’ (Zimmermann and Brittin, 1957)assumes that there is a small fraction ofhighly immobilized cell water on the sur-face of macromolecules (ice-like) and alarge fraction of cell water that behaves likebulk water. Rapid exchange between thetwo populations yields reduced averageproperties. This two-fraction model can bewritten as:

1�

X�

(1 � X)T * Tslow TH2O

(27)

where T* is measured (average) relaxationtime; X and (1�X) are the fraction ofimmobilized water and the fraction of cellwater that is like bulk water, respectively;Tslow and TH2O are the relaxation times ofthe slow fraction and bulk water. In thisequation, there is only one measured para-meter (T*), but three unknown quantities(X, Tslow and TH2O). To estimate X, Tslowand TH2O must be arbitrarily assigned. Thismodel represents a simplistic view on thedynamics of water in biological systems,which is still in use by some workers. Ifone intends to solve Tslow, then X must beestimated through other methods. When Xis equated to the ‘non-freezable fraction’ or‘osmotically inactive fraction’, additionalassumptions are made. By redefining X asan adjustable parameter in different sys-tems, a new model is established (Beall,1983). This example clearly shows theuncertainty of biophysical interpretation.Simply because the model is easy to useand fits the data well it does not necessarily



mean that it represents the true state ofwater in a system. Of course, all models areopen to interpretation.

However, it is possible to measurechanges in the properties of water in livingsystems that correlate with physiologicalfunctions (Clegg et al., 1982; Clegg, 1986;Bruni et al., 1989). Changes in dynamicproperties of water at different hydrationlevels indicate the existence of differentfractions of water, which may vary in struc-ture and property and presumably play dif-ferent biological roles. Studies haveidentified the existence of at least four orfive fractions of water, presumably relatingto different interactions between water andcellular constituents (Clegg, 1986;Ratkovic, 1987; Vertucci, 1990; Pissis et al.,1996; Sun, 2000). Hydration levels corre-sponding to these fractions of cellularwater are associated with the onset of vari-ous metabolic activities in organisms(Clegg, 1986).

2.6.2. Biophysical techniques(see also Chapter 4)

Kinetic properties and functions of waterhave been studied, using calorimetry(Ruegg et al., 1975; Bakradze and Balla,1983; Vertucci, 1990; Sun, 1999), infrared(IR) and Raman spectroscopy (Careri et al.,1979; Cameron et al., 1988), NMR spec-troscopy (Fung and McGaughy, 1974;Mathur-de Vre, 1979; Seewaldt et al., 1981;Rorschach and Hazlewood, 1986; Ratkovic,1987), quasi-elastic neutron-scatteringspectroscopy (Lehmann, 1984; Trantham etal., 1984) and dielectric relaxation tech-niques (Harvey and Hoekstra, 1972;Kamiyoshi and Kudo, 1978; Clegg et al.,1982; Pissis et al., 1987, 1996; Bruni andLeopold, 1992). These techniques differgreatly in how and what they measure withrespect to the dynamic properties andstructures of water and other biomolecules.A great deal of confusion over the physicalstate of water in biological systems hasresulted from the separation of informationobtained with diverse techniques appliedto similar systems. The nature of different

measurements is briefly summarized inTable 2.1. Readers are advised to consultother references, including those citedabove, and Chapter 4 in this volume. In thischapter, only a brief introduction will beprovided on several techniques that havebeen increasingly used in recent years.

2.6.2.1. Differential scanning calorimetry

Differential scanning calorimetry (DSC) isprobably the most commonly used thermalanalysis technique. It has been used by anumber of workers to study the possiblerelationship between freezing, desiccationtolerance and water properties in plant tis-sues (Williams and Leopold, 1989;Vertucci, 1990; Pammenter et al., 1991;Berjak et al., 1992; Sun et al., 1994; Vertucciet al., 1994, 1995; Buitink et al., 1998b;Pritchard and Manger, 1998; Sun andDavidson, 1998; Sun, 1999). DSC measuresthe heat flow of plant tissues associatedwith various thermal events during coolingand/or heating scans. Such thermal eventsinclude phase transitions (e.g. freezing,melting, glass transition, etc.), polymor-phism, thermochemistry and the kineticsfor a variety of complex reactions (e.g. invivo protein denaturation). The key ideainvolved in DSC measurement of water sta-tus is that thermal changes of water andtheir corresponding quantities of energyare greatly affected by the presence of otherbiomaterials in plant tissues, and that ther-mal behaviours of other biomaterials inplant tissues are affected by water content.For example, as water content decreases,the onset freezing and melting temperatureof water decreases due to solute concentra-tion, while at the same time glass transitiontemperature of the tissue increases due tothe reduced plasticization effect by water.By analysing thermal behaviours of waterand biomaterials as a function of watercontent, temperature and time, the status ofwater in plant tissues can be studied andthe water status correlated to its biologicalfunctions.

Technically, DSC is really a quite simplemethod. There are two cells in the DSCdetector, one reference cell and one sample

74 W.Q. Sun


cell. An empty crucible is placed into thereference cell and a crucible containing thetissue sample is placed into the samplecell. During a DSC experiment, both refer-ence cell and sample cell are cooled and/orheated at a constant rate over a range oftemperatures. When a thermal event occursin the tissue, it releases or absorbs heatenergy (i.e. heat flow). A plot of heat flowas a function of temperature is called athermogram, from which the thermalbehaviour of the tissue can be deduced.Glass transition is usually marked by astepwise shift in the baseline of a thermo-gram. This distinguishes it from freezingand melting transitions, which producepeaks. A melting event is accompanied byan endothermic peak and a freezing eventis accompanied by an exothermic peak.The area under the particular peak repre-sents the total heat energy or enthalpychange (�H) for the event. DSC is a highlyinformative tool for analysing biologicalmaterials. Other information such as transi-tion temperature and heat capacity change(�Cp) of the tissue upon cooling or heatingcan also be calculated from a thermogram.The difficulty in analysing DSC thermo-grams lies in the correct identification oforigin for thermal events in heterogeneousbiological samples. For example, lipid tran-sition in seeds can mask the actual glasstransition (Williams and Leopold, 1989)and interfere with the accurate calculationof freezing and melting enthalpies (Sun,1999).

2.6.2.2. Thermally stimulated current (TSC)method

Different dielectric relaxation techniqueshad been used previously to study theproperties of water in biological systems(Harvey and Hoekstra, 1972; Kamiyoshiand Kudo, 1978; Clegg et al., 1982; Careriand Giansanti, 1984). More recently, theTSC technique has been employed to studythe mode of hydration and water organiza-tion in plant tissues (Pissis et al., 1987,1996; Bruni and Leopold, 1992; Sun et al.,1994; Sun, 2000). This technique is capableof providing information concerning themobility and rotational freedom of hydra-tion water, hydration sites and mechanisms(Mascarenhas, 1980; Pissis et al., 1987,1996; Pissis, 1990; Bruni and Leopold,1992). The TSC technique is based upon:(i) the dependence of the microdynamics ofwater dielectric relaxation on their sur-roundings resulting in different dielectricrelaxation times for water in different frac-tions; and (ii) the influence of water on thedielectric relaxation mechanisms of otherbiomolecules (similar to those used forDSC measurements).

The TSC method measures the tinycurrent generated by the thermally acti-vated release of stored dielectric polariza-tion during controlled heating andbasically consists of three steps: (i) thepolarization of a sample by a strong d.c.electric field at a particular temperature;(ii) ‘freeze-in’ the polarization by coolingdown to a sufficiently low temperature


Table 2.1. Biophysical techniques used to study the dynamic and structural properties of water andmacromolecules in biological systems.

Type of information Information about

Time scale Time-Techniques (s) average Dynamic Structural Water Macromolecule

Thermal analysis (DSC, DTA) 101 ~103 + +X-ray diffraction 101 ~102 + + + +Spectroscopy (NMR, EPR) 10�4 ~100 + (+) (+) +Relaxation (NMR, EPR, dielectric) 10�11 ~100 + + (+)Ultrasonic absorption 10�10 ~10�5 + + +Quasi-elastic neutron scattering 10�13 ~10�7 + + +Infrared and Raman spectroscopy 10�16 ~10�12 + + + +

DSC, differential scanning calorimetry; DTA, differential thermal analysis; NMR, nuclear magnetic resonance;EPR, electron paragmagnetic resonance.


(e.g. liquid nitrogen temperature) whilethe field is still on; and (iii) the measure-ment of the TSC spectrum during heatingafter the d.c. field is disconnected (Bruniand Leopold, 1992). When a polarized tis-sue reaches a temperature at which dipolemolecules (such as water) relax (lose theirfixed orientation), a tiny current is gener-ated and recorded. From a TSC spectrum,several important physical parameters canbe obtained, including the intensity ofdepolarization charge (peak size, relatedto the size of the water pool), depolariza-tion temperature, its activation energy andstatic permittivity. The measurement ofdielectric relaxation properties of waterand water-plasticized biomolecules offersvaluable insight into the organization ofwater in plant tissues and the molecularinteractions between water and other bio-molecules during desiccation (Bruni andLeopold, 1992; Sun, 2000).

2.6.2.3. Nuclear magnetic resonance (NMR)

NMR spectroscopy studies the interactionof electromagnetic radiation with matter. Itis a powerful tool for the studies of kineticmotion of water in tissues and of macro-molecule/water or membrane/water inter-actions. Solid-state NMR can be used todetermine the molecular structure of solidtissue samples. Solid-state H-NMR is oftenused to investigate the relaxation character-istics of the protons of water molecules inlow-moisture biological systems (Mathur-de Vre, 1979; Seewaldt et al., 1981;Rorschach and Hazlewood, 1986; Ratkovic,1987; Chapter 4). The basic principle of H-NMR is that each of two hydrogen nucleiin a water molecule possesses a single spinproton, which will cause the nucleus toproduce an NMR signal. When an atom isplaced in a magnetic field, the spin of itselectrons will orient toward the directionof the applied magnetic field. This orienta-tion produces a small local magnetic fieldat the nucleus that opposes the externallyapplied field, resulting in a smaller mag-netic field (i.e. effective field) at thenucleus than the applied field. (Note thatin some cases it might also enhance the

magnetic field at the nucleus.) Since theelectron density around each nucleus in amolecule varies according to the type ofnuclei and its molecular environment, theopposing field and thus the effective fieldat each nucleus will differ, which is called‘chemical shift’. The chemical shift of anucleus is the difference between the reso-nance frequency of the nucleus and a stan-dard (relative to the standard, expressed inp.p.m., �). The chemical shift is a very pre-cise measure of the chemical environmentaround a nucleus.

The major frustration for many biolo-gists wishing to understand and to useNMR is the complexity of the subject.However, as with other physical tech-niques used in studies of biological sys-tems, NMR may be used in an ‘empirical’mode, simply examining the variation ofan NMR parameter with the change ofexperimental variable (e.g. water content)(James, 1993). Figure 2.11 shows 1H-NMRspectra of mung bean seeds at three watercontents. The 1H-NMR spectra were broad,with the line width in the order of 103 Hz.Two NMR peaks were easily identifiable.The peak of water in the immobile fractionhad a peak maximum � (chemical shift)value of 4.3 p.p.m. relative to the proton inD2O, which was used as a standard refer-ence. The peak of water in the mobile frac-tion had a peak maximum � value at thesame place as the proton in D2O (i.e. � = 0p.p.m.). At a water content of 0.07 g g�1

dw, water appeared to exist primarily inthe immobile fraction. The very small pro-portion of water in the mobile fractionappeared as a shoulder in the spectrum.The amount of water in the mobile fractionincreased rapidly as water contentincreased. At 0.24 g g�1 dw, two peaksmerged almost completely as one peak,centred at � = 0 p.p.m. The relative propor-tion of the immobile and mobile water frac-tions may be estimated using the standardsignal processing techniques.

Two important spin relaxation parame-ters are T1, the spin–lattice relaxation, andT2, the spin–spin relaxation time. Thespin–lattice relaxation (T1) involves theexchange of energy with the environment

76 W.Q. Sun


(the lattice), and is caused by fluctuatinglocal magnetic fields arising from themotion of the molecules. The spin–spinrelaxation (T2) characterizes interactionsbetween spins and is related to the widthof the NMR peak (Fig. 2.11). T1 and T2 canbe used to study chemical kinetics androtational and conformational motion ofmolecules.

2.6.2.4. Electron spin resonance(see also Chapter 4)

Electron spin resonance (ESR) or electronparamagnetic resonance (EPR) is related toNMR. It is the study of molecules withunpaired electrons (free radicals, transitionmetal complexes, triplet states, etc.) byobserving the magnetic fields at which they

come into resonance with monochromaticradiation. The magnetic field of most com-mercial ESR spectrometers is about 0.3 T,corresponding to resonance with an elec-tromagnetic frequency of ~10 GHz andwavelength of ~3 cm. Therefore, the rangeof applicability of ESR is narrower thanthat of NMR. ESR is basically a microwavetechnique, and is one of the fastest-growingareas in analytical instrumentation becauseof recent and remarkable achievements inmicrowave technology. ESR consists of amicrowave source, a cavity, a microwavedetector and an electromagnet. The sampleis placed in a glass or quartz tube, which isinserted into the cavity. The ESR spectrumis obtained by measuring the microwaveabsorption as the magnetic field strength iscontinuously changed. This method


Sig

nal a

mpl

itude

–16 –8 0 8 16 24

Chemical shift (�, p.p.m.)

D2O

0.07 g g–1

0.14 g g–1

0.24 g g–1

1

2

Fig. 2.11. The 1H-NMR spectra of water in mung bean seeds at three water contents. The width of allspectra was 4000 Hz. D2O was used as a standard reference. The inset shows the assignment of 1H-NMRsignal into two different water fractions: mobile water (fraction 1) and immobile water (fraction 2). Thespectrum was recorded with FX90Q�NMR (JEOL Ltd, Japan). The powdered sample, weighing approx. 1–2 g, was loaded into the standard 5 mm NMR tube. A 90° 36-µs electromagnetic pulse was applied to the sample. A total of eight scans were used to improve the resolution. A repeat time was 20 s to re-establishthe equilibrium via spin–lattice and spin–spin relaxation before the next scan (W.Q. Sun, unpublished data).


detects the number of ‘unpaired spins’ ofelectronic charges. The ‘strange’ ESR spec-trum is the first derivative of themicrowave energy absorption (Fig. 2.12).The hyperfine structure (splitting of indi-vidual resonance lines into components) ofan ESR spectrum is a fingerprint that helpsto identify free radical species in the sam-ple and characterize their environments.

The ESR technique does not directlymeasure water properties in tissues; how-ever, it can be used to study many ques-tions that are related to desiccation. Usingan ESR spin-labelling technique, Belagyi(1975) reported that a portion of cell waterin muscles exhibited a restricted motion. Inrecent years, Hoekstra and his co-workershave used this technique to study mem-brane behaviours, molecular mobility, cyto-plasmic viscosity and partitioning ofamphiphilic molecules of desiccation-tol-erant and desiccation-intolerant plant tis-sues upon desiccation (Golovina et al.,1998; Buitink et al., 1999, 2000a,b,c;Leprince et al., 1999; Chaper 4). A varietyof molecular spin probes (stable free radi-

cals) is commercially available. By incor-porating some probes such as nitroxidederivatives into tissues before drying,detailed studies may be undertaken on thechanges in the aqueous and non-aqueousintracellular environments upon desicca-tion. The ESR technique provides a fairlydirect measurement of the change in cyto-plasmic viscosity, which probably playsan important role in metabolic down-regulation in desiccation tolerance (seeChapter 10).

2.7. Concluding Remarks

Comparative studies play a key role inunderstanding the mechanisms or strate-gies of various organisms in the survival ofdesiccation. The water status of tissues indesiccation tolerance studies should beexpressed precisely by preferred thermody-namic parameters to permit the compari-son of data from different biologicalsystems. The commonly used parameter,water content, is not adequate for the

78 W.Q. Sun

(a)

(b)

Magnetic field (B)

Firs

t der

ivat

ive

(dA

/dB

)M

icro

wav

e ab

sorp

tion

(A)

Fig. 2.12. (a) Microwave energy absorption. (b) The peculiar appearance of the electron spin resonance(ESR) spectrum. The ESR spectrum is the first derivative signal of microwave energy absorption. The peak ofabsorption corresponds to the point where the first derivative passes through zero (dashed lines). (SeeFigures in Chapter 4.)



expression of tissue water status in mostcases. Whenever possible, the researchershould first study the components of thewater relations of cells or tissues andobtain important reference parametersabout their water status. The Höfler dia-gram and PV curve can be applied to mostwell-hydrated plant tissues, whereas theisothermal sorption study can be applied tointermediate- to low-moisture systems.Drying rate and desiccation stress can bequantified by introducing thermodynamicconcepts into the study of water-lossdynamics during desiccation (dryingcurve). The quantitative (instead of qualita-tive) analysis of physico-chemical aspects

of desiccation stresses would certainlyimprove the mechanistic studies of desic-cation tolerance. Water plays an importantrole in maintaining the structural integrityof biological systems. Although the kineticproperties of water in many biological sys-tems have been extensively studied, theorganization of cellular water and its rela-tion to desiccation tolerance or desiccationdamage are not fully understood. Furtherstudies on the interactions between waterand macromolecular structures by biophys-ical techniques are essential to identifyfundamental cellular or metabolic compo-nents that are associated with desiccationdamage or desiccation tolerance.

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Appendix: Solutions for ControlledDehydration and Rehydration

Saturated salt solutions

In a closed container, a saturated salt solu-tion (with excess salt present) produces aconstant water vapour pressure at a giventemperature. Relative humidity (RH) in thecontainer is calculated by:

RH � A exp(B/T) (1)

where A and B are constants, and T is thetemperature in kelvin (Wexler, 1997). Thevalues of A and B as well as the valid tem-perature range are given in Table A2.1 forvarious salts. For example, RH of saturatedKCl solution at 25°C is equal to 49.38 �exp(159/298) = 84.2 (%). Calculated values

are generally accurate within � 2%. RHs ofcommonly used salt solutions between 5and 40°C are compiled in Table A2.2. RHsof additional salt solutions at 25°C aregiven in Table A2.3. These data are takenfrom earlier works of Rockland (1960),Winston and Bates (1960) and Young(1967), tested and corrected by the author.Users are advised to avoid salts that releasesalt vapours into the atmosphere. Whenautoclaving is required, the decompositiontemperature of a salt should be checked.After autoclaving, the closed containershould be allowed sufficient time to equili-brate (i.e. avoiding condensation andsupersaturation). The supersaturation inthe liquid phase and the condensation ofwater on the wall in the container affectRH significantly.

Table A2.1. Commonly used salts, their vapour pressure constants A and B, and the valid temperaturerange. Data are taken from Wexler (1997).

Temperature range Relative humidity Compound (°C) at 25°C A B

NaOH.H2O 15–60 6 5.48 27LiBr.2H2O 10–30 6 0.23 996ZnBr2.2H2O 5–30 8 1.69 455KOH.2H2O 5–30 9 0.014 1924LiCl.H2O 20–65 11 14.53 �75CaBr2.6H2O 11–22 16 0.17 1360LiI.3H2O 15–65 18 0.15 1424CaCl2.6H2O 15–25 29 0.11 1653MgCl2.6H2O 5–45 33 29.26 34NaI.2H2O 5–45 38 3.62 702Ca(NO3) 2.4H2O 10–30 51 1.89 981Mg(NO3) 2.6H22O 5–35 53 25.28 220NaBr.2H2O 0–35 58 20.49 308NH4NO3 10–40 62 3.54 853KI 5–30 69 29.35 254SrCl2.6H2O 5 –30 71 31.58 241NaNO3 10–40 74 26.94 302NaCl 10–40 75 69.20 25NH4Cl 10–40 79 35.67 235KBr 5–25 81 40.98 203(NH4) 2SO4 10–40 81 62.06 79KCl 5–25 84 49.38 159Sr(NO3) 2.4H2O 5–25 85 28.34 328BaCl2.2H2O 5–25 90 69.99 75CsI 5–25 91 70.77 75KNO3 0–50 92 43.22 225K2SO4 10–50 97 86.75 34

84 W.Q. Sun


Non-saturated salt solutions

Non-saturated salt solutions can also beused. This method allows for the creation ofa precise and evenly graded series of RHs (orwater potentials) with the same salt. The dis-advantage of using non-saturated salt solu-tions is that they have limited bufferingcapacity relative to saturated salt solutions.RHs inside the container are not constant

during equilibration, because the equilibra-tion between the tissue, vapour phase andsolution phase results in a slight decrease orincrease in salt concentration. This problemcan be minimized using high mass ratiosbetween the solution and the sample. Amass ratio of 150–200 (i.e. 150–200 g solu-tion g�1 tissue) is sufficient. Water potentialsof NaCl solutions at different concentrationsand temperatures between 5 and 40°C are


Table A2.2. Equilibrium relative humidities of saturated salt solutions at different temperatures. Data aretaken from Rockland (1960), Winston and Bates (1960) and Young (1967).

SaturatedTemperature

salt solution 5°C 10°C 15°C 20°C 25°C 30°C 35°C 40°C

H2SO4 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0ZnCl2 5.5 – 5.5 – 5.5 – 5.5 –NaOH 6.0 – 6.0 6.0 7.2 – 7.5 –LiBr 9.0 – 8.0 – 7.0 – 7.0 –KOH 13.0 – 9.0 – 8.0 – 8.0 –LiCl.H2O 14.0 13.5 13.0 12.5 12.0 11.5 11.5 11.0CaBr2 23.0 – 20.0 18.5 16.5 – 15.0 –KAc 24.8 24.0 23.5 23.0 23.0 23.0 22.0 23.0MgBr2 32.0 31.0 31.5 31.0 30.5 30.0 30.0 30.0MgCl2 34.0 33.5 33.5 33.0 32.5 32.0 32.5 32.0CaCl2 40.0 38.0 35.0 32.5 30.0 – 30.0 –K2CO3 43.0 47.0 44.0 44.0 43.0 42.0 41.5 40.0NaI 43.5 – 38.0 38.5 38.0 36.0 34.0 32.5Zn(NO3) 2 45.0 43.0 40.7 40.0 32.5 24.0 21.0 19.0KCNS 54.0 52.0 50.0 47.0 46.5 43.5 41.5 41.0Mg(NO3) 2 55.0 53.0 53.7 53.0 52.5 52.0 50.5 51.0Na2Cr2O7.H2O 59.5 60.0 56.5 54.5 53.0 52.5 51.0 50.0NaBr 60.5 58.0 58.0 57.8 57.2 57.0 57.0 57.0Ca(NO3) 2 61.0 66.0 58.0 56.0 52.2 51.0 45.5 46.0NaBr.2H2O 63.0 61.0 59.0 57.5 56.0 54.5 53.0 51.5CuCl2 66.7 68.0 68.0 68.5 67.0 67.0 67.0 67.0LiAc 72.0 72.0 71.0 70.0 68.0 66.0 65.0 64.0NH4NO3 73.0 75.0 70.0 65.5 62.5 59.5 56.8 53.0NaCl 76.0 75.8 75.5 75.3 75.1 75.0 75.0 75.0NaNO3 79.0 77.5 76.5 76.0 74.0 72.5 71.0 70.5(NH4) 2SO4 81.7 81.2 80.0 79.8 79.7 79.5 79.2 79.0NH4Cl 82.0 79.0 79.5 79.0 78.0 77.5 75.5 74.0Li2SO4 84.0 84.0 84.0 85.0 85.0 85.0 85.0 81.0KBr 85.0 86.0 85.0 83.5 83.0 82.0 81.0 80.0KCl 87.8 86.7 86.0 85.3 85.0 83.5 83.0 83.0K2CrO4 89.0 89.0 88.0 88.0 87.0 86.0 84.0 82.0BaCl2 95.0 93.0 92.0 90.7 90.0 89.0 88.0 87.0ZnSO4 95.0 93.0 92.0 90.0 88.0 86.0 85.0 84.0KNO3 96.5 95.5 95.0 94.0 92.5 91.5 89.5 88.5K2SO4 98.0 97.0 97.0 97.0 97.0 97.0 96.0 96.0Na2HPO4 98.0 98.0 98.0 98.0 97.0 96.0 93.0 91.0Pb(NO3) 3 99.0 98.5 98.5 98.5 96.2 95.5 95.2 94.7


86 W.Q. Sun

Table A2.3. A list of saturated salt solutions (with the presence of excess salt) and their equilibriumrelative humidities (RHs) at 25°C.

Saturated salt solution RH (%) Saturated salt solution RH (%)

ZnBr2 8.6 SrBr2.6H2O 58.5H3PO4 9.0 FeCl2.4H2O 60.0CaAc2.H2O + sucrose 13.0 NaMnO4.3H2O 61.5CaAc2.H2O 17.0 NH4NO3+ AgNO3 61.5Ca(CNS) 2.3H2O 17.5 CuBr2 62.5LiI.3H2O 18.0 CoCl2 64.0KHCO2 (Formate) 21.5 NaNO2 64.2KAc.1.5H2O 22.2 K2S2O3 66.0NiBr2.3H2O 27.0 CuCl2.2H2O 67.7MgBr2.6H2O 31.5 NaCl + NaNO3 69.0Sr(CNS) 2.3H2O 31.5 SrCl2.6H2O 71.0SrI2.6H2O 33.0 SrCl2 71.0MnBr2.6H2O 34.5 NH4Cl + KNO3 71.2Cu(NO3) 2.6H2O 35.0 NaCl + KCl 71.5Ca(MnO4) 2.4H2O 37.5 NaAc.3H2O 73.0FeBr2.6H2O 39.0 NaCl + Na2SO4.7H2O 74.0NaI.2H2O 39.2 BaBr2 74.5Mg(ClO4) 2.6H2O 41.0 K tartrate 75.0CoBr2 41.5 NH4Br2 75.0CrCl3 42.5 Zn(CNS) 2 80.5BaI2.2H2O 43.0 NaH2PO4 81.0K2CO3.2H2O 43.0 AgNO3 82.0CeCl3 45.5 KCl + KClO3 85.0LiNO3.3H2O 47.0 KNa tartrate 87.0Mg(CNS) 2 47.5 Na2CO3.10H2O 87.0KNO2 48.1 MgSO4.7H2O 89.0K4P4O7.3H2O 49.5 BaCl2.2H2O 90.3Co(NO3) 2.6H2O 49.8 Na tartrate 92.0NH4NO3 + NaNO3 50.0 (NH4)H2PO4 92.7KBr + urea 51.0 NH4HPO4 93.0Zn(MnO4) 2.6H2O 51.0 CaH4(PO4) 2.H2O 96.0NiCl2.6H2O 53.0 KH2PO4 96.0Na2Cr2O7.2H2O 53.7 CaHPO4.2H2O 97.0Ba(CNS) 2.2H2O 54.5 CuSO4.5H2O 97.2Pb(NO3) 2 + NH4NO3 55.0 K2Cr2O7 98.0MnCl2.4H2O 56.0 KClO3 98.0

given in Table A2.4. Besides NaCl solutions,CaCl2 and KCl solutions offer good RH con-trol. Water potentials of CaCl2 and KCl solu-tions are listed in Table A2.5.

Polyethylene glycol (PEG) solutions

PEG solutions are widely used in controlleddehydration and rehydration. PEG solutionshave several advantages over salt solutions.Salt solutions at high specific water poten-

tial or RH are often not available. PEG isinexpensive and not corrosive, whereasmany salt solutions are corrosive andvolatile. Effects of PEG concentration andtemperature on water potential were studiedby Michel and Kaufmann (1973). An empiri-cal equation has been derived to calculatethe water potential of PEG solutions at givenconcentrations and temperatures:

� � (�1.18 � 10�3C) – (1.18 � 10�5 C2) �(2.67 � 10�5CT) � (8.39 � 10�8C 2T) (2)



where C is the concentration of PEG (molec-ular weight 6000) in g kg�1 water and T istemperature (°C). Water potential of PEGsolutions is curvilinearly related to the con-centration and increases linearly with tem-perature. The error of calculated waterpotential is generally within �0.03 MPa incomparison with the psychrometric mea-surements. Water potentials of PEG-6000solutions at concentrations ranging from 10to 400 g kg�1 water and at temperaturesbetween 5 and 40°C are given in Table A2.6.PEG solutions are very viscous, especially athigh concentrations; hence it takes a longertime to achieve the equilibrium between thePEG solution and the tissue. Caution isneeded to prevent bacterial and fungal cont-amination during the study. The equilibra-tion can be accelerated by placing closedcontainers in a gently shaking incubator andin some cases submerging the tissue in solu-tion. The change in PEG concentration afterthe experiment can be determined by thegravimetric method, and the equilibrium

water potential is calculated with Equation(2). The PEG solution can be dried at 105°Cin an oven to a constant dry weight. Theproblem with submerging the tissue in aPEG solution is that PEG can enter the inter-cellular spaces. PEG is considered a non-penetrating polymer because it does notcross the membrane! Our calorimetric studyreveals a PEG melting peak in submergedtissues, even after extensive washing.

Glycerol solutions

Glycerol can also be used to create a pre-cise and evenly graded series of RHsbetween 30 and 98%. Glycerol solutionsare safe to use and less corrosive (exceptfor tin) than salt solutions. Microbialgrowth can be effectively inhibited byadding four drops of saturated CuSO4 solu-tion to each 100 ml of glycerol solution(ASTM, 1983). The CuSO4-treated solutioncan be used repeatedly after the required

Table A2.4. Water potentials of sodium chloride (NaCl) solutions at different concentrations andtemperatures. Mass (%); g solute per 100 g solution.

Molality MassWater potential (MPa)

(mol kg�1) (%) 5°C 10°C 15°C 20°C 25°C 30°C 35°C 40°C

0.05 0.29 �0.22 �0.22 �0.23 �0.23 �0.23 �0.24 �0.24 �0.250.1 0.58 �0.43 �0.44 �0.45 �0.45 �0.46 �0.47 �0.48 �0.490.2 1.16 �0.85 �0.87 �0.88 �0.90 �0.92 �0.93 �0.95 �0.960.3 1.72 �1.27 �1.30 �1.32 �1.34 �1.37 �1.39 �1.42 �1.440.4 2.28 �1.69 �1.73 �1.76 �1.79 �1.82 �1.86 �1.89 �1.920.5 2.84 �2.12 �2.16 �2.20 �2.24 �2.28 �2.32 �2.36 �2.400.6 3.39 �2.54 �2.59 �2.64 �2.69 �2.74 �2.79 �2.84 �2.890.7 3.93 �2.97 �3.03 �3.09 �3.15 �3.21 �3.27 �3.33 �3.390.8 4.47 �3.40 �3.47 �3.54 �3.61 �3.68 �3.75 �3.82 �3.890.9 5.00 �3.83 �3.92 �4.00 �4.08 �4.16 �4.23 �4.31 �4.391.0 5.52 �4.27 �4.37 �4.46 �4.55 �4.64 �4.73 �4.82 �4.901.1 6.04 �4.71 �4.82 �4.92 �5.03 �5.13 �5.23 �5.32 �5.421.2 6.55 �5.16 �5.28 �5.39 �5.51 �5.62 �5.73 �5.84 �5.941.3 7.06 �5.61 �5.74 �5.87 �5.99 �6.12 �6.24 �6.35 �6.471.4 7.56 �6.07 �6.21 �6.35 �6.49 �6.62 �6.75 �6.88 �7.011.5 8.06 �6.53 �6.68 �6.84 �6.99 �7.13 �7.28 �7.41 �7.551.6 8.55 �7.00 �7.16 �7.33 �7.49 �7.65 �7.81 �7.95 �8.111.7 9.04 �7.46 �7.64 �7.82 �8.00 �8.17 �8.33 �8.49 �8.651.8 9.52 �7.94 �8.13 �8.33 �8.52 �8.70 �8.88 �9.04 �9.211.9 9.99 �8.43 �8.63 �8.84 �9.04 �9.24 �9.43 �9.60 �9.782.0 10.46 �8.92 �9.13 �9.36 �9.57 �9.78 �9.98 �10.16 �10.35


88 W.Q. Sun

Table A2.5. Water potentials of potassium chloride (KCl) and calciumchloride (CaCl2) solutions at 20 and 25°C. Mass (%): g solute per 100g solution.

KCl CaCl2

Mass (%) 20°Ca 25°Cb 20°Ca 25°Cb

0.5 �0.28 �0.31 �0.27 �0.281 �0.56 �0.61 �0.54 �0.572 �1.12 �1.22 �1.07 �1.163 �1.68 �1.85 �1.62 �1.814 �2.26 �2.48 �2.22 �2.505 �2.83 �3.13 �2.87 �3.246 �3.42 �3.80 �3.58 �4.027 �4.02 �4.48 �4.36 �4.898 �4.64 �5.18 �5.23 �5.779 �5.25 �5.90 �6.15 �6.73

10 �5.87 �6.65 �7.16 �7.7612 �7.18 �8.21 �9.40 �10.0914 �9.89 �12.00 �12.8516 �11.68 �14.99 �16.1318 �13.62 �18.45 �20.0220 �15.69 �22.34 �24.6022 �26.5024 �20.34 �30.89 �36.2026 �36.2628 �42.37 �51.6730 �50.0632 �60.68 �71.7836 �97.3340 �129.12

a Derived from Handbook of Chemistry and Physics, 78th edn(Lide, 1997). Water potential is calculated according to thefreezing-point depression.b Data were derived from Robinson and Stokes (1959).

concentration adjustment. The composi-tion of glycerol solutions can be accuratelydetermined using specific gravity or therefractive index. Equilibrium RHs of glyc-erol solutions at 24°C were reported byBraun and Braun (1958). Using the sameset of data, Forney and Brandl (1992)derived an empirical equation to calculateequilibrium RHs of glycerol solutions.Equilibrium RHs and water potentials ofglycerol solutions at 20°C were derivedaccording to the freezing-point depression.These data are listed in Table A2.7. Onecan calculate the required concentration ofa glycerol solution for a desired RH at agiven temperature, using the ASTM’s stan-dard recommended practice (ASTM, 1983).

The ASTM’s method uses the refractiveindex at 25°C to express glycerol concen-tration. The relationship between RH,refractive index and temperature isdescribed by the following equations:

(R0 � A)2 � (100 � A)2 � A2 � (RH � A)2 (3)

A � 25.6 � 0.195T � 0.0008T 2 (4)

R � 1.3333 � (1.398 � 10�3 R0) (5)

where RH is the desired relative humidity(%), R is the refractive index of the glycerolsolution, and T is temperature (°C). Thevalue of A is calculated using Equation (4).Ro is calculated by substituting A and RHin Equation (3). The refractive index at25°C is calculated using Equation (5). For a



glycerol solution of the known refractiveindex (R), equilibrium RH at different tem-peratures can be calculated by rearrangingEquations (3), (4) and (5) (Sun and Gouk,1999). The relationship is:

where A is defined by Equation (4), and Rois calculated from Equation (5). The mea-surement of refractive index is quitetedious. The concentration (mass %) of thedesired glycerol solution has been derivedfrom concentrative properties of aqueousglycerol solutions (Lide, 1997) by theRH = (6)100

2 20

2+( ) + − +( ) −A A R A A

Table A2.6. Water potentials of polyethylene glycol (MW 6000) solutions. Data were derived accordingto Michel and Kaufmann (1973).

PEGWater potential (MPa) at different temperatures

(g kg�1 H2O) 5°C 10°C 15°C 20°C 25°C 30°C 35°C 40°C

10 �0.012 �0.010 �0.009 �0.007 �0.006 �0.005 �0.003 �0.00220 �0.025 �0.023 �0.020 �0.017 �0.014 �0.011 �0.008 �0.00630 �0.042 �0.037 �0.033 �0.028 �0.024 �0.020 �0.015 �0.01140 �0.060 �0.054 �0.048 �0.042 �0.036 �0.030 �0.024 �0.01850 �0.081 �0.073 �0.065 �0.058 �0.050 �0.042 �0.034 �0.02760 �0.104 �0.094 �0.085 �0.075 �0.066 �0.056 �0.047 �0.03770 �0.129 �0.118 �0.106 �0.095 �0.083 �0.072 �0.061 �0.04980 �0.157 �0.143 �0.130 �0.116 �0.103 �0.090 �0.076 �0.06390 �0.186 �0.171 �0.156 �0.140 �0.125 �0.109 �0.094 �0.078

100 �0.22 �0.20 �0.183 �0.166 �0.148 �0.131 �0.113 �0.096110 �0.25 �0.23 �0.21 �0.194 �0.174 �0.154 �0.134 �0.114120 �0.29 �0.27 �0.25 �0.22 �0.20 �0.179 �0.157 �0.135130 �0.33 �0.30 �0.28 �0.26 �0.23 �0.21 �0.182 �0.157140 �0.37 �0.34 �0.32 �0.29 �0.26 �0.24 �0.21 �0.181150 �0.41 �0.38 �0.35 �0.32 �0.30 �0.27 �0.24 �0.21160 �0.46 �0.43 �0.39 �0.36 �0.33 �0.30 �0.27 �0.23170 �0.51 �0.47 �0.44 �0.40 �0.37 �0.33 �0.30 �0.26180 �0.56 �0.52 �0.48 �0.44 �0.41 �0.37 �0.33 �0.29190 �0.61 �0.57 �0.53 �0.49 �0.45 �0.41 �0.37 �0.33200 �0.66 �0.62 �0.58 �0.53 �0.49 �0.45 �0.40 �0.36210 �0.72 �0.68 �0.63 �0.58 �0.54 �0.49 �0.44 �0.40220 �0.78 �0.73 �0.68 �0.63 �0.58 �0.53 �0.48 �0.43230 �0.84 �0.79 �0.74 �0.68 �0.63 �0.58 �0.53 �0.47240 �0.91 �0.85 �0.79 �0.74 �0.68 �0.63 �0.57 �0.51250 �0.97 �0.91 �0.85 �0.79 �0.73 �0.67 �0.62 �0.56260 �1.04 �0.98 �0.92 �0.85 �0.79 �0.73 �0.66 �0.60270 �1.11 �1.05 �0.98 �0.91 �0.85 �0.78 �0.71 �0.65280 �1.19 �1.11 �1.04 �0.97 �0.90 �0.83 �0.76 �0.69290 �1.26 �1.19 �1.11 �1.04 �0.96 �0.89 �0.82 �0.74300 �1.34 �1.26 �1.18 �1.10 �1.03 �0.95 �0.87 �0.79310 �1.42 �1.34 �1.25 �1.17 �1.09 �1.01 �0.93 �0.85320 �1.50 �1.41 �1.33 �1.24 �1.16 �1.07 �0.99 �0.90330 �1.58 �1.49 �1.41 �1.32 �1.23 �1.14 �1.05 �0.96340 �1.67 �1.58 �1.48 �1.39 �1.30 �1.20 �1.11 �1.01350 �1.76 �1.66 �1.56 �1.47 �1.37 �1.27 �1.17 �1.07360 �1.85 �1.75 �1.65 �1.54 �1.44 �1.34 �1.24 �1.13370 �1.95 �1.84 �1.73 �1.62 �1.52 �1.41 �1.30 �1.20380 �2.04 �1.93 �1.82 �1.71 �1.60 �1.48 �1.37 �1.26390 �2.14 �2.02 �1.91 �1.79 �1.68 �1.56 �1.44 �1.33400 �2.24 �2.12 �2.00 �1.88 �1.76 �1.64 �1.52 �1.40


90 W.Q. Sun

Table A2.7. Equilibrium relative humidity (RH) and water potential of glycerol solutions at 20°C and24°C. Mass (%): g glycerol per 100 g solution.

20°C 24°C

Mass (%) Specific gravity a % RH b � b (MPa) % RH c � (MPa)

10 1.0215 97.9 �2.79 98.0 �2.7712 1.0262 97.4 �3.45 97.5 �3.4714 1.0311 96.9 �4.16 97.0 �4.1816 1.0360 96.4 �4.89 96.5 �4.8918 1.0409 95.8 �5.69 95.9 �5.7420 1.0459 95.2 �6.52 95.4 �6.4624 1.0561 93.9 �8.35 94.1 �8.3428 1.0664 92.4 �10.42 92.6 �10.5532 1.0770 90.7 �12.71 91.0 �12.9436 1.0876 88.9 �15.28 89.2 �15.6840 1.0984 86.9 �18.19 87.2 �18.7944 1.1092 84.9 �22.4648 1.1200 82.5 �26.3952 1.1308 79.7 �31.1356 1.1419 76.6 �36.5760 1.1530 73.2 �42.7864 1.1643 69.4 �50.1168 1.1755 65.2 �58.6872 1.1866 60.6 �68.7176 1.1976 55.5 �80.7780 1.2085 49.8 �95.6484 1.2192 43.6 �113.8888 1.2299 36.7 �137.51

a Taken from Handbook of Chemistry and Physics, 78th edn (Lide, 1997).b Calculated according to the freezing-point depression.c The relationship between concentration and equilibrium RH was reported by Braun and Braun (1958).

The equation derived by Forney and Brandl (1992) was used to determine equilibrium RH of solutionsat other concentrations.

author. RHs of glycerol solutions at concen-trations between 10 and 92% (mass %) andat temperatures between 5 and 35°C hasbeen calculated according to the ASTM’smethod (Table A2.8). The accuracy of

ASTM’s method is �0.2% at 25°C, andincreases as temperature deviates from25°C (ASTM, 1983). Data in Table A2.8 areconsistent with those in Table A2.7, whichwere derived by different methods.



References

ASTM (1983) Maintaining constant relative humidity by means of aqueous solutions. In: 1983Annual Book of ASTM Standards (Standard E104). American Society for Testing and Materials,Philadelphia, pp. 572–575.

Braun, J.V. and Braun, J.D. (1958) A simplified method of preparing solutions of glycerol and waterfor humidity control. Corrosion 14, 117–118.

Forney, C.F. and Brandl, D.G. (1992) Control of humidity in small controlled environment chambersusing glycerol–water solutions. HortTechnology 2, 52–54.

Lide, D.R. (ed.) (1997) Handbook of Chemistry and Physics, 78th edn. CRC Press, New York, pp. 8,57–81.

Michel, B.E. and Kaufmann, M.R. (1973) The osmotic potential of polyethylene glycol 6000. PlantPhysiology 51, 914–916.

Robinson, R.A. and Stokes, R.H. (1959) Electrolyte Solutions, 2nd edn. Butterworths ScientificPublications, London, 559 pp.

Rockland, L.B. (1960) Saturated salt solutions for static control of relative humidity between 5 and40°C. Analytical Chemistry 32, 1375–1376.

Sun, W.Q. and Gouk, S.S. (1999) Preferred parameters and methods for studying moisture content ofrecalcitrant seeds. In: Marzalina, M., Khoo, K.C., Jayanti, N., Tsan, F.Y. and Krishnapillay, B.(eds) Recalcitrant Seeds. Proceedings of IUFRO Seed Symposium 1998. Forest Research InstituteMalaysia, Kuala Lumpur, pp. 404–430.

Wexler, A. (1997) Constant humidity solutions. In: Lide, D.R. (ed.) Handbook of Chemistry andPhysics, 78th edn. CRC Press, New York, pp. 15, 24–25.

Winston, P.W. and Bates, D.H. (1960) Saturated solutions for the control of humidity in biologicalresearch. Ecology 41, 232–237.

Young, J.F. (1967) Humidity control in the laboratory using salt solutions – a review. Journal ofApplied Chemistry 17, 241–245.

Table A2.8. Equilibrium relative humidity (RH) of glycerol solutions at differenttemperatures. Values were derived, using the refractive index of the solution at 25°C.Mass (%): g glycerol per 100 g solution.

RH (%) at different temperatures

Mass (%) 5°C 10°C 15°C 20°C 25°C 30°C 35°C

10 98.1 98.1 98.2 98.2 98.3 98.3 98.312 97.6 97.7 97.7 97.8 97.8 97.9 97.914 97.1 97.2 97.2 97.3 97.4 97.4 97.516 96.6 96.6 96.7 96.8 96.9 96.9 97.018 96.0 96.1 96.1 96.2 96.3 96.4 96.420 95.4 95.5 95.5 95.6 95.7 95.8 95.924 94.0 94.1 94.2 94.3 94.4 94.5 94.628 92.5 92.6 92.8 92.9 93.0 93.1 93.232 90.8 91.0 91.1 91.3 91.4 91.5 91.736 88.9 89.1 89.3 89.4 89.6 89.7 89.940 86.8 87.0 87.2 87.4 87.6 87.7 87.944 84.5 84.7 84.9 85.1 85.3 85.5 85.748 82.0 82.2 82.5 82.7 82.9 83.1 83.352 79.2 79.5 79.7 80.0 80.2 80.4 80.656 76.2 76.5 76.7 77.0 77.2 77.4 77.760 72.8 73.0 73.3 73.6 73.8 74.1 74.364 69.0 69.3 69.6 69.9 70.1 70.4 70.668 64.9 65.2 65.5 65.8 66.1 66.3 66.672 60.3 60.6 60.9 61.2 61.5 61.8 62.176 55.2 55.6 55.9 56.2 56.5 56.8 57.180 49.5 49.8 50.2 50.5 50.8 51.1 51.484 43.0 43.4 43.7 44.0 44.4 44.7 45.088 35.5 35.9 36.2 36.5 36.9 37.2 37.5



3 Experimental Aspects of Drying andRecovery

Norman W. Pammenter,1 Patricia Berjak,1 James Wesley-Smith1 andClare Vander Willigen2

1School of Life and Environmental Sciences, University of Natal, Durban 4041, South Africa; 2Department of Botany, University of Cape Town, Private Bag,

Rondebosch 7701, South Africa

3.1. Introduction 943.2. Drying Rate 94

3.2.1. Commonly employed drying techniques 943.2.1.1. Seed material 953.2.1.2. Vegetative tissue 97

3.2.2. Quantification and modelling of drying rates 983.2.3. Effects of different drying rates 99

3.2.3.1 Desiccation-sensitive tissue 993.2.3.2. Desiccation-tolerant tissue 100

3.2.4. ‘Ultradrying’ of desiccation-tolerant material 1013.3. Influence of Rehydration Technique 1023.4. Length of Time in the Partially Dehydrated State 1023.5. Methods of Assessing Response to Rehydration 103

3.5.1. ‘Germination’ 1033.5.2. Resurrection plants 1043.5.3. Electrolyte leakage 1043.5.4. Tetrazolium test 1043.5.5. Other responses 104

3.6. Expression of Water Content Data 1053.6.1. Mass basis 1053.6.2. Water potential 1053.6.3. Relative water content 106

3.7. Conclusion 1063.8. References 106


Dessication - Chap 03 18/3/02 1:55 pm Page 93

3.1. Introduction

When plant material is subjected to dehy-dration, the observed response (in terms ofdamage accumulation and survival) canvary with the techniques used to assess theresponse. This has the potential to causeconfusion in the interpretation of experi-mental data. It is the objective of this chap-ter to outline the range of techniques thathave been used, to note the effects each canhave on the observed response and, as faras possible, to suggest underlying causes ofthese effects.

Experimental or technical aspects thatcan influence the response to dehydrationinclude drying rate, the length of time thetissue is maintained in the (partially) drystate, the rehydration and recovery tech-niques, and the method used to assess theresponse. The developmental status of thetissue may also influence the response todrying. The information presented in thischapter is derived almost exclusively fromstudies on seeds and vegetative tissue (vas-cular and non-vascular species). The dis-cussion has not been extended to pollenand spores, largely because their size issuch that they normally dehydrate rapidlyand so effects of drying conditions are lessfrequently studied (see Chapter 6).

3.2. Drying Rate

During drying, water molecules diffusefrom the tissue to the surrounding air.There are a number of factors that canaffect the rate at which this diffusionoccurs and hence the rate at which the tis-sue will dry. Some of these factors are:

1. The vapour pressure of the surroundingair; this affects the difference in free energyof water between the tissue and the air, andhence the drying rate. Tissue will obvi-ously dehydrate faster in dry than inhumid air.2. Temperature; this will also influence thewater free energy difference, which will begreater at higher temperatures. However,use of elevated temperatures during dehy-

dration may have adverse metabolic andother deleterious consequences unrelatedto drying.3. The rate of air movement across the tis-sue; this affects the thickness of the bound-ary layer and the ability of water vapour todiffuse through it. Forced ventilation willlead to faster drying than will still air.4. The size and shape of the tissue; thisaffects the surface area-to-volume ratio andthe distance water must diffuse from theinterior of the tissue to the surface. Smalltissue pieces will dry faster than largeones, and ‘flat’ tissue will dry faster than‘bulky’ tissue.5. The chemical and physical compositionof the outer layer of the tissue; this willaffect the permeability of the tissue towater, in either the liquid or vapour form.Tissues with lignified, suberized or waxyouter layers will dry more slowly thanthose without such layers.6. The amount of material to be dried; thiswill affect factors such as surface area-to-volume ratios and boundary layers.Generally, small quantities of material willdry faster than will a large mass.

These factors are, to a greater or lesserextent, under the control of the experi-menter, who can thus alter (but not controlwith precision) the rate at which the mater-ial dries. It should be noted that the terms‘rapid’, ‘intermediate’ and ‘slow’ are com-parative only within a single study; thereare no absolute boundaries to these terms.A drying rate that in one study might bedescribed as ‘rapid’ might be considered tobe ‘slow’ in another.

3.2.1. Commonly employed dryingtechniques

Commonly used drying techniques vary inthe degree to which temperature andhumidity are controlled and the extent ofair movement across the material beingdried. In the case of seeds they also vary inwhether or not outer coverings areremoved. The choice of method is ofteninfluenced by the amount of material that

94 N.W. Pammenter et al.


is being dried, the facilities available andthe reason for which the material is beingdried. Some of the methods are specific forseeds, and some for resurrection plants, butmany can be used for any small piece orpieces of tissue. Commonly used tech-niques are summarized in Table 3.1. Asdrying conditions can have marked effectson the response of tissue to drying (Section3.2.3), it is critical that the drying condi-tions (e.g. temperature, relative humidity(RH), light conditions, air flow) used in anyinvestigation are reported.

3.2.1.1. Seed material

Techniques that have been used to dry seedmaterial are outlined below:

1. Air drying of seeds or fruits in sun orshade (e.g. Albrecht, 1993). Of all the tech-niques used this one offers the least controlof the various factors affecting drying, butit is commonly used in the field wherefacilities are limited, samples are large anddrying shortly after collection is desired.When drying seeds, the usual practice is tospread them in an even layer, but, if thelayer is more than one seed deep, the sam-ple must be turned frequently to preventuneven drying. A similar practice isemployed for the whole fruits, for particu-lar species. Drying using this technique cantake several days.2. Drying under ambient laboratory condi-tions (e.g. Wu et al., 1998) has the samedisadvantages as the first method, exceptthat ambient conditions probably vary lessin the laboratory than outside. Dryingtimes will be similar to those achieved inthe first method.3. Walk-in chambers with temperatureand RH control. These facilities are nor-mally available only in large-scale com-mercial or research organizations and aregenerally used to dry substantial quanti-ties of material. Recommended practice isto dry at 10–25°C and 10–15% RH(International Plant Genetic ResourcesInstitute (IPGRI), 1994). This approachpermits standard and repeatable dryingconditions, but the drying rate achieved

will vary amongst species. Cromarty et al.(1985) have given details of the design ofseed bank facilities for orthodox seeds,including detailed consideration of dryingprotocols. Drying times using these proto-cols are of the order of days.4. Air flow in a laminar flow cabinet(Grout et al., 1983; Normah et al., 1986;Pence, 1992; Chandel et al., 1995). Thistechnique can be used for small seeds andparts of seeds (excised embryos or embry-onic axes) and has the advantages that itintroduces an air flow over the material(forced ventilation) and reduces the intro-duction of further microbial contaminants.However, the temperature and RH of the airare not controlled, and will vary withlocality and between seasons, but dryingrates of the order of hours can be achievedwith small tissue pieces.5. Drying seeds by burying them in acti-vated silica gel (Pammenter et al., 1998).This will yield the most rapid rate of dry-ing in the absence of forced ventilation,and has been adopted as the standard tech-nique by the IPGRI–Danida Forest SeedCentre sponsored project on the handlingand storage of recalcitrant tropical treeseeds (IPGRI/DFSC, 1996).6. Rapid air flow over material in a smallchamber, i.e. flash drying. A common prac-tice is to place the material on a grid in asmall container and pass air into the bot-tom, over the sample, and vent from thetop of the chamber (Berjak et al., 1990;Pammenter et al., 1991). The air may ormay not be dried. If air from a gas cylinderor a compressor is used, it should be dry.(As a word of warning, not all compressorsare properly maintained, and a compressedair line to a laboratory bench may havedroplets of water in it and is obviouslyunsuitable.) An improved system designedby one of us (J.W.-S.) in which air is circu-lated through silica gel and over excisedembryonic axes is illustrated in Fig. 3.1;this equipment yields the fastest dryingrates of all the techniques we have used(several minutes to hours).7. Drying excised axes under partial vac-uum (Fu et al., 1993). Although this tech-nique yielded rapid drying (faster than

Experimental Aspects of Drying and Recovery 95



Tab

le 3

.1.

Sum

mar

y of

dry

ing

met

hods

and

app

roxi

mat

e dr

ying

tim

es u

sed

in s

tudi

es o

n re

spon

ses

to d

esic

catio

n.

Qua

ntity

of

Dry

ing

Dry

ing

met

hod

mat

eria

ltim

eR

efer

ence

See

dsS

un d

ryin

gK

ilogr

ams

Day

sA

lbre

cht,

1993

Stil

l air

at c

ontr

olle

d te

mpe

ratu

re a

nd R

HD

ays

to w

eeks

Cro

mar

ty e

t al.,

198

5; IP

GR

I, 19

94;

Wu

et a

l., 1

998

For

ced

vent

ilatio

n at

con

trol

led

tem

pera

ture

and

RH

Mon

olay

erH

ours

to d

ays

Ntu

li et

al.,

199

7

Bur

ied

in s

ilica

gel

Tens

to h

undr

eds

Gro

ut e

t al.,

198

3; P

amm

ente

r et

al.,

of

see

ds19

98

Exc

ised

axe

sLa

min

ar fl

ow h

ood

Mon

olay

erH

ours

Gro

ut e

t al.,

198

3; N

orm

ah e

t al.,

198

6

Fla

sh-d

ryin

g –

forc

ed v

entil

atio

n w

ith o

r w

ithou

t Te

ns o

f min

utes

P

amm

ente

r et

al.,

199

1si

lica

gel

to h

ours

Veg

etat

ive

tissu

eW

ithdr

awal

of w

ater

ing

from

who

le p

lant

Who

le tr

ache

ophy

tes

Day

sG

aff e

t al.,

199

2; V

ande

r W

illig

en, e

t al.,

2001

Exc

ised

pla

nt p

arts

or

mos

s cl

umps

on

benc

h to

pS

egm

ents

of l

eave

s,

Hou

rsB

arte

ls e

t al.,

199

0; F

arra

nt e

t al.,

199

9tw

igs,

mos

s cl

umps

Sm

all c

ham

bers

at c

ontr

olle

d te

mpe

ratu

re a

nd R

HH

ethe

ringt

on a

nd S

mill

ie, 1

982;

S

eel e

t al.,

199

2

For

ced

vent

ilatio

n in

lam

inar

flow

hoo

d or

sm

all

Boc

hicc

hio

et a

l., 1

998;

Far

rant

et

al.,

cham

bers

1999


storage above silica gel), none of the axessurvived the treatment. Consequently, thistechnique is not recommended withoutfurther investigation.8. Drying to equilibrium at constant RH.This is generally done by placing the mate-rial over saturated solutions of salts, withina small chamber. The air can be still or maybe stirred by a fan in the chamber (Ntuli etal., 1997). The advantage of this techniqueis that the equivalent water potential at equilibrium is known (� =(RT/V)*ln(RH/100), where R is the univer-sal gas constant, T the absolute tempera-ture, V molal volume of water and RHrelative humidity). However, under differ-ent RH conditions the material will dry atdifferent rates and will reach equilibriumafter different times, ranging up to severaldays.

Whatever drying method is adopted, therate of drying is often ultimately deter-mined by the size of the tissue; large seedswill always dry more slowly than smallones. To overcome this limitation, it hasbecome common practice to dry embryonic

axes excised from seeds (Grout et al., 1983;Normah et al., 1986; Pritchard andPrendergast, 1986; Berjak et al., 1990). Thishas the advantage of permitting dryingrates of the order of tens of minutes to afew hours, but can be very laborious iflarge numbers of axes are required. If thisapproach is adopted, it is important tominimize damage whilst excising the axes.

3.2.1.2. Vegetative tissue

A similar wide range of drying techniqueshas been applied to vegetative material,although in this case the drying is gener-ally for experimental purposes and onlysmall quantities are dried. One of the fewexamples of drying large quantities con-cerns the alga Porphyra (Ooshua, 1993). Asis the case with seeds, the various tech-niques differ in the degree of control andthe drying rates achieved.

In experiments on tracheophytes, a com-mon method is to induce desiccation by thesimple expedient of withholding water (e.g.Gaff et al., 1992; Reynolds and Bewley,1993; Quartacci et al., 1997; Vander


Embryonic axes

Plastic mesh

Fan

Silica gel

Fig. 3.1. The improved apparatus for flash-drying of excised embryonic axes. A computer fan circulates airthrough the silica gel and over the axes.


Willigen et al., 2001). This technique mim-ics conditions and drying rates (usuallydays to weeks) occurring in the naturalhabitat of the plants, most drying appearingto occur only after the soil has lost virtuallyall its water (Sherwin et al., 1998; Norwoodet al., 1999). The size and nature of thematerial (younger leaves enclosed or cov-ered by older ones) often results in unevendrying within a single plant.

Faster drying rates (hours to days) havebeen achieved by using excised tissues(twigs, leaves or callus) on the laboratorybench top, with forced ventilation in a lam-inar flow cabinet, or rapid air flow in asmall chamber (e.g. Bartels et al., 1990;Reynolds and Bewley, 1993; Quartacci etal., 1997; Farrant et al., 1999). Drying insmall chambers over silica gel or saturatedsalt solutions, with or without stirring theair, has also been employed (Hetheringtonand Smillie, 1982; Bochicchio et al., 1998;Farrant et al., 1999). Non-vascular plantsdry in the field within a few hours(although this rate could be retarded by theclumped nature of the growth form).Laboratory drying techniques are oftenchosen to simulate natural rates, withinsmall enclosed chambers at RHs rangingfrom 50 to 85% at constant temperature (e.g. Seel et al., 1992; Oliver et al., 1993;Tuba et al., 1996). Very rapid drying hasbeen achieved by the use of silica gel or ofa lyophilizer (Oliver and Bewley, 1984;Oliver et al., 1998).

An extremely important experimentalaspect of drying vegetative tissue is thelight conditions during dehydration, asphoto-oxidation can be an important com-ponent of desiccation damage in photosyn-thetic tissue (Seel et al., 1992; Sherwin andFarrant, 1998; Tuba et al., 1998). Althoughmany seeds have green cotyledons that pre-sumably contain chlorophyll, the light con-ditions during drying have not attractedthe attention of seed biologists. It is alsopossible that temperature could affect theresponse; seeds of Zizania palustris aremore tolerant of desiccation when dried at25°C than at lower (Kovach and Bradford,1992; Berjak et al., 1994; Ntuli et al., 1997)or higher (Ntuli et al., 1997) temperatures.

3.2.2. Quantification and modelling ofdrying rates

The factors determining drying rate aremanifold, many of them are difficult toquantify and they change during the dryingprocess. Consequently, mechanistic model-ling of the drying process is extremely diffi-cult. During the drying process, water isconverted from the liquid form in the tissueto the vapour phase in the surrounding air.The driving force is the difference in thefree energy of water between the tissue andthe air. As the tissue dries the free energy ofthe water decreases, leading to a decrease inthe free energy differential and so to non-linear drying kinetics. There are a numberof resistances retarding the diffusion ofwater from the tissue to the air. Theseinclude the boundary layer (the layer of stillair in immediate contact with the tissue),the outer layer of the tissue (the nature andpermeability of which vary) and the resis-tance to the movement of water from theinterior of the tissue to the surface. The siteof evaporation is not known, and so it isunclear whether water moves from the inte-rior to the tissue surface in the liquid orvapour phase. The boundary layer will beinfluenced by the speed of the air movingover the tissue and by the size and shape ofthe tissue. With forced ventilation, the airflow is probably turbulent, rather than lami-nar, and so difficult to model. The size andshape of seeds and excised axes are oftenirregular and variable, and so their effect onthe boundary layer is also difficult to model.During the drying process, as the tissuedehydrates, the resistance to the transfer ofwater from the interior to the surface willprobably change, complicating the analysis.All this complexity means that mechanisticmodelling of drying rates must, by necessity,be based on simplifying assumptions.Cromarty et al. (1985) have suggested amodel for use in seed banks, based on satu-ration vapour pressure at seed temperature,velocity of air over the seed lot, seed massand oil content. The model assumes a thinlayer of seeds and a spherical shape.

Empirical models of drying rates are alsonot always successful. Depending on the



methods used, and the drying ratesobtained, different factors could be deter-mining the rate of drying, and the rate-deter-mining factor could be changing during thedrying process. Although exponential rela-tionships sometimes can be fitted to the datafrom a drying time course, particularly forseeds or excised axes, it is our experiencethat in many cases the same form of equa-tion cannot be used to describe the data fordifferent drying rates (e.g. Pammenter et al.,1998). As a generalization, if tissue is driedrelatively slowly, the relationship betweenwater content and drying time is exponen-tial. However, for material dried rapidly, theinitial water loss is considerably faster thanthat predicted by an exponential relation-ship. It must be re-emphasized that theterms ‘slowly’ and ‘rapidly’ are relative.There is no drying rate common acrossspecies where drying changes from fasterthan exponential to exponential; a fast dry-ing rate in one experiment could be theequivalent of slow in another.

For example, during ‘slow’ drying ofwhole seeds of Landolphia kirkii(Pammenter et al., 1991), and of Camelliasinensis (Berjak et al., 1993), the water con-tent of the axes within the seeds followedan exponential relationship with time, but‘rapid’ drying of excised axes did not(curve-fitting exercises were not reportedin the original publications; the data havebeen re-analysed). Similarly, initial faster-than-exponential drying rates have beenobserved in rapidly dried excised axes ofSyzigium guiniense, Castanospermum aus-trale, Trichilia dregeana, Artocarpus het-erophyllus, Azadirachta indica, and theradicle tips of axes of Podocarpus henkelii.By way of contrast, axes of Avicenniamarina and entire axes of P. henkelii (theembryonic axes of both species are rela-tively large) show exponential drying(N.W. Pammenter, P. Berjak and J. Wesley-Smith, unpublished observations). It mightbe argued that the same relationship can-not be used to describe ‘slow’ and ‘rapid’drying because different material is oftenused to obtain different drying rates: seedsfor slow drying and excised axes for rapiddrying. However, when excised axes of A.

heterophyllus (J. Wesley-Smith, unpub-lished data), C. australe (Govindasamy,1997) and T. dregeana (Govindasamy,1997; Pammenter et al., 1999) were dried at96% RH (slowly), drying was exponential;when axes of these species were dried oversilica gel (rapidly), the initial drying wasfaster than predicted by an exponentialrate. Similarly, when seeds of Ekebergiacapensis were dried slowly (seeds withendocarp buried in silica gel; dehydrationover 10 days), drying was exponential;when seeds were dried more rapidly (seedswithout endocarp buried in silica gel;dehydration over 1 day), initial drying wasfaster than predicted by an exponentialrelationship (Fig. 3.2; Pammenter et al.,1998). These authors suggested that thedrying kinetics indicated that uneven dry-ing of the tissue might be occurring underrapidly dehydrating conditions. It doesappear, however, that excised axes ofTheobroma cacao show exponential dryingover a range of drying rates (see Chapter 2).

3.2.3. Effects of different drying rates

It is beyond the scope of this chapter todiscuss in detail the consequences of dehy-drating plant tissue. However, referencemust be made to the subject to understandthe influence of drying rate on the responseto dehydration. The effect appears todepend on whether the tissue is inherentlydesiccation-sensitive or -tolerant.

3.2.3.1. Desiccation-sensitive tissue

Certainly with desiccation-sensitive (recal-citrant) seeds, or embryonic axes excisedfrom these seeds, material that is driedrapidly (of the order of tens of minutes tohours) can survive to lower water contentsbefore viability is lost than material that isdried slowly (over a period of days)(Normah et al., 1986; Pritchard andPrendergast, 1986; Farrant et al., 1989;Pammenter et al., 1991, 1998; Pritchard,1991; Berjak et al., 1993; Pritchard andManger, 1998). The rapid drying is notactually increasing desiccation tolerance as



such; it is simply that, if the tissue is driedfast enough, low water contents can beachieved before sufficient time elapses forit to die. It has been suggested that some ofthe processes leading to viability loss areaqueous-based and so occur at relativelyhigh (intermediate) water contents, of theorder of 1.0–0.3 g water g�1 dry mass, cor-responding to water potentials of about�1.5 to �14 MPa (Vertucci and Farrant,1995; Farrant et al., 1997; Pammenter andBerjak, 1999; Walters et al., 2001). Materialthat is dried very rapidly passes throughthese intermediate water contents so fastthat the damage caused by the deleteriousprocesses does not have time to accumu-late; thus viability loss does not occur as aconsequence (Pammenter et al., 1998;Pammenter and Berjak, 1999). Althoughrapid drying does permit viability retentionto low water contents, those tolerated bydesiccation-sensitive seeds or axes (mini-mum of about 0.2 g water g�1 dry mass) arenever as low as water contents usuallyoccurring naturally in dry desiccation-tol-erant (orthodox) seeds (< 0.05 g water g�1

dry mass). Also, survival of these lowwater contents by embryonic axes of recal-citrant seeds is apparent only if the mater-ial is assessed for survival immediatelyafter drying. If the material is maintained(at room temperature) in the partially dry

state, it rapidly loses viability (Walters etal., 2001).

Studies on the effects of dehydration ofdesiccation-sensitive vegetative tissue aremore limited. However, if much of the vol-ume of the cells is occupied by fluid-filledvacuoles, mechanical damage associatedwith volume reduction consequent upondrying might be important (Iljin, 1957;Vertucci and Farrant, 1995; Farrant et al.,1997), in which case drying rate mighthave little effect.

3.2.3.2. Desiccation-tolerant tissue

In tissue that is, or becomes, desiccation-tolerant, the effect of drying rate dependsupon the stage of development and/or thenature of the tolerance mechanisms.Developing orthodox seeds start toacquire desiccation tolerance concomitantwith, or slightly preceding, reserve accu-mulation, and the population as a wholeis generally tolerant by the end of thisstage (e.g. Bewley and Black, 1994). Theresponse to rate of drying of orthodoxseeds that are still in the desiccation-sen-sitive stage of development is in almostdirect contrast to the response of recalci-trant seeds. If, after histodifferentiation,prior to the acquisition of desiccation tol-erance, a developing orthodox seed is


2.5

2.0

1.5

1.0

0.5

0.0

Axi

s w

ater

con

tent

(g

wat

er g

–1 d

ry m

ass)

0 2 4 6 8 10

Time (days)

0 10 20 30 40 50

Time (h)

(a) (b)

Fig. 3.2. Data illustrating that although the rate of water loss of slowly dried seeds (a) can be described byan exponential equation, that of rapidly dried seeds (b) is not exponential. In (a) the line is an exponential fitto the data; in (b) the dotted line is an exponential fit, the solid line is drawn by eye. Note the different timescales. Data from Pammenter et al. (1998).


dried rapidly, it will not survive; if it isdried slowly, it will (Kermode and Bewley,1985; Bewley and Black, 1994). This isprobably because, during slow drying, suf-ficient time elapses for the development ofthe tolerance mechanisms.

The response of desiccation-tolerantvegetative tissue (‘resurrection’ plants) todrying rate appears to vary with thenature of the tolerance mechanism(reviewed by Oliver and Bewley, 1997). Innon-vascular resurrection plants, toler-ance seems to be achieved predominantlyby an ability to repair damage caused bydesiccation, and is thus primarily basedon constitutive mechanisms (Oliver andBewley, 1997). Drying rate appears to havelittle influence on ultimate survival, butrecovery takes longer in rapidly dried tis-sue, perhaps suggesting the existence ofsome inducible protection mechanisms(Schonbeck and Bewley, 1981a). It has beenobserved that non-vascular resurrectionplants tend to become less desiccation-tolerant if kept in the hydrated state forextended periods compared with dailydehydration/rehydration ‘hardening’ cycles(Schonbeck and Norton, 1979; Schonbeckand Bewley, 1981b).

Resurrection tracheophytes have beenclassified as ‘modified desiccation-tolerantplants’, in comparison with non-vascularresurrection plants (‘fully desiccation-tol-erant plants’), because their ability to sur-vive desiccation is rate-dependent (Oliverand Bewley, 1997). The responses of resur-rection angiosperms to drying techniqueappears to be complex. Rapid desiccationis generally lethal, although Bochicchio etal. (1998) have shown that it is water con-tent, rather than drying rate, that affectssurvival of detached leaves of the resurrec-tion plant Boea hygroscopica. The generaleffect of drying rate on the response of res-urrection tracheophytes is a consequenceof an inducible desiccation tolerance, thistolerance being based on protection duringdesiccation, rather than repair on rehydra-tion. This group can be further subdividedinto two, based on the strategy to preventlight-associated damage on drying. Thehomoiochlorophyllous species retain

chlorophyll during drying, whereasspecies that are poikilochlorophyllous losechlorophyll and dismantle thylakoid mem-branes (Hetherington and Smillie, 1982;Sherwin and Farrant, 1998), although it ispossible that some of the observed abnor-malities are a consequence of the fixationmethod (see Platt et al., 1997).Poikilochlorophylly appears to precluderapid drying rates and, although somehomoiochlorophyllous plants can survivefaster drying rates, there are other speciesthat cannot (Farrant et al., 1999).Excluding old leaves, which would natu-rally senesce, all tissues of most resurrec-tion angiosperms are tolerant; however,there are species in which only the young,immature tissues are tolerant (Gaff andEllis, 1974; Vander Willigen et al., 2001),suggesting that developmental stage isanother compounding factor. Leaf tissuesof some species show tolerance whetherattached or detached from the parentplant, whereas others survive onlyattached, or after an initial drying phaseon the parent plant, during which theypresumably acquire the necessary signalsfor tolerance (Gaff and Loveys, 1992). Adetailed discussion of these responses,and their underlying causes, is beyond thescope of this chapter. Suffice it to say thatthese complexities must be borne in mindby the investigator when designing andinterpreting the results of experiments.

3.2.4. ‘Ultradrying’ of desiccation-tolerantmaterial

There has been recent interest in, andlively debate on, the effects of storingorthodox seeds at water contents belowthose normally used in gene banks. It is notintended to review that debate here, butsimply to point out that, even in desicca-tion-tolerant tissue, the experimental tech-nique – the extent to which the material isdried – may have an influence on theresponse to drying (and subsequent stor-age). For more information, the reader isreferred to Ellis (1998), Walters (1998) andWalters and Engels (1998).



3.3. Influence of Rehydration Technique

It is well known that if dry biological mate-rial is immersed in water a number of sub-stances of low molecular weight will leakfrom the tissue (discussed by Hoekstra etal., 1999). If the tissue is desiccation-toler-ant, this leakage will subside with rehydra-tion, although, if the tissue is initially verydry, leakage can be extensive and could bedamaging, this damage being exacerbatedby imbibition at low temperatures (Pollock,1969; Hobbs and Obendorf, 1972). Becauseof this, it is common in studies on seeds orexcised axes to ‘prehumidify’ tissue bymaintaining it in a saturated atmosphere orplacing on damp paper, before immersionin water. However, many recalcitrant seedsare damaged at water contents far in excessof those at which ‘imbibitional damage’occurs, and there is little evidence to sug-gest that such damage upon imbibition isan important factor in desiccation-sensitiveseed material. Kovach and Bradford (1992)initially ascribed the response to desicca-tion of seeds of Z. palustris to imbibitionaldamage, although Vertucci et al. (1995)suggested that the damage was a directresult of desiccation, rather than imbibi-tion. Sacandé et al. (1998) have demon-strated imbibitional damge exacerbated bylow-temperature imbibition and increasedstorage time in seeds of neem (A. indica) atwater contents < 8% (fresh mass basis), butthis is a water content considerably lowerthan most recalcitrant seeds will survive.

In vegetative tissues, rehydration tech-niques are generally even less welldescribed and assessed than are the dehy-dration techniques, and consequently theeffects of various methods of rehydrationare relatively unknown. In the tracheo-phytes, whole plants are generally rewa-tered to field capacity with (Norwood etal., 1999) and with or without (Sherwinand Farrant, 1998) additional aerial spray-ing. Dehydrated excised leaves are rehy-drated by floating on or in water (Gaff andLoveys, 1992; Reynolds and Bewley, 1993;Bochicchio et al., 1998; Dace et al., 1998).Mosses are rehydrated by immersion indistilled water (Oliver and Bewley, 1984)

or more slowly in misting chambers (Seelet al., 1992, Tuba et al., 1996). As withdehydration, lighting and temperaturemust be carefully controlled.

3.4. Length of Time in the PartiallyDehydrated State

When desiccation-sensitive tissue is dehy-drated, it is subjected to a number ofstresses as it dries. The type of damage thatpotentially can occur will change as thewater content decreases (see Chapter 9); asthe intensity of the stress increases, theeffect on the tissue generally becomes moresevere. However, the effect of a stress, par-ticularly a mild stress, is not instantaneous.If a stress induces a metabolic disorder, ittakes time for the damage consequent uponthat disorder to accumulate. Thus, theeffect of a stress depends not only on itsintensity, but also on the time for which thestress is applied. It is this concept of ‘inten-sity’ versus ‘duration’ of a stress that under-lies the confusion that has obscured theinterpretation of the effects of drying rateson desiccation-sensitive seed material.

To unravel the confounding issues ofwater content and time in drying experi-ments, it is necessary to dry tissue almostinstantaneously to a range of water con-tents and then to maintain the material atthese water contents. In practice, this is nota simple experimental achievement. Mostseeds are too large to dry rapidly and, ifisolated embryonic axes are dried, it is dif-ficult to ‘store’ them in the partially dehy-drated state without some other deleteriousconditions (such as anoxia or microbialproliferation) occurring. Maintaining vege-tative tissue in the partially dehydratedstate is probably even more difficult and noexperiments are known where this hasbeen attempted.

Despite these difficulties, some informa-tion is available. Walters et al. (2001) haveshown that partially dehydrated embryonicaxes of tea lose viability within a few days,and that the rate at which viability is lostdepends on the water content. Similarly,isolated axes of T. dregeana dried over sil-



ica gel lost viability as the water contentreached the level in equilibrium with thedesiccant, whilst axes dried at 96% RH lostviability some days after the tissue hadreached equilibrium (Pammenter et al.,1999). Similarly, in the relatively long-livedseeds of Araucaria huntsteinii, longevity at6°C was reduced as water content wasreduced below about 45% (Pritchard et al.,1995). As an aside, this raises questionsconcerning the concept of ‘degree of recal-citrance’. Would this be assessed on thebasis of the minimum water content towhich the seed (or embryonic axis) can bedried without loss of viability, or on thetime for which it survives in equilibriumwith some predetermined water potential?The concept of ‘intensity’ versus ‘duration’of a stress has practical applications. It hasbeen suggested that, in the case of recalci-trant seeds that germinate in storage, partialdehydration may prevent this and soincrease storage life span (Hong and Ellis,1996). However, it appears that even a mildwater stress applied to seeds of the tropicalspecies T. dregeana is deleterious (Drew etal., 2000), and relatively mild partial dryingof the temperate seeds of A. huntsteinii(Pritchard et al., 1995) and Aesculus hip-pocastanum (Tompsett and Pritchard, 1998)can reduce longevity. Thus ‘sub-imbibedstorage’ should be approached with cautionas it can actually reduce life span.

In passing, it should be noted that evendesiccation-tolerant organisms do not havean infinite life span in the dehydratedstate; they accumulate damage in this state,and so desiccation could be considered tobe a stress, even in tolerant tissues.

3.5. Methods of Assessing Response toDehydration

A variety of techniques have been used toassess damage in response to drying.Different techniques measure different phe-nomena (membrane characterisics, respira-tory competence, photosynthetic activity),but the ultimate test is whether an indepen-dent functioning organ(ism) can be re-estab-lished after dehydration and rehydration.

3.5.1. ’Germination’

With seeds, the most common assessmentmethod is to set them out to germinate.However, as pointed out by Hong and Ellis(1996), it is possible that the treatment mayinduce a dormancy and that seeds that donot germinate may not actually be dead.Those authors recommended that the dura-tion of germination tests be extended untilall non-germinated seeds have been posi-tively identified as dead by the fact that theyrot. Another problem is that a seed may pro-duce a radicle, and so be scored as havinggerminated, but be so damaged as to beunable to establish a viable seedling (Fu etal., 1993; Berjak et al., 1999). The precisionof a germination test can be increased by fol-lowing the time course of germination. Anincreased lag before the first seed germi-nates or a decrease in the rate of germina-tion (increased time to 50% germination)may indicate damage that is repaired duringthe lag phase. A simple assessment of finalgermination would not reveal this damage.A number of suggestions have been madeconcerning fitting equations to germinationdata (Brown and Mayer, 1988), but they gen-erally require more samples than are oftenavailable when undertaking studies onrecalcitrant seeds from wild species.

When experiments are conducted onexcised embryonic axes, ‘germination’ canbe assessed by placing the axes on damppaper in an enclosed chamber (such as aPetri dish), although it is common to use invitro growth media. As many recalcitrantseeds, particularly from the tropics, har-bour fungal propagules (Berjak, 1996;Calistru et al., 2000), their removal by suit-able pretreatment is essential under theseconditions. As with seeds, care must betaken in assessing ‘germination’. Swellingand/or greening of an axis suggests that itis not dead (although a dehydrated axiswill swell on rehydration), but it does notnecessarily imply that it is capable of pro-ducing an independent plantlet.Assessment can also be complicated bychoice of the medium, as this may have aninfluence on the ‘growth’ of the tissue (as itdoes in normal in vitro multiplication and



propagation). Material that has been dam-aged, but not killed, by the dehydrationtreatment may take a considerable time toshow signs of growth, and so care shouldbe taken not to discard it too early.

3.5.2. Resurrection plants

With whole resurrection plants, the criteriafor determining whether the organism is‘functional’ may vary. This may simply beon physical appearance (particularly thegreening of poikilochlorophyllous tissue),measurement of chlorophyll fluorescencecharacteristics (Fv/Fm), which indicates thephotochemical efficiency of photosystemII), or assessment of the ability to assimi-late carbon dioxide photosynthetically, orto respire.

3.5.3. Electrolyte leakage

A technique that is commonly employedwith small pieces of tissue (small seeds,excised axes, leaves of resurrection tra-cheophytes, ‘pieces’ of non-vascularplants) is to measure leakage of electrolytesor of specific ions such as K+. An advan-tage of this technique is its simplicity andrapidity, especially using multiple-cellelectrical conductivity meters, which arecommercially available. The extent of elec-trolyte leakage is considered to assess thedegree of membrane damage (Bramlage etal., 1978; McKersie and Tomes, 1980). Asthe rate of leakage over the first few min-utes is often higher than the steady rateestablished later, the steady-state rate ofleakage is often taken as an indication ofmembrane damage (McKersie and Stinson,1980). An alternative approach is to assessleakage (or rate of leakage) after a giventime as a proportion of total leakage (ormaximum rate), which occurs when allmembranes are fully disrupted by treat-ments such as homogenizing, boiling, auto-claving or repeated freeze/thaw cycles. It iscommon practice to prehumidify tissue ina saturated atmosphere or on damp filterpaper prior to immersion to reduce any

damage associated with imbibition. Withseeds or excised axes there is generallygood agreement between leakage character-istics and other signs of damage such asloss of vigour or viability (McKersie andTomes, 1980; Pammenter et al., 1991;Berjak et al., 1992, 1993). Older techniquesto assess membrane integrity involve theuse of vital dyes, which leak from damagedcells (Gaff and Loveys, 1992).

3.5.4. Tetrazolium test

The reduction of colourless tetrazoliumchloride (2,3,5-triphenyl tetrazolium chlo-ride (TTZ)) to a pink/red formazan dye istaken as a measure of respiratory activity, asTTZ is reduced by components of the mito-chondrial electron transport chain.Although the tetrazolium test is used exten-sively to assess the quality of orthodoxseeds, its use in the study of desiccationresponse has been limited. Ntuli et al.(1997) showed that considerable differencesoccurred between the ability to germinateand apparent viability as a result of tetra-zolium tests, when investigating the desic-cation response of Z. palustris, indicatingthat the two tests were not equivalent or notnecessarily measuring the same thing. Afurther caveat in using the TTZ test as a via-bility assay is that a dead seed supporting avigorous mycelium internally will test posi-tive as a result of fungal respiration.

3.5.5. Other responses

A number of biochemical and biophysicalresponses to dehydration have beenassessed by a variety of workers. Examplesinclude the activities of antioxidants(Tommasi et al., 1999), accumulation of lateembryogenesis abundant proteins (Finch-Savage et al., 1994; Gee et al., 1994), ethyl-ene production, respiration and proteinsynthesis (Salmen Espindola et al., 1994),partitioning of amphipathic moleculesbetween cytosol and membranes (Golovinaet al., 1998) and changes in cytoplasmic vis-cosity (Leprince and Hoekstra, 1998). These



are not covered here because often it wasthe details of these responses that were theobjectives of the investigations, and so theydo not fall within the scope of a discussionof general experimental approaches.

3.6. Expression of Water Content Data(see also Chapter 2)

3.6.1. Mass basis

The amount of water in plant tissue hasbeen expressed in a number of differentways, but generally most commonly onsome form of ‘mass’ basis. This is probablybecause it is the simplest measure toobtain; the hydrated/partially hydrated tis-sue is weighed, dried for some predeter-mined time, and reweighed. TheInternational Seed Testing Association rec-ommends drying at 103°C for 17 h (ISTA,1999); an alternative approach, particularlyif using small amounts of tissue, is to dry ata lower temperature (to reduce loss of non-water volatile material) to constant weight.The data can then be expressed on a freshmass basis (mass of water per unit freshmass, often presented as a percentage); thisindicates the proportion of the hydrated orpartially dehydrated tissue that is water.Alternatively, the data can be expressed ona dry mass basis (mass of water per unitdry mass). Strictly speaking, when express-ing the data on a mass basis, the term‘water content’ is incorrect; this should bereserved for expressing the absoluteamount of water in the tissue, irrespectiveof the quantity of tissue. What is generallydescribed as ‘water content’ is actually a‘water concentration’ (the amount of waterper unit amount of fresh or dry tissue).Thus, water content on a dry mass basisshould be termed ‘dry mass-specific waterconcentration’. However, use of the term‘water content’ to describe a ‘water concen-tration’ is so deeply entrenched that it isunlikely to change. We prefer data to beexpressed on a dry mass basis. In this case,the basis to which values are being normal-ized does not change as the amount ofwater changes, and the proportional

change in ‘water content’ reflects the pro-portional change in the amount of water inthe tissue; if the water content changesfrom 1.0 to 0.5 g water g�1 dry weight, thetissue has lost half its water. If the data areexpressed on a fresh mass basis, for tissueat 1.0 g water g�1 dry mass that loses halfits water, water content on a fresh massbasis changes from 50% to 33.3%.

3.6.2. Water potential

The responses of tissue to drying are deter-mined by the processes that occur duringdrying. The processes that occur at anywater content are influenced by the freeenergy status of the water, and so it is bio-logically more meaningful to express tissuewater in terms of water potential ratherthan water content. Water potential ofsmall pieces of tissue can be measured bythermocouple psychrometry, but this islimited to higher potentials (above about�5 MPa). However, recent technicaladvances permit dewpoint psychrometricmeasurements to much lower water poten-tials. Sorption isotherms (equilibrating tis-sue at known fixed RH to constant watercontent) can be used to establish the rela-tionship between water content and waterpotential (Vertucci et al., 1994), althoughthis is difficult at high water potentialsbecause the relationship between RH andwater potential changes rapidly in thisregion. Soaking tissue in solutions ofknown concentrations (and hence knownwater potentials) of non-penetrating solutessuch as polyethylene glycol (PEG) 8000 hasbeen used to assess the watercontent/water potential relationship athigh water potentials (Vertucci et al., 1994;Pritchard et al., 1995; Tompsett andPritchard, 1998). A complication of estab-lishing sorption isotherms is the phenome-non of hysteresis; the equilibrium watercontent at any RH depends on whether drytissue is being hydrated or hydrated tissueis losing water (for a discussion, see Eira etal., 1999). There is an additional problemwhen working with recalcitrant seeds oraxes of tropical species. This material gen-



erally harbours very high levels of fungalpropagules, and fungal proliferation almostinvariably accompanies attempts to equili-brate tissue with atmospheres of RH aboveabout 80%; it is therefore impossible todiscriminate between the contribution ofthe seed material and the fungal myceliumto the derived isotherm. Under these con-ditions, use of concentrated solutions ofPEG 8000 is advised.

3.6.3. Relative water content

Relative water content, RWC (amount ofwater in the tissue/amount of water at fullturgor), has also been used to express thewater status of tissue (see Grange and Finch-Savage (1992) for seed tissue, and VanderWilligen et al. (2001) for vegetative tissue).It is more meaningful than simple watercontents, although relative cell volume(which is based on symplastic water only) isprobably a better measure of direct stress towhich the tissue is subjected. To assess theproportions of apoplastic and symplasticwater in tissue requires the construction ofpressure–volume curves. Not only is thisdifficult (because of the difficulty of measur-ing water potential at low water contents),but it is possible that the assumptionsunderlying the analysis of pressure–volumecurves (Tyree and Hammel, 1972) may nothold at low water contents. An additional

complication when calculating RWC is theestimate of water content at ‘full turgor’. Ifrecalcitrant seeds are immersed in water,they will take up water as they germinate,and so full turgor cannot be equated withwater content of tissue imbibed in water.Data could be expressed relative to thewater content at shedding, but this can bevery variable among seeds within a harvest,as well as between collections. With vegeta-tive tissue it is possible to over-hydrate tis-sue such that liquid water occupies some ofthe intercellular air spaces in leaves, orexists as intercellular or surface water innon-vascular plants (Beckett, 1997). Theseeffects lead to overestimates of water con-tent at full turgor.

3.7. Conclusion

When investigating the response of tissueto dehydration, a range of techniques areavailable. However, the observed responseis likely to depend on factors such as thedrying method, and possibly the rehydra-tion technique and method of assessment.The techniques adopted will depend onthe size or amount of tissue being dried,the facilities available and, importantly, thepurpose of the investigation. The investiga-tor should be aware of the technical com-plications when assessing and interpretingthe data obtained.


3.8. References

Albrecht, J. (ed.) (1993) The Tree Seed Handbook of Kenya. GTZ, Nairobi, Kenya, 264 pp.Bartels, D., Schneider, K., Terstappen, G., Piatkowski, D. and Salamini, F. (1990) Molecular cloning

of abscisic acid-modulated genes which are induced during desiccation of the resurrection plantCraterostigma plantagineum. Planta 181, 27–34.

Beckett, R.P. (1997) Pressure–volume analysis of a range of poikilohydric plants implies the exis-tence of negative turgor in vegetative cells. Annals of Botany 79, 145–152.

Berjak, P. (1996) The role of microorganisms in deterioration during storage of recalcitrant and inter-mediate seeds. In: Ouédraogo, A.S., Poulsen, K. and Stubsgaard, F. (eds) Intermediate/Recalcitrant Tropical Forest Tree Seeds. IPRGI, Rome, pp. 121–126.

Berjak, P., Farrant, J.M., Mycock, D.J. and Pammenter, N.W. (1990) Recalcitrant (homoiohydrous)seeds: the enigma of their desiccation-sensitivity. Seed Science and Technology 18, 297–310.

Berjak, P., Pammenter, N.W. and Vertucci, C.W. (1992) Homoiohydrous (recalcitrant) seeds: develop-mental status, desiccation sensitivity and the state of water in axes of Landolphia kirkii Dyer.Planta 186, 249–261.

Berjak, P., Vertucci, C.W. and Pammenter, N.W. (1993) Effects of developmental status and dehydra-


tion rate on characteristics of water and desiccation-sensitivity in recalcitrant seeds of Camelliasinensis. Seed Science Research 3, 155–166.

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4 Biochemical and Biophysical Methods forQuantifying Desiccation Phenomena in Seeds

and Vegetative Tissues

Olivier Leprince1 and Elena A. Golovina2,31UMR Physiologie Moléculaire des Semences, Institut National d’Horticulture, 16 Bd

Lavoisier, F49045, Angers, France; 2Laboratory of Plant Physiology, Department ofPlant Sciences, University of Wageningen, Arboretumlaan 4, 6703 BD Wageningen,

The Netherlands; 3Timiryazev Institute of Plant Physiology, Botanicheskaya 35,Moscow 127276, Russia

4.1. Introduction 1124.2. Caveats: the Consequences of Being Dry 1124.3. How to Study Biochemical Responses to Drying 114

4.3.1. Responses of gas exchange and volatile emission to drying 1144.3.1.1. Headspace analysis 1144.3.1.2. Laser photoacoustic spectroscopy (PA) 114

4.3.2. NMR applications to measure steady-state concentrations and to assess metabolic responses to drying 115

4.3.3. Photosynthesis studies 1164.3.4. Oxidative stress and anhydrobiosis 116

4.4. Spectroscopy Techniques 1194.4.1. Electron paramagnetic resonance (EPR) 119

4.4.1.1. General description 1194.4.1.2. Applications of EPR methods 120

4.4.2. Nuclear magnetic resonance 1274.4.2.1. General description 1274.4.2.2. The NMR study of water in living systems 1284.4.2.3. NMR imaging 1304.4.2.4. High-resolution multinuclear NMR spectroscopy 1314.4.2.5. Structure and dynamics of cellular membranes 133

4.4.3. Fourier transform infrared (FTIR) spectroscopy 1344.4.3.1. General description of infrared spectroscopy 1344.4.3.2. Biological applications 134

4.5. Additional Techniques to Study Biochemical and Biophysical Aspects of Desiccation Tolerance 1364.5.1. Differential scanning calorimetry (DSC) 1364.5.2. Electron microscopy 136

4.6. Acknowledgements 1374.7. References 137



4.1. Introduction

In recent years, the development of physi-cal techniques has brought substantialinsights into the physical state of water andcellular components of desiccated systems.These techniques cover a large range ofspectroscopic techniques such as nuclearmagnetic resonance (NMR), electron para-magnetic resonance (EPR, also referred toas electron spin resonance (ESR)), Fouriertransform infrared (FTIR) spectroscopy aswell as dielectric measurements andcalorimetry. The success of these tech-niques and spectroscopy in particular orig-inated in their versatility and in theirability to assess the physical state of desic-cated systems by non-invasive means.Below, the resourcefulness and limitationsof the physical techniques are reviewed.

In contrast to physical aspects, most ofthe fundamental questions pertaining tothe biochemical aspects of desiccationtolerance in anhydrobiotes remain to beanswered. The lack of non-destructiveand sensitive techniques has greatlyimpeded our understanding of the role ofmetabolism in desiccation tolerance.Furthermore, all of our biochemical assaysand isolation of organelles have been setup in dilute solutions using water ororganic compounds as solvents. However,they have been indiscriminately applied todrying and desiccated specimens. Here itis argued that the experimental approachof grinding dry or nearly dried specimensin aqueous buffers and measuring meta-bolic activities and biological markers ofoxidative stress in vitro in dilute solutionsis unlikely to reflect the in vivo situation.This methodology has complicated theinterpretation of data regarding biochemi-cal activities associated with differenthydration levels (Lynch and Clegg, 1986;Vertucci and Leopold, 1986). Hoekstra andvan Roekel (1983) have clearly illustratedhow isolation-inflicting injury of isolatedmitochondria in germinating pollen canconfuse the interpretation of resultsobtained in vitro. To partially overcomethis technical bottleneck, two strategiescan be adopted: (i) whenever possible,

resort to non-invasive techniques thatenable the investigator to assess any bio-chemical phenomenon in drying tissueswithout introducing water during theanalysis. The few techniques available arebriefly presented; and (ii) characterizemutants and/or transgenic plants the phe-notypic traits of which can be associatedboth with a particular biochemical path-way and the level of desiccation tolerance(Chapter 12; Wolkers et al., 1998a; Shiotaand Kamada, 2000; Weber et al., 2000;Wehmeyer and Vierling, 2000). For exam-ple, the antisense inhibition of ADP-glu-cose pyrophosphorylase, a key enzyme instarch synthesis, was found to increase thesucrose and nitrogen content of matureseeds of transgenic Vicia narbonensis.Also, the decrease in ADP-glucosepyrophosphorylase activity altered the cel-lular volume and water relations duringthe seed-filling phase (Weber et al., 2000).These observations show that a specificalteration in carbon metabolism haspleiotropic effects on seed developmentand illustrate the potential of molecularbiology to assess non-destructively the roleof various biochemical and biophysical phe-nomena related to desiccation tolerance.

4.2. Caveats: the Consequences of BeingDry

Since water acts as a solvent and substratein the cell in a variety of ways, its reducedavailability in dried tissues will induce aset of physical and biochemical responsesthat may disappear during an invasivemeasurement, thereby confusing the inter-pretation of the data. Before ascertaining acause–effect relationship between desicca-tion tolerance and a specific biochemicaland biophysical process, the followingremarks should be taken into account:

1. To adequately link the response ofmetabolism to drying with desiccation tol-erance, it is necessary to map metabolicactivities as a function of water content orwater potential of the drying cell and notas a function of time of drying (Vertucci

112 O. Leprince and E.A. Golovina


and Leopold, 1986; Leprince et al., 1999,2000). This necessity is more acute whenaccumulation of dry matter occurs duringdevelopment. Often a sensitive microbal-ance is needed to determine fresh and dryweights of milligram quantities of samples.Furthermore, fast measuring techniquesshould be preferred over time-consumingassays so that the rates of water loss matchthe time necessary to acquire the data.Furthermore, Hendry (1993) argued thatattempts to characterize desiccation phe-nomena in drying tissues should be setagainst a range of desiccation-induceddamage and not only against percentages ofsurvival after drying. 2. It is important to recognize that duringdrying the cytoplasmic viscosity increasesdramatically until glass formation(Leprince and Hoekstra, 1998; Buitink etal., 2000e; Chapters 2 and 10). Unlessenzymatic activities are assessed in anenvironment similar to that found in thedrying cells, we do not know how the risein viscosity during drying will affect meta-bolic rates and/or pathways. We canalready predict that O2-processing systemswill be altered by the loss of water sinceO2 solubility is known to decrease withrising viscosity (Gros et al., 1992; Leprinceand Hoekstra, 1998). Biochemical eventsduring drying should obey different lawsof diffusion since the cytoplasm willundergo a physical transformation from aliquid state to a solid-like state (i.e a glass).The moisture contents at which thesechanges in diffusion characteristics occurduring drying should preferably be deter-mined. It has been suggested that thismoisture content corresponds to the glassformation that is measured by the tempera-ture at which the drying cytoplasm forms aglass (Tg). Below Tg, solid statephysics/chemistry prevails (Chapter 10).However, Buitink et al. (2000f) suggestedthat the most important change in thephysical properties of the cytoplasmoccurs 50°C above Tg, at a temperature cor-responding to the so-called collapse tem-perature (Tc). A distinction between liquidstate and solid state should be made whenchemical and biochemical events (such as

oxidative reactions) occurring during nat-ural ageing (i.e. below Tg) are comparedwith those occurring during acceleratedageing (between Tc and Tg (for example,75–85% relative humidity (RH) and tem-peratures between 35 and 50°C) or aboveTc (100% RH, 41°C)). This is illustrated inthe study of Lievonen et al. (1998) on non-enzymatic browning reaction rates aroundthe Tg of mixtures made of water, glyceroland maltodextrin. It is also clearly illus-trated in the kinetics of seed and pollenageing (see, for example, Buitink et al.,2000g and references therein).3. To be quantified and characterized,metabolites, proteins, DNA, organelles,etc., must be extracted and purifiedbeforehand. This procedure stronglydepends on the tissue water content. Thisis valid for both water-soluble compoundsusing aqueous extraction and lipid-solu-ble compounds using organic solvents.For lipid extraction and separation usingthe Folch’s method, Hamilton et al. (1992)recommended that the amount of waterpresent in the tissues should be calculatedand taken into account during the differ-ent washing and partitioning procedures.For aqueous extraction, we do not knowwhether the extraction and purification ofwater-soluble metabolites or proteins dif-fer quantitatively and qualitatively whentissues are ground either fresh or dried.This point is particularly relevant foramphiphilic molecules such as phenoliccompounds, which are likely to partitioninto the membranes and/or oil bodies dur-ing drying and vice versa during rehydra-tion (Chapter 10; Golovina et al., 1998;Buitink et al., 2000e). Thus, the desicca-tion-induced changes in metabolite con-centrations and in protein conformationshould be interpreted with caution if thesechanges are assessed from crude extracts. 4. Most of the methods used so far areaveraging techniques. However, it islikely that there is a gradient of waterwithin seed tissues during dehydrationand rehydration. Therefore, a qualitativeand quantitative gradient of responseswithin the tissues submitted to dryingmight be expected.

Methods for Quantifying Desiccation Phenomena 113


4.3. How to Study BiochemicalResponses to Drying

Two technically different strategies areavailable to probe the responses of metabo-lism to drying by non-invasive means: thedetection and analysis of gases thatemanate from, or are absorbed by, the dry-ing tissues (so-called headspace analysis)and the resort to in vitro or in vivo NMR. Inphotosynthetic anhydrobiotes, fluorescencespectroscopy is also a convenient tool tocharacterize energy metabolism during dry-ing (see Chapter 7). The methods thatassess free-radical-induced damage in des-iccation tolerance are also reviewed.

4.3.1. Responses of gas exchange andvolatile emission to drying

4.3.1.1. Headspace analysis

A wide variety of gaseous metabolites canbe studied in drying tissues: O2 and CO2exchange as markers of respiration or photo-synthesis, ethanol and acetaldehyde asmarkers of fermentation, alkanes, alkones,alkenes, aldehydes (volatile) as markers ofoxidative stress. Several instruments suchas infrared CO2 analysers, gas-phase O2 elec-trodes or O2 analysers can detect andanalyse CO2 and O2 exchanges. These typesof analysers have mainly been used todescribe the effects of desiccation on photo-synthetic activities of resurrection plants(Schwab et al., 1989) and photosymbioticlichens (Nash et al., 1990; Scheidegger etal., 1995). However, they are not alwayssuited to assaying low respiration rates fromnearly dried material because of their lowsensitivity (O. Leprince, unpublished data).

Gas exchange rates and volatile produc-tion can also be measured by gas chro-matography (GC) (Kimmerer andKozlowski, 1982; Gorecki et al., 1984; Kleinand Sachs, 1992; Leprince and Hoekstra,1998; Leprince et al., 1999), gas chromatog-raphy–mass spectrometry (GC-MS) (Zhanget al., 1994), high-pressure liquid chro-matography (HPLC) (Degoussée et al., 1995)or by using a Gilson differential respirome-

ter (Rogerson and Matthews, 1977; Vertucciand Leopold, 1986, 1987). In these tech-niques, the gas to be analysed has to accu-mulate over time in a closed environmentbefore taking the measurement (i.e. staticheadspace analysis). To reach a detectableconcentration, the gas accumulation maytake some time, particularly in drying sam-ples in which the metabolism and gas diffu-sion are greatly reduced by the lack ofwater. Consequently, the assay may be tooslow in comparison with the rate of waterloss. Furthermore, an additional problem isreliably maintaining the specimen at thesame water content during the measure-ment. Thus, unless the kinetics of waterloss matches the time frame needed toassess the metabolic rates, the relationbetween hydration levels and metabolicactivities in drying tissues may not be accu-rate. Therefore, it is best to adapt a flow-through system coupled to an activetrapping system (i.e. dynamic headspaceanalysis). A flow of dry or humidified airpasses over the sample acting as both a gascarrier and dehydrating agent. The volatilesare then absorbed by a compound locatedin the exit flow and analysed by GC (Wilsonand McDonald, 1986; Zhang et al., 1994).For dried tissues that are in a glassy state,the release of volatiles may take severaldays or weeks because of the slow diffusionof molecules. To overcome this problem,several authors have used a thermal desorp-tion technique consisting of heating the dryspecimens to at least 60°C. This procedureis thought to purge the volatiles that aretrapped in the glassy matrix (Wilson andMcDonald, 1986; Hailstones and Smith,1989; Zhang et al., 1994; Degoussée et al.,1995). However, it is not always possible todetermine whether the production ofvolatiles after desorption results from theheat treatment per se or not (Wilson andMcDonald, 1986).

4.3.1.2. Laser photoacoustic spectroscopy(PA)

PA is an emerging technique that has over-come the problems associated with head-space analysis. A PA set-up consists mainly



of two components: a line-tunable CO laserthat excites gas molecules specificallyaccording to their infrared fingerprintabsorption, and parallel resonators. Eachresonator is coupled to a sensitive micro-phone in which the concentration of gasesis sequentially measured based on anacoustic phenomenon (Harren and Reuss,1997; see Zuckerman et al., 1997,www.sci.kun.nl/tracegasfac/experime.htmfor technical details of the experimentalset-up). This technique permits the probingof metabolic processes non-invasively thatresult in the emission and/or absorption ofethylene, acetaldehyde, ethanol and CO2from drying tissues without altering thewater content (Leprince et al., 2000).Furthermore, technical improvements arebeing made to allow the analysis of lipidperoxidation products such as ethene,ethane, pentane and hexane. PA techniquesoffer two important advantages over con-ventional headspace analysis: (i) biologi-cally relevant gases can be detected with asensitivity limit of 100- to 1000-fold higherthan GC; and (ii) the time response is lessthan 1 min. The PA is set up as a flow-through system. The gas employed to drythe tissues is also the carrier of thegas/volatile to be analysed. The disadvan-tages are: (i) to our knowledge, the access toPA is restricted to a handful of laboratoriesin The Netherlands (www.sci.kun.nl/tracegasfac/), Germany, Italy and the US; (ii)the equipment is not commercially avail-able and the current prototypes require theassistance of physicists and engineers totake and process the measurements; (iii) alimited range of biologically relevant gasescan be measured; and (iv) water vapourstrongly interferes with the measurementand must be totally removed from the gas.

4.3.2. NMR applications to measure steady-state concentrations and to assess metabolicresponses to drying (see also Section 4.4.2)

To determine the effects of desiccation onthe dynamics of metabolic pathways, theflux of metabolites through the differentpaths must be known. NMR spectroscopy

appears to be the most appropriate tech-nique for this purpose (Shachar-Hill andPfeffer, 1996; Roberts, 2000). Several strate-gies can be adopted using 13C-, 31P-, 14N- or15N-NMR, depending on the nature of themetabolite to be analysed. NMR studiesmay or may not be destructive. The princi-ples of NMR spectroscopy and the advan-tages and disadvantages of in vivoapplications as a non-invasive techniquewill be described below in Section 4.4 (seealso Shachar-Hill and Pfeffer, 1996).

A first strategy is to monitor dynamicchanges of natural or enriched nuclei in thesamples over different intervals during dry-ing. This approach may or may not bedestructive. In both cases, it yields dynamicinformation about metabolic fluxes and fastresponses of metabolism to physiologicalperturbations. The natural abundance of 13Cis only 1.1%. Thus 13C-NMR is not a sensi-tive method and requires concentrations inthe mM range. However, one can takeadvantage of this low sensitivity to tracesome metabolic changes by the detection ofcompounds that accumulate to high levels.These metabolites include compatiblesolutes that accumulate in cyanobacteria(Reed et al., 1985), sugars and oil in seeds(Rutar, 1989; Ishida et al., 1990, 1996;Koizumi et al., 1995), and trehalose in fun-gal spores (Bécard et al., 1991). The non-invasive character of NMR may allow thetime-course of metabolic events to bedirectly followed and the subcellular local-ization of some metabolites to be deter-mined, for instance, in maturing orgerminating seeds (Colnago and Seidl,1983; Ishida et al., 1990, 1996).

Alternatively, since the natural abun-dance of 13C is low, it is possible to labelspecific metabolites and monitor their fatethrough the cellular network of metabolicpathways in vivo or in vitro with crudeextracts (Dieuaide-Noubhani et al., 1995;Roberts, 2000; Roscher et al., 2000).Commercially n-13C-labelled sugars areavailable for almost every carbon positionof sucrose, glucose and fructose molecules.Similarly 31P- and 14N-labelled compoundscan be used to monitor the dynamics ofphosphorylated metabolites and amino



acids, respectively. Whether in vivo NMRis applicable to drying tissues remains tobe ascertained. A particular problem is tomaintain reliably the specimen in the samefunctional state and with the same watercontent during the NMR measurements.

A second strategy, which is not mutu-ally exclusive to that above, is to screenchemical fingerprints of crude extractsobtained from different hydration levels.This application will yield analytical infor-mation about metabolites in a steady state.By comparing spectra of crude extractsobtained during drying, it should be possi-ble to pinpoint the metabolites, the concen-trations of which are mostly sensitive tochanges in water content during drying(Fan, 1996; Noteborn et al., 2000). Roughly0.5–1 g of fresh material is often requiredto take an NMR spectrum, which could bea limiting factor if the amount of biologicalmaterial is restricted.

4.3.3. Photosynthesis studies

The recent methods to assess photosyn-thetic activities have become non-destruc-tive. They exploit the interactions betweenlight, gas exchange, operation of the photo-synthetic electron transport and ambientconditions. For these reasons, they arewidely used to study the photosyntheticresponses to environmental stresses(Bukhov et al., 1989; Foyer et al., 1994).They have also been applied to compare thephotosynthetic responses of anhydrobioteswith those of desiccation-sensitive plants(Schwab and Heber, 1984; Schwab et al.,1989; see Chapter 7). The most appliedtechnique is chlorophyll a fluorescence,which assesses the efficiency of electrontransport through photosystem II and non-photochemical quenching processes associ-ated with it. Light-induced electrontransport in photosystem II can be studiedusing fluorescence induction kinetics(Vertucci and Leopold, 1986). Light-scatter-ing measurements at 535 nm are also usefulto gain insights into the physical and chem-ical events associated with the formation ofa membrane potential in thylakoids.

4.3.4. Oxidative stress and anhydrobiosis

Whether oxidative stress is a cause or aneffect of desiccation sensitivity has yet tobe resolved due to a large body of conflict-ing evidence in the literature. The core ofthe problem is two fold. From a physiologi-cal point of view, Hendry (1993) arguedthat attempts to correlate free radicalprocesses with desiccation tolerance mustbe done in relation to the characterizationof other desiccation-induced damage. Thismust be done in order to assess whetherfree radicals generated during drying are acause or an effect of the loss of viability.Echoing the earlier review of Gutteridgeand Halliwell (1990) and Leprince et al.(1990), he pointed out that free-radical-mediated injury can occur before or afterthe time of death during drying (Hendry,1993). Thus, great caution should be exer-cised in ascertaining whether oxidativestress plays a role in desiccation-inducedinjury and loss of desiccation tolerance.Oxidative injury in both animals and plantsgenerally results from stress-induced meta-bolic disturbances, particularly in the elec-tron transport chains. Considering thetechnical difficulties in estimating meta-bolic activities during drying (see Section4.3, p. 114), even greater care should thenbe taken in attempts to link oxidative dam-age to desiccation-induced metabolic per-turbation. Various pathologies anddegenerative diseases have been linked tomitochondrial dysfunction and generationof reactive O2 species (ROS) in mammals(Yates and van Houten, 1997; Esposito etal., 1999; Wallace, 1999). Thus, it shouldbe interesting to see whether parallels existbetween animal and plant anhydrobiotes.

The second problem regarding the puta-tive role of oxidative stress in desiccationtolerance and ageing is the methodologythat has been employed so far. A survey ofmethods employed to detect oxidative stressin seeds can be found in Benson (1990).

1. The array of techniques that have beenemployed to assess the role of free-radical-induced injury in anhydrobiotes is verysmall. A survey of the literature on molecu-



lar markers used to estimate free-radical-induced damage during drying or in thedry state shows that the results are basedmostly on a thiobarbituric acid-reactivesubstances (TBARS) assay and a non-inva-sive EPR spectroscopy technique (Table4.1). The former measures malonyldialde-hyde (MDA) as a breakdown product oflipid peroxidation using a simple andrapid assay (Heath and Packer, 1968) andthe latter estimates a carbon free radical ofunknown origin (Hendry, 1993). As dis-cussed below, doubts have been cast as towhether these two assays are reliableenough for all anhydrobiotic material. 2. Table 4.1 also shows that, in 98% ofthese studies, lipid peroxidation was mea-sured as a marker of free-radical-inducedinjury. It must be recognized that proteins(Dean et al., 1993; Berlet and Stadtman,1997) and DNA (von Sonntag andSchuchmann, 1987; Hageman et al., 1992;Wiseman and Halliwell, 1996) are also sen-sitive to free radicals, albeit less than fattyacids. Interestingly, mitochondrial DNA ismore sensitive to ROS than nuclear DNA.Free-radical-induced DNA damage can sig-nificantly contribute to mitochondrial dys-

function and cell death (Yates and vanHouten, 1997). Oxidation of proteins byROS can induce protein fragmentation orenzyme inactivation, leading to the disrup-tion of glycolysis (Hyslop et al., 1988) andthe Calvin cycle (Kaiser, 1979). Protein oxi-dation has been linked to various patholo-gies in humans (Dean et al., 1993; Berletand Stadtman, 1997) and to seed ageing(Zhang et al., 1997). 3. Analytical procedures to estimate lipidhydroperoxides in crude extracts arefraught with potential artefacts (Gutteridgeand Halliwell, 1990; Hageman et al., 1992;Meagher and Fitzgerald, 2000). The prob-lems are numerous, ranging from the pres-ence of contaminants (metal ions) thatinitiate lipid peroxidation during tissuegrinding to instability of peroxidized lipidsduring the extraction and lack of sensitiv-ity. Furthermore, the nature of peroxidativedamage depends on the type of free-radicalinitiator and the membrane or lipid compo-sition (McKersie et al., 1990). Thus, thelipid peroxidation assays currently used inseed science cannot be indiscriminatelyapplied to all seeds. Unfortunately, thestudies surveyed in Table 4.1 did not


Table 4.1. Occurrence and types of methods employed to determine free-radical damage in drying and/or ageing seeds, pollen and vegetativetissues. Examples of free-radical damage specific to DNA are oxidizednucleotides such as thymine glycol, 8-hydroxy-2�-deoxyguanine andmethylguanine (Hageman et al., 1992). Examples of free-radical damage toprotein are iminopeptides, carbonyl content and glutamyl-semialdehyderesidues of oxidatively modified proteins (Berlet and Stadtman, 1997).

Number of studies over the past three

Methods decades

MDA or TBARS determinationa 17Determination of an organic free radical by

in vivo EPR spectroscopy 11Free fatty acid determination 6Chemical modifications of lipids (e.g. conjugated

dienes, fatty acid composition) 5Other techniques (determination of breakdown

products resulting from lipid peroxidation, chemiluminescence, fluorescent probes) 9

Free-radical damage specific to DNA 0Free-radical damage specific to protein 2

aMDA, malonyldialdehyde; TBARS, thiobarbituric acid-reactive substances.


assess whether the marker used as an indexof oxidative injury was sensitive or appro-priate for the biological material or experi-mental conditions. For example, in oilyseeds, lipid peroxidation is dependent onthe triacylglycerol composition and struc-ture of oil reserves (Neff et al., 1992).Furthermore, in an assay of peroxidizedlipids of a crude extract obtained from oilyseeds, the triacylglycerol fraction maymask more important and significantchanges occurring in membrane lipids.Another example further illustrates thepoint. Table 4.2 compares two methodsestimating the level of peroxidized lipidsin germinating pea axes in relation to theloss of desiccation tolerance. Using theassay developed by Jiang et al. (1991),results suggest that there is a link betweenan increase in oxidative damage followingdrying and the loss of desiccation toler-ance. In contrast, the TBARS assay pro-vides inconclusive evidence. 4. The limitations of the TBARS test havebeen known for several years (Gutteridgeand Halliwell, 1990; Hodges et al., 1999)and include a lack of sensitivity and speci-ficity and a tendency to overestimate MDAcontents. Recently, it has been shown thatseveral compounds commonly found inplant extracts (e.g. sugars, oligosaccharides,anthocyanins) also react with thiobarbi-turic acid, thereby interfering strongly withthe peroxidized products (Table 4.3). Non-reducing sugars and oligosaccharides are

known to accumulate in desiccation-toler-ant systems. Anthocyanins accumulate tolarge concentrations in leaves of resurrec-tion plants. Hodges et al. (1999) have intro-duced several modifications to overcomethis interference in leaf extracts. However,the suggested improvements did notappear to be reliable for the results shownin Table 4.2.5. An organic stable free radical has beenlinked to respiration, oxidative stress anddesiccation tolerance (Hendry et al., 1992;Hendry, 1993; Leprince et al., 1995).However, measurements of this organicradical using non-invasive EPR have gener-ated conflicting evidence concerning itslink to desiccation tolerance (Hendry,1993). Its chemical nature and localizationshould be identified to ascertain whetherthis is a reliable method to estimate oxida-tive stress in seeds. The fact that the EPRsignal is sensitive to liquid water makes itdifficult to use in drying tissues. Studyingfrozen specimens could lessen the problembut this approach will not be able to tellwhether the radical is generated by dryingor by freezing.

From these remarks, we conclude thatpositive results from a lipid peroxidationassay will provide evidence that a free-radi-cal reaction has occurred either duringdrying or during drying and extraction. Anegative result will not provide any evi-dence one way or the other. Therefore, it issuggested that a range of assays should be


Table 4.2. Comparison of two methods measuring lipid peroxidation as a marker of oxidative damage incrude extracts of germinating axes of pea before and after fast drying. TBARS were measured as inLeprince et al. (1990) and calculated as in Du and Bramlage (1992) to take into account interference bysugars. Lipid hydroperoxide levels were measured using the xylenol orange/ammonium sulphate reagentaccording to the method of Jiang et al. (1991) and quantified using H2O2 as a standard. Data are theaverage (± SE) of 3–5 replicates (O. Leprince, J. Fajerman and F.A. Hoekstra, unpublished observations).

TBARSa Lipid peroxideSensitivity to drying Treatment (�mol mg�1 dw) (�mol equiv. H2O2 mg�1 dw)

Tolerant Fresh 2.76 ± 1.93 15 ± 3Dried 9.89 ± 1.79 16 ± 2

Intolerant Fresh 33.05 ± 15.01 22 ± 2Dried 27.12 ± 9.00 71 ± 15

aTBARS, thiobarbituric acid-reactive substances.dw, dry weight.


tested as a necessary prerequisite toaddress the role of free-radical processesin desiccation tolerance and seedlongevity. To be validated, these assaysshould be carried out on material that hasbeen treated with agents to generate oxida-tive stress such as ultraviolet (UV) radia-tion and free-radical generators such asparaquat or H2O2.

An increasing number of methodologiesare currently being developed to analysefree-radical-induced damage in vitro and invivo. Most of these methods are derivedfrom studies on animals, humans or food-stuffs. The challenge will be to adapt thesetechniques to seed science and anhydrobi-ology, keeping in mind the caveatsdescribed in this chapter and the guide-lines provided by studies aimed at unravel-ling the free-radical chemistry occurring inliving tissues. Among emerging methodolo-gies, PA (see above), the use of specific flu-orescent probes (LeBel et al., 1992) andfluorescence spectroscopy, spin-trappingtechniques and non-invasive EPR spec-troscopy (see Section 4.4) and new spec-troscopy assays (Jiang et al., 1991; LeBel etal., 1999; Junqua et al., 2000) warrant fur-ther investigation.

4.4. Spectroscopy Techniques

As follows from a consideration of quan-tum mechanics, an atom or molecule hasdiscrete energy states. Spectroscopy is themeasurement of the energy differencesbetween these states. The energy differ-ences �E can be measured by the absorp-tion spectra of electromagnetic radiation.In conventional spectroscopy, the fre-quency is varied and the frequency atwhich maximal absorption occurs reflectsthe difference between the states. The fre-quencies vary from the MHz range for NMRto the GHz (microwave) range for EPRspectroscopy. The frequencies for absorp-tion spectroscopy range from 1012 Hz for IRto 1016 Hz for UV light. The frequencies ofX-rays and �-irradiation are 1019 Hz and1021 Hz, respectively.

4.4.1. Electron paramagnetic resonance (EPR)(see also Chapter 2)

4.4.1.1. General description

The energy differences that are studiedwith EPR spectroscopy are the result of theinteraction of unpaired electrons with a


Table 4.3. Interference of various compounds in the thiobarbituric acid-reactivesubstances (TBARS) assay in crude extracts of plant and seed tissues. The TBARSassay was performed using the procedure of Heath and Packer (1968) in thepresence of various amounts of sugars and anthocyanins. TBARS concentrationswere calculated from the difference between absorbance values at 532 and 600 nmand expressed as a relative increase compared to extracts without interferingcompounds.

Absorbance peak of the interfering Relative

Compound compound (nm) increase (%)

Apple peel extract +a

2.5 mM sucrose 440 + 9%1 mM fructose 440 + 5%

Cabbage leaves extract +a

anthocyanins 540 + 272%Germinating pea axes +b

0.3% (w:v) raffinose 441 + 35%1% (w:v) raffinose + 79%

aData derived from Du and Bramlage (1992) and Hodges et al. (1999).bUnpublished data of O. Leprince, J. Fajerman and F.A. Hoekstra.


static magnetic field (Zeeman splitting). InEPR spectroscopy the electromagnetic fre-quency is kept constant and the magneticfield is scanned (due to limitations ofmicrowave electronics). There are four fre-quencies available in EPR spectrometers:1.1 GHz (L-band), 3.0 GHz (S-band), 9.5GHz (X-band) and 35 GHz (Q-band).Among them, the X-band is the most com-monly used. The interaction betweennuclei and the electron (hyperfine interac-tions) causes the hyperfine splitting of theEPR spectrum. The spectral shape can giveinformation about the sample under study.

Only systems that contain non-pairedelectrons will give an EPR signal. Pairinggives zero net electron magnetic moment.Since a paired spin system is energeticallyfavourable, chemical bonding normallyresults in molecules that have no unpairedelectrons and, hence, no EPR signal occurs.Exceptions to this rule are transition-metalions, free radicals and free electron centressuch as those produced by high-energyirradiation of macromolecules. Free radi-cals produced in biological systems usuallycannot be detected by EPR because of theirshort half-life times, resulting in lowsteady-state concentrations.

The introduction of spin-label/probemethods has considerably increased thepossibilities for the application of EPR inbiological systems. The spin-label groupthat is almost exclusively used is thenitroxide moiety synthesized by Rozantzev(1970) and introduced by McConnell(McConnell and McFarland, 1970).

There is a limitation to the hydrated sam-ple size that can be used for spectra record-ing because of dielectric losses caused bywater. However, the high sensitivity of themethod allows the use of very small samplesizes of less than 1 mg. The relative simplic-ity of sample preparation and spectrarecording enables the operator to handlelarge numbers of samples per day.

4.4.1.2. Applications of EPR methods

GENERAL REMARKS. The great variety of spinprobes allows a multiplicity of informationabout biological systems to be obtained.

Sample preparation is relatively easy. Thesample is incubated in an aqueous solutionof spin-probe or spin-labelled moleculesfor a short time and then transferred intothe resonator of an EPR spectrometer forspectra recording. Usually, neutral spin-probe molecules can readily pass throughmembranes and partition within cells overthe polar and apolar phases according totheir partition coefficients. The EPR signalof the spin-probe molecules outside cellscan be eliminated by broadening agents viaspin–spin interaction. Paramagnetic metalion complexes such as chromium oxalateor ferricyanide are often used as broaden-ing agents. Thus, the EPR spectrum of spinprobes in the sample in the presence ofbroadening agents is exclusively derivedfrom the inside of cells. Different aspects ofdesiccation tolerance can be studied by theanalysis of EPR spectra of spin probesintroduced into the cells.

Spin labels are stable free radicals. Theunpaired electron belongs to the nitroxidegroup, which is flanked by quaternary car-bon atoms of methyl groups, protectingthe radical from recombination andaccounting for the high stability of thelabel. The EPR spectra of nitroxide spinlabels have a three-line nitrogen hyperfinestructure and are environmentally sensi-tive. The variety of spin probes of differ-ent properties and the possibility of theattachment of nitroxides to biologicalmolecules of interest have created exten-sive applications of this method in biol-ogy (Marsh, 1981; Morse, 1985).

A problem that often arises in experi-ments with biological samples is chemicalreduction of spin labels and spin probes.Despite the high stability in aqueous andother media, nitroxides are susceptible to(reversible) reduction by some biologicalmetabolites (such as ascorbic acid and thi-ols), electron transport chains and otherredox systems, resulting in the disappear-ance of paramagnetism. Ferricyanide isusually effective at limiting the rate ofreduction or reoxidizing reduced label(Kaplan et al., 1973). Oxygenation, aerationor the use of specific inhibitors can also beused to protect nitroxides against reduction



or to restore them from the reducedhydroxylamines (Marsh, 1981).

Another problem with the application ofthe spin-label technique is the disturbancethat might be caused by the guest moleculesthemselves (e.g. membrane spin labels).This is inherent in all methods usingreporter groups, in contrast to spectroscopicmethods that do not use guest molecules(e.g. NMR, FTIR). Because of the high sensi-tivity of the spin-label method, very lowconcentrations of label can be used, whichminimizes possible disturbance. However,the possibility of obtaining informationabout a specific environment of interestmakes the EPR method attractive in thestudy of desiccation tolerance. In contrast,NMR and FTIR give information that isaveraged along the sample, which can becomplementary to the information fromreporter group methods.

CELLULAR VIABILITY BASED ON MEMBRANE INTEGRITY.The principle of the EPR spin-probemethod for the estimation of the relativeamount of viable cells is based on the factthat membranes of viable cells are imper-meable to some broadening agents,whereas the membranes of damaged cellsare not (Keith and Snipes, 1974). Thus, theEPR signal of spin-probe molecules insidecells with disrupted membranes isquenched by a broadening agent, and thetotal amplitude of the EPR signal from thesample will correlate with the amount ofviable cells in a sample (Dobrucki et al.,1990). Because of the high sensitivity of themethod, it is possible to determine smallamounts of viable cells in mostly dead tis-sue. This approach has been applied in thestudy of desiccation tolerance acquisitionof proembryonic cells in wheat kernels,which were slowly dried on the ear at anearly stage when proembryos could not bedetected morphologically (Fig. 4.1)(Golovina et al., 2001). Such an approachallows the developmental death of wheatendosperm cells during kernel develop-ment (Golovina et al., 2000) and theprogress of cell death after cold (Fig. 4.2) orimbibitional stress in neem seeds to be fol-lowed (Sacandé et al., 2001).

Many of the anhydrobiotic systems con-tain oil bodies as storage material. In thiscase, amphiphilic spin-probe moleculeswill partition into lipid bodies as well, andEPR spectra are composed of two compo-nents originating from spin-probe mole-cules in aqueous (cytoplasm) andhydrophobic (oil) environments. Thesespectra differ in the distance between lines(the isotropic splitting constant aiso isaround 16 G for an aqueous environmentand around 14 G for a lipid environment)and in the position of the central line


4 daa wheat kernel

Deadtissue

Difference (viable cells)

(a)

(b)Oil

Cytoplasm

Fig. 4.1. (a) The electron paramagnetic resonance(EPR) spectrum of 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPONE) in developing wheatkernel that was harvested at 4 days after anthesis (4daa) and dried on the ears. The thick line representsthe broad component of the spectrum andoriginates from TEMPONE in dead tissue; the thinline represents the total spectrum. (b) Spectrumshowing the difference between the total spectrumand broad component, representing TEMPONElocated in viable cells. Peaks originating fromTEMPONE in the aqueous cytoplasm of viable cellsand from oil bodies are indicated. Total scan widthis 100 gauss. Spectra are reproduced from Golovinaet al. (2001).


(g-factor), and can be resolved in the highfield (right-side) part of the spectrum(Marsh, 1981). In the case of loss of mem-brane integrity, ferricyanide ions that havepenetrated the cell only broaden the signalof TEMPONE (4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy) molecules localized in theaqueous cytoplasm. The signal of TEM-PONE from oil bodies remains unbroad-ened, because ferricyanide cannot partitioninto the lipid phase. The intensity of thishydrophobic signal can then be used as ameasure of the total amount of cells in thesample, whereas the intensity of the polarcytoplasmic component represents theamount of cells with intact membranes(Golovina et al., 1997a,b). The ratio Rbetween the heights of the aqueous cyto-

plasmic (hcyt) and oil peaks (hoil) can beused for the quantitative assessment of theproportion of viable cells in a sample(Golovina and Tikhonov, 1994; Golovina etal., 1997a,b; Leprince et al., 1999).

PLASMA MEMBRANE PERMEABILITY. Changes inplasma membrane permeability can be esti-mated by using the water-soluble nitroxideradicals in the presence of a broadeningagent (Miller and Barran, 1977; Golovina etal., 1998; Hoekstra et al., 1999). Themethod is based on the presence of tempo-rary defects in membranes that allow ferri-cyanide ions to penetrate into the cell andbroaden the signal arising from spin probeslocated in the cytoplasm. The line-heightratio of the lipid peak to the water peak(L/W) will correlate with the number of fer-ricyanide ions that have penetrated the cellthrough the plasma membrane and can beused to characterize plasma membranepermeability (Fig. 4.3).

CELL VOLUME AND OSMOTIC EFFECT. The heightratio of cytoplasmic to lipid peaks in EPRspectra can be used to determine cell vol-ume changes under osmotic stress. Thisfollows from the fact that spin-probe mol-ecules are equally distributed inside andoutside the cells. The total cellular vol-ume that is not accessible to broadeningagents will determine the amount of spinprobe that is separated from the broaden-ing agent and, hence, the line-height ofthe cytoplasmic component in the EPRspectrum. This total volume is the prod-uct of the number and volume of viablecells. Cell division and enlargement ofcells during imbibition and germination(Golovina et al., 2001) and osmoticallyinduced changes in cell volume (Miller,1978) can cause changes in the line-height of the cytoplasmic component.The ratio between the line-heights ofcytoplasmic and lipid peaks can be usedto quantify the osmotic effect. However,when the amount of oil changes duringseed development (Golovina et al., 2001)or during germination (Sacandé et al.,2001), the height of the cytoplasmic linecan be used instead.


Axis ofneem

(a)

(b)

(c)

Oil

Cytoplasm

Control

7 days

28 days

Fig. 4.2. Electron paramagnetic resonance (EPR)study of chilling damage of neem (Azadirachtaindica) seeds using 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPONE) as a spin probe.Spectrum (a), mature, fresh axes used as control;spectra (b) and (c), whole embryos after,respectively, 7 and 28 days of storage under humidconditions at 5°C. Spectra are plotted in the samescale to allow comparison. The oil and cytoplasmicpeaks are indicated in the high-field region (rightside) of spectrum (a). Total scan width is 100 gauss.Spectra are reproduced from Sacandé et al. (2001).


CYTOPLASMIC VISCOSITY. Because the shape ofEPR spectra of spin probes is sensitive tomolecular motion, cytoplasmic viscositycan be studied with spin-probe techniques(Keith and Snipes, 1974). To characterizethe shape of the spectrum originating fromthe cytoplasmic location of the spin probe,other components (lipid, starch-like) of thespectrum have to be subtracted (Golovinaet al., 2000, 2001). Alternatively, chargedspin probes that do not partition into thelipid phase can be applied (Buitink et al.,

1998). Line-height differences arising fromdifferentially broadened lines due toslowed motion of spin-probe molecules(Fig. 4.4, spectra a and b) are used to esti-mate viscosity (Keith and Snipes, 1974).The line-height ratio between lines can beconverted to viscosity. Based on such anapproach, the changes in cytoplasmic vis-cosity with drying of desiccation-tolerantand sensitive samples (Leprince et al.,1999) and with the acquisition of desicca-tion tolerance during seed development(Golovina et al., 2001) have been estab-lished.

When spin-probe motion slows further,not only is a progressive increase in differ-ential broadening observed, but also a dis-tortion of the line shape (Fig. 4.4, spectrumc). Rigidly immobilized, randomly orientedradicals give a powder spectrum (Marsh,1981), which can be used to characterizebiological glasses (Buitink et al., 1998, 1999,2000b,c,d,f). In this case, the viscosity mustbe estimated using saturation transfer EPRor pulsed EPR methods (see below).

PARTITIONING OF AMPHIPHILES INTO THE LIPID PHASE

WITH DRYING. The shape of spin-probe spec-tra depends on properties of the environ-ment; therefore amphiphilic spin probescan be used to follow their partitioningwith drying (Golovina et al., 1998; Buitinket al., 2000e; Hoekstra and Golovina, 2000;Golovina and Hoekstra, 2002). The samplesare preloaded with spin probes and allowedto dry. The EPR spectra recorded from thesamples at different moisture content willbe composed of spectra originating fromspin-probe molecules at different locations(Fig. 4.5). They can be decomposed, and therelative proportion of spin probes at the dif-ferent locations can be estimated (Hoekstraand Golovina, 2000; Golovina andHoekstra, 2001).

PHYSICAL PROPERTIES OF MEMBRANES. The small,water-soluble spin probe TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or spin-labelled fatty acids, steroids orphospholipids are used to study the physi-cal properties of membranes (for refer-ences, see Berliner, 1976; Marsh, 1981;


Typha latifolia

(a)

(b)

(c)

(d)L

W

0 s

7 s

30 s

60 s

Fig. 4.3. Electron paramagnetic resonance (EPR)study of imbibitional leakage of Typha latifoliapollen using 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPONE) as a spin probe. Pollenwas either directly incubated in a solution ofTEMPONE/ferricyanide (spectrum a) or after aprevious rehydration in liquid germination mediumfor 7 s (spectrum b), 30 s (spectrum c) and 60 s(spectrum d). All the spectra exhibit contributionsfrom the aqueous cytoplasm (W) and from lipid (L)(oil bodies) environments, which are resolved in thehigh-field region (right side). The spectra werenormalized to the height of the lipid (L) peak. Theratio W/L was taken as a measure of plasmamembrane permeability (see explanation in the text,Section 4.4.1). Total scan width is 80 gauss. Spectraare based on data from Hoekstra et al. (1999).


Morse, 1985). Isolated membranes can belabelled with TEMPO, which partitionsbetween water and the membranes. Thepartitioning depends on membrane fluidityand aqueous-phase viscosity and can beused for the determination of the mem-brane phase transition. However, the possi-ble partitioning of TEMPO into oil bodiescomplicates the in vivo membrane investi-gations. The use of spin-labelled fatty acids

provides valuable information about mem-brane dynamics, because the stable free-radical doxyl group can be placed atdifferent positions along the acyl chain.Thus information can be obtained from dif-ferent depths in membranes, from the sur-face to the core. Labelling model membranesposes no problem. The spin label is mixed at1 mol % with the lipids in organic solvent,which is subsequently removed by evapora-tion. The anhydrous mixture is then dis-persed in the appropriate amounts ofaqueous phase. Labelling isolated biological


Root

Axis

Sucroseglass

2Amax

(a)

(b)

(c)

h0 h–1

h0/h–1= 1.19

h0/h–1= 1.75

Fig. 4.4. Electron paramagnetic resonance (EPR)spectra of 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPONE) in the cytoplasm ofwheat seedling root (spectrum a) and hydratedwheat axis (spectrum b). The ratio of the height ofthe central line (h0) to the height of the high-fieldline (h�1) reflects cytoplasmic viscosity. Thecytoplasmic viscosity is less in seedling root(h0/h�1= 1.19) than that in hydrated wheat axis(h0/h�1= 1.75). The EPR spectrum of TEMPONE inair-dried sucrose glass (spectrum c) is typical forrigidly immobilized, randomly oriented spin-probemolecules. The distance between the outer extremes2Amax (in gauss) can be used to characterize theslow motion of spin-probe molecules. Total scanwidth is 100 gauss. Spectra are based on data fromGolovina and Hoekstra (2001).

(a)

(b)

(c)

(d)

Cytoplasm

Oil bodies

Membranesurface

Combined

a:b:c = 5:10:85

Fig. 4.5. Typical electron paramagnetic resonance(EPR) spectra of 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPONE) in aqueous cytoplasm(spectrum a), oil bodies (spectrum b), and of spin-probe molecules immobilized at the membranesurface (spectrum c). Spectrum (d) is the sum ofspectra (a), (b) and (c). Spectra were combined insuch a proportion that the relative amounts of spinprobe in spectra (a), (b) and (c) were 5%, 10% and85%, respectively. Spectrum (d) simulates the latestage of TEMPONE partitioning during drying. Totalscan width is 100 gauss. Spectra are based on datafrom Golovina and Hoekstra (2001).


membranes poses more problems. Spin-labelled fatty acids have first to be dis-solved in ethanol and then added to themembranes in such a quantity that the finalconcentration of ethanol does not exceed 1mol %. Other ways of membrane labellinghave been reviewed by Marsh (1981).Labelling membranes with doxyl stearatesin vivo following the same approach can beused but is more tricky than with isolatedmembranes. A small number of in vivo stud-ies have been published. Among them onlya few investigations have been conductedon desiccation-tolerant systems (Golovinaand Tikhonov, 1994; Vishnyakova et al.,2000; Golovina and Hoekstra, 2001).

ADDITIONAL EPR TECHNIQUES APPLIED TO DESICCATION-TOLERANT SYSTEMS. When conventional EPRspectroscopy as described above yields therigid-limit powder line shape (see Fig. 4.4,spectrum c), it is insensitive to the rate ofmolecular motion. The following two EPRmethods have been designed to overcomethis problem and adapted to anhydrobioticmaterial. They are particularly suitable tocharacterize a glassy state.

Saturation-transfer EPR. Saturation-transferEPR (ST-EPR) allows the measurement ofvery slow molecular motion with rotationalcorrelation times between 10�7 and 10�3

s. For comparison, conventional continuouswave (CW) EPR enables rotational correla-tion times below 10�7 s to be resolved. Inconventional EPR, motion averaging of thespectral anisotropy occurs within the timeof spin–spin relaxation T2. In ST-EPR, thisaveraging occurs within the time ofspin–lattice relaxation T1, which is 300times longer than T2 for slow-movingnitroxide molecules (Marsh, 1981). Thus,ST-EPR extends the motional sensitivity ofthe spin-label technique to one that moni-tors a 300-fold slower motion than withconventional EPR. ST-EPR spectra ofnitroxide spin probes can be analysed byindependent line shape parameters. Using areference material of known viscosity, themolecular rotation can be calculated in anempirical way (Hemminga, 1983). ST-EPRhas been used to study the motion of pro-

teins and lipids in biological membranes(for references, see Marsh, 1981; Hemminga,1983) and glasses (Roozen et al., 1991). Themethod has been successfully applied inthe study of biological glasses in anhydrobi-otic systems (Buitink et al., 1998, 1999;2000b,c,d,f). This approach of measuringslow rotational motion has given stunninginsight into the differences between biologi-cal glasses and sugar or polymer glasses.For example, a remarkable observation orig-inating from the ST-EPR measurements wasthe occurrence of a second kinetic changein mobility at a definite temperature abovethe glass transition temperature (Buitink etal., 2000f), which may have physiologicalrelevance for survival in the dry state (Fig.10.3 in Chapter 10).

Pulsed EPR. CW-EPR often cannot givetrue values for the relaxation times of thespin label, because of the inhomogeneousbroadening of the lines. However, pulsedEPR (electron spin echo technique) pro-vides a direct method for the measurementof relaxation times that give insight intothe molecular dynamics of spin probes(Morse, 1985). This method has been usedto identify the glassy state in wheat seeds(Dzuba et al., 1993) and to characterize themotion of guest molecules in biologicalglasses of different moisture content(Buitink et al., 2000a).

EPR imaging (EPRI). The potential of usingboth NMR and EPR imaging was suggestedby Lauterbur (1973). However, while NMRimaging (NMRI) has progressed into clinicalusage, the application of EPRI is restricted,particularly in biological systems. This iscaused by the severe dielectric losses andconsequent heating that occurs in aqueoussamples at conventional EPR frequencies (X-band). The dimension of the hydrated sam-ple that can be used for imaging is limited toa few millimeters. This problem can bepartly overcome by using low-frequency EPR(L-band) and a surface coil (Berliner andFujii, 1985). EPRI is used for visualizingparamagnetic centres in a sample. There areseveral biological applications in whichEPRI has a significant advantage over NMRI:the spatial distribution of O2 and redox



metabolism, mapping viable and non-viablecells, the diffusion of paramagneticallylabelled solutes, and mapping native free radi-cals that are stable or trapped by incorporatedspin traps at the sites of transient radical pro-duction (Berliner and Fujii, 1986; Bacic et al.,1989; Dobrucki et al., 1990). In spite of thepotential advantage of EPRI, there are only afew cases in which the method has beenapplied to desiccation-tolerant systems. Thepathways of bulk water penetration into wheatkernels during imbibition have been studiedusing the nitroxide radical TEMPOL (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy)(Smirnov et al., 1988; Golovina et al., 1991).To avoid the problem of dielectric losses, thekernels were placed in liquid nitrogen.Using perdeuterated 15N nitroxides as imag-ing substances, tens of micrometre resolu-tion can be achieved. Such resolution wassufficient to obtain EPRI of viable cells inhydrated lettuce seeds in the presence of fer-ricyanide (Walczak et al., 1987). The imageenabled contrast between embryo and stor-age tissue to be observed. The EPRI of thepenetration and distribution of natural spinprobes (humic substances) in wheat kernelshas also been demonstrated (Smirnov et al.,1991).

POTENTIAL APPROACHES FOR STUDYING ANHYDRO-BIOSIS. Additional EPR methods have beendesigned to study several biochemical andbiophysical aspects of biology. However,these methods have not yet been appliedspecifically to seeds or other types of anhy-drobiotic tissues.

Intracellular O2 concentration. IntracellularO2 affects the shape of EPR spectra of spinprobes because, as a paramagnetic mole-cule, it causes line broadening. Broadeningis proportional to O2 concentration, therebyallowing the intracellular concentration tobe calculated (Swartz, 1987). However, theapplication of this method may not bestraightforward for drying biological mater-ial because the rise of viscosity also intro-duces changes in line broadening. Anotherapproach is to use the effects of O2 on themicrowave power saturation: the presenceof O2 diminishes power saturation, and

this effect is proportional to the O2 concen-tration (Swartz, 1987).

pH measurements. Spin-labelled amineand carboxylic acids have been used todetermine the pH in vesicles and cells(Mehlhorn et al., 1982). The method isbased on the differential membrane perme-ability for charged and neutral forms ofthese spin probes. Because the equilibriumbetween charged and neutral amines andacids depends on pH, the intracellularEPR signals of these spin probes can beused to calculate the intracellular pH.Unfortunately, such an approach can onlybe applied in the presence of a solution ofa broadening agent. To study the changesin pH in a sample during drying, specificpH-sensitive spin probes can be applied.The reversible effect of pH on EPR spectrais associated with proton exchange in theradicals. Protonated and non-protonatedforms have different EPR parameters. Theprotonable group in the radical structurehas to be close to the unpaired electron.Iminonitroxides are the most promising inthis respect (Khramtsov and Weiner, 1988).

Spin trapping. The free radicals that areproduced in anhydrobiotic organisms dur-ing water loss (Section 4.3.4) cannot bedetected by EPR because of their short half-life resulting in low steady-state concentra-tion, or the short relaxation times leading tovery broad lines. These radicals can betrapped specifically by spin traps anddetected by EPR in organic extracts or invivo (Knecht and Mason, 1993). Four differ-ent traps are commonly used in biologicalsystems: 2-nitrosopropane (MNP), phenyl-N-tert-butylnitrone (PBN), �-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), and5,5-dimethyl-1-pyrroline-N-oxide (DMPO).The primary free radicals interact with thedouble bond of diamagnetic spin-trap mole-cules and form radical adducts that aremuch more stable than the primary freeradicals. The radical adducts of these spintraps are nitroxide radicals. The primaryfree radical can be identified either from thespectra of radical adducts, or after purifica-tion of radical adducts and further identifi-cation by mass spectroscopy. Various



shortcomings complicate the application ofspin trapping in vivo in drying organisms:oxidation of the spin traps and reduction ofthe radical adducts, and amphiphilicbehaviour which may relocate spin trapsinto the membranes (Knecht and Mason,1993).

4.4.2. Nuclear magnetic resonance(see also Chapter 2)

4.4.2.1. General description

Analogous to EPR spectroscopy, NMR spec-troscopy is based on the resonance absorp-tion of electromagnetic radiation by thesystem during the transition between twodiscrete energy states. The energy differ-ences studied in NMR spectroscopy are dueto the interaction of nuclear magneticmoments with the magnetic field (Zeemansplitting for nuclei). The energy differencesare smaller than those in EPR because ofthe smaller magnetic moment of nuclei.This explains why electromagnetic radia-tion in the radiofrequency range is requiredto excite the transitions that produce theNMR signal, whereas that in the microwaverange is used in EPR spectroscopy.

Because the energy differences in NMRare small, the differences in number ofnuclei at different energy levels are alsosmall. As a consequence, the signalstrength is weak, which makes NMR aninherently insensitive technique. Onlythose nuclei that have a non-zero spinquantum number resulting in non-zeromagnetic moment can be used. The split-ting between energy levels depends on thestrength of the magnetic field and the mag-netogyric ratio of the nucleus. The highestmagnetogyric ratio and the almost 100%natural abundance make proton (1H) NMRthe most sensitive. The reasonably highmagnetogyric ratio and 100% natural abun-dance of 31P nuclei give moderately goodreceptivity for in vivo phosphorus NMR. Incontrast, the 13C nucleus has very lowreceptivity because of its low natural abun-dance, but, as a label, this isotope could beuseful (Schneider, 1997; Roberts, 2000).

NMR signals can be characterized byintensity, frequency, line shape and relax-ation times. All these characteristics areaffected by the physical and chemical envi-ronment of the magnetic nucleus and canbe used to obtain information of biologicalinterest such as the state of water, intracel-lular pH and membrane dynamics. Signalintensity is related to the number of mole-cules that produce the signal. In relaxationexperiments, the intensity depends on thetime of signal registration and on the rateof magnetization decay. For quantitativeestimation of peaks in NMR spectra, inte-gration of the lines should be used becauseof the different relaxation times of the sig-nals. The limits of integration are deter-mined by the signal-to-noise ratio of thesignal and the overlapping with other sig-nals in a spectrum.

Local fields originating from the localelectron density modify the external fieldimposed on magnetic nuclei. As a result, theresonance frequency of a nucleus dependson its chemical environment, which iscalled chemical shift. Magnetization relaxesexponentially, and the faster the decay, thebroader the line in the spectrum. Broadlines have lower amplitudes and overlapwith other lines, which leads to poorlyresolved spectra. In living systems, the vari-ations of magnetic susceptibility across thesample cause line broadening, which makesit difficult to record high-resolution spectrafrom dense heterogeneous tissue, such asseeds, and from tissues containing air-spaces (leaves and roots).

The T1 (spin–lattice or longitudinal) andT2 (spin–spin or transverse) relaxationtimes characterize the magnetization decaybecause of the interaction of the nuclearmagnetic moments with the environment(T1) and with each other (T2). Relaxationtimes are mostly determined by themotional properties of the nucleus.Measurements of relaxation are particu-larly important in NMR studies of tissuewater when information about the exis-tence of different water fractions in the tis-sue is required. In practice, themeasurements are easier to conduct than tointerpret (Ratcliffe, 1994).



In basic NMR experiments, the sample isplaced in a magnetic field, and the NMR sig-nal is generated by irradiation of the samplewith a radio-frequency field, given as pulsesof different sequences. A single pulse cre-ates a net magnetization, which is regis-tered. The magnetization decays to zero,and the time-dependence of the decay (freeinduction decay) is recorded. In low-fieldstudies, this decay is analysed directly. Inhigh-field NMR, the decay is converted intospectra by Fourier transformation. The NMRspectrum is the plot of intensity against fre-quency of the radio-frequency field. AllNMR applications developed for studyingliving systems can be divided into fourgroups: (i) detection of water signal; (ii)NMR imaging; (iii) high-resolution multinu-clear NMR spectroscopy; and (iv) solid-stateNMR spectroscopy (Ratcliffe, 1994).

To exploit the advantage of a non-inva-sive technique, NMR experiments need tominimize the physiological perturbationand maintain the tissue in a physiologi-cally controllable state. In this respect, thewhole plant, cell suspensions and intactseeds are the easiest tissues, and excisedtissues the most demanding (Ratcliffe,1994). Often, it is necessary to submerge asample in water to avoid differences inmagnetic susceptibility between air andcellular material, a practice that is incom-patible with drying organisms. Proper O2supply and illumination have to be main-tained, especially in densely packed sam-ples. In solid-state NMR, when magic anglespinning is applied, it is impossible to con-trol the physiological state because of theextreme conditions (more than 1000 rota-tions per minute) imposed on the sample.

4.4.2.2. The NMR study of water in livingsystems

GENERAL REMARKS. The study of water inanhydrobiotes is of particular interestbecause with drying and rehydration bothwater content and water properties change.NMR is a powerful tool to study water invivo. There is no problem with the sensitiv-ity of detecting the water signal in biologi-

cal systems because of the generally highwater content, the high natural abundanceand the high magnetogyric ratio of 1H. Thisallows the use of low-field NMR instead ofexpensive high-field NMR magnets.

MEASUREMENTS OF WATER CONTENT. 1H low-fieldNMR allows the non-destructive measure-ment of the water content in biological sys-tems with high precision. There are twotypes of analytical NMR commonly used inthis respect – continuous wave (CW) NMR(wide-line) (Pohle and Gregory, 1968) andpulsed NMR (Martin et al., 1980), the latternow being generally adopted. In CW-NMRthe amount of liquid water is estimated fromthe area under the absorption peak. The sig-nal from water strongly ‘bound’ to biopoly-mers is not visible because of broadening.The signals from liquid water and oil are notresolved, but the contribution of oil to thesignal can be estimated by drying.

Pulsed NMR can be used to analyse thedifferent water fractions. In pulsed NMRall protons are excited by a short intenseradio frequency (RF) pulse resulting in afree induction signal, which decays whenthe pulse is switched off. The initialamplitude of free induction decay (FID) isproportional to the total number of pro-tons in a sample. The signals due to nucleiin different physical states decay at differ-ent rates: signals due to protons in solidstate decay faster (microseconds) thanthose in liquid phase (from millisecondsto seconds). This signal decay can beanalysed to reveal the contribution of dif-ferent proton fractions. To avoid the influ-ence of inhomogeneity of magnetic fieldand water diffusion on the rate of decay,special sequences of pulses such as spin-echo (SE) or Carr–Purcell–Meiboom–Gill(CPMG) are used (Farrar and Becker,1971). In air-dry samples, the signal decayfrom water associated with polymers(mainly starch) can be distinguished easilyfrom that of oil protons on the basis of theconsiderable differences in spin–spinrelaxation time T2. Such an approach iswidely used for rapid and non-destructivedetermination of moisture and oil contentin air-dry seeds (e.g. Tiwari et al., 1974;



Gambhir and Agarwala, 1985; Brusewitzand Stone, 1987; Gambhir, 1992; Rubel,1994; Warmsley, 1998). In hydrated seeds,drying or D2O exchange can be used toseparate the NMR signal of free water fromthat of oil (Ratkovic et al., 1982a).

Because the different water fractionshave the same chemical shift, only pulsedNMR can be used to characterize them inliving tissues. The changes in water frac-tions with different relaxation characteris-tics can be followed during thedehydration or rehydration of anhydrobi-otic systems. This gives insight into therole of the different water fractions in bio-logical systems (Seewaldt et al., 1981;Ratkovic et al., 1982a; Aksyonov andGolovina, 1986a,b; Ishida et al., 1987,1988b; Bacic et al., 1992; Golovina andAksyonov, 1993; Marconi et al., 1993).However, data on different water fractionsmust be interpreted with extreme caution(Ratcliffe, 1994). Different water fractionswith specific relaxation times can be dis-criminated only if there is no fast exchangeof protons between the fractions in theNMR time window. In the case of fastexchange between protons, only one relax-ation time is observed. The number of pro-tons of different mobility and theirrelaxation times will determine theobserved effective relaxation time. Whenassociated with macromolecules, waterprotons have shorter relaxation times,which will influence the overall relaxationtime. This is the reason why T1 (spin–latticerelaxation time) and T2 (spin–spin relax-ation time) values are lower in cellularwater than in bulk water and decrease fur-ther with water loss. Thus, T2 values canalso be used to measure moisture content(MC) (Ratkovic, 1987). Below 0.2 g H2O g�1

dry weight, the relationship between relax-ation times (T1 and T2) and moisture con-tent is reversed (Clegg et al., 1982; Ratkovicet al., 1982b; Wolk et al., 1989). Becausethe increase in T1 and T2 at low water con-tents has also been observed instarch/water systems besides anhydrobioticorganisms, the increase might be attributedto water molecules jumping from one sorp-tion site to another.

Compartmentation is the reason whymore than one fraction of water is generallyobserved in hydrated living systems. A the-ory of transverse relaxation in compart-mented systems has been developed, basedon the chemical exchange and diffusionproperties of the water (Belton andRatcliffe, 1985). Two to three water frac-tions have been shown in hydrated tissueoriginating from different plant cell com-partments (Bacic and Ratkovic, 1984;Belton and Ratcliffe, 1985; Snaar and vanAs, 1992). However, it appears that there isno simple relationship between the multi-exponential character of T2 and the com-partmentation of the water (Ratcliffe, 1994).The heterogeneity in cellular size and com-position, subcellular compartmentation,and plasmalemma and tonoplast permeabil-ity could have influenced the multi-expo-nential decay curves (Snaar and van As,1992). The detection of the simultaneouspresence of water of different relaxationbehaviour in anhydrobiotes with reducedMC may have been caused by the inhomo-geneous water distribution within theorganisms. Thus, the water with long T2 (orslow-relaxing water) observed in wheat ker-nels during the first hours of imbibition isthought to be localized around the embryoand in the vascular bundle, whereas thefast-relaxing water is thought to be associ-ated with starchy endosperm (Golovina andAksyonov, 1993).

WATER SELF-DIFFUSION COEFFICIENT. The behav-iour of water in living systems can also becharacterized by the water self-diffusioncoefficient. The diffusion coefficient ismeasured by the pulsed (spin-echo) NMRtechnique in the presence of a (pulsed)field gradient (Fukushima and Roeder,1981). In addition to nuclear magneticrelaxation, the spin-echo amplitudedecreases in the presence of a field gradientif water changes its position during themeasurement. Diffusion coefficients as ameasure of water mobility can be calcu-lated from the signal decay in the presenceof a field gradient. As in the case of relax-ation times, self-diffusion coefficients ofcellular water are lower than those of bulk



water and decrease with drying (Clegg etal., 1982). This can be caused by the pres-ence of diffusion barriers (membranes orcell walls) or macromolecules. Thesemacromolecules can cause either obstruc-tion of the diffusion or water binding (Seitzet al., 1981; Back et al., 1991). As a result,the diffusion coefficient in hydrated anhy-drobiotes has been shown to be 2–5 timessmaller than that of bulk water (Clegg etal., 1982; Fleischer and Werner, 1992). InArtemia cysts the diffusion coefficient hasbeen measured from 0.02 to 1.49 g waterg�1 dry weight, the minimum value beingalmost 50 times lower in the dry cysts thanin the hydrated cysts (Seitz et al., 1981).

MEMBRANE PERMEABILITY. Paramagnetic ions(Mn2+) cause a decrease in relaxation timesdue to their interaction with nuclei. Conlonand Outhred (1972) proposed a method ofmeasuring membrane permeability to water,based on the change in relaxation time ofintracellular water that is in diffusionalexchange with an extracellular MnCl2 solu-tion. From the estimated water-exchangetime and the cell dimension, the diffusionpermeability coefficient Pd can be calcu-lated (Stout et al., 1977, 1978; Bacic andRatkovic, 1984). Unfortunately, thisapproach cannot be applied to the systemsthat are subjected to drying, because the tis-sue has to be in Mn2+ solution.

The pulsed-gradient spin-echo methodproposed by Stejskal and Tanner (1965)can be used to study the in situ membranepermeability for water during drying. Themethod allows the water diffusion to bemeasured over the time between twopulses of field gradient. The presence ofpartly permeable barriers causes thedecrease in the apparent diffusion coeffi-cient for water, so that the permeability ofmembranes for water and the size of watercompartments can be calculated (Tanner,1978; von Meerwall and Ferguson, 1981).This approach has been applied to followthe changes in membrane properties indeveloping barley seeds (Ishida et al.,1995) and to calculate the size of oil bodiesin rape seeds (Fleischer et al., 1990;Fleischer and Werner, 1992).

4.4.2.3. NMR imaging

GENERAL REMARKS. NMRI is mainly based onthe detection of the water signal. 1H reso-nance frequency is independent of thelocation of the water in a tissue, so that tis-sue water signal is averaged across thewhole sample. The spatial distribution ofthe water signal can be obtained if a mag-netic field gradient is applied, which arisesfrom the dependence of the resonance fre-quency of NMR signals on the magneticfield strength. In spite of the simplicity ofthe principle of NMRI, its practical appli-cation is rather complicated. Informationon the spatial distribution of water or waterproperties (relaxation times or diffusioncoefficients) can be obtained. Dynamicinformation can be obtained from time-dependent properties of the image. Thereare two different experimental approachesin NMRI: imaging large objects (roots,stems or whole plants) with low spatialresolution, and imaging small samples(seeds, excised tissues) with high spatialresolution (NMR microscopy) (Ratcliffe,1994; Ishida et al., 2000).

Spatial resolution is mainly determinedby the signal/noise ratio, but other factorssuch as short relaxation times and the pres-ence of air space cause intensity loss and adecrease in spatial resolution. The develop-ment of NMRI has led to a resolution thatapproaches the dimension of single cells inplant tissues (Connelly et al., 1987). Thetheoretical limit is considered as 10 � 10 �10 µm (Ratcliffe, 1994). While NMR is notyet able to compete with optical microscopyin its resolution of cellular structures, it hasthe great advantage of being non-invasiveand, thus, can be used to monitor function-ing plant tissue. The ability to resolve struc-tures depends not only on resolution butalso on the image contrast, which is deter-mined by the differences in signal intensitybetween different regions of the sample.Knowledge of relaxation properties of thetissue water is central to the understandingof image contrast. Nitroxide radicals(Magin et al., 1986; Swartz et al., 1986) andparamagnetic ions (Ishida et al., 2000) canbe used as contrasting agents.



WATER DISTRIBUTION IN SEEDS DURING MATURATION

AND GERMINATION. It is possible to map sta-tionary, diffusing and flowing water inplant tissue (Ratcliffe, 1994). NMRI enablesthe water distribution inside seeds to bedetermined. The brightness of the image isproportional to the proton density.Experiments with seeds of different specieshave shown that the signal/noise ratio inthe image is sometimes limited by the shortrelaxation time for tissue water (T2 < 10ms) (Connelly et al., 1987). The sensitivityproblem can be overcome to some extentby signal averaging, since the time scale fordetectable structural changes in germinat-ing seeds is long in comparison with thetime required to obtain an image. In NMRimages of seeds, a clear distinctionbetween axis and storage tissue can beobtained (Connelly et al., 1987; Kano et al.,1990; Hou et al., 1996; Fountain et al.,1998; Carrier et al., 1999). The changes inwater distribution during drying and rehy-dration have shown the transfer routes forwater (Ruan and Litchfield, 1992; Ruan etal., 1992; Song et al., 1992; Kovacs andNemenyi, 1999).

The water content may be more uni-formly distributed in seeds than protonNMRI indicates. This discrepancy arisesfrom the inhomogeneity of the susceptibil-ity of the sample associated with the pres-ence of cell walls and storage substances(Back et al., 1991). Eccles et al. (1988)applied pulsed gradient spin-echo andsteady gradient NMRI to maturing wheatkernels and found the spatial distributionof the self-diffusion coefficient of water.The diffusion was slowest in endospermand highest in the vascular bundle. Back etal. (1991) used the dependence betweenthe self-diffusion coefficient of water andthe relative water content obtained byCallaghan et al. (1979) to correct the protonmap for wheat grain and showed the moreuniform distribution of water in the cor-rected image.

For experiments in which germination ofseeds has to be followed over many hours inthe magnet, it is necessary to maintain a con-tinuous water supply to the seeds, while atthe same time minimizing the spectroscopic

signal of the externally supplied water(Connelly et al., 1987). The changes in relax-ation times of tissue water during seed matu-ration or germination cause changes in theimage contrast. Relaxation times of waterdepend on the interaction of water withmacromolecules. The synthesis of storagesubstances during maturation and theirhydrolysis during germination result in anapparent decrease or increase in brightnessof the NMR image (Ishida et al., 1990, 1995;McIntyre et al., 1995), so that solubilizedparts of the storage tissue can become visi-ble. The changes in image contrast duringprecocious germination of Phaseolus vul-garis seeds after ethylene treatment havebeen attributed to changes in the water sta-tus and water redistribution from the cotyle-don to the axis (Fountain et al., 1998).

THE DISTRIBUTION OF OIL AND SUCROSE IN SEEDS.The spatial image of other compounds,mainly lipids and carbohydrates that accu-mulate in storage tissue, can be mapped invivo using the chemical-shift imaging (CSI)technique (Bottomley et al., 1984). The 1HNMR spectra of water, oil and sugars havedifferent chemical shifts, but the peaks arenot resolved unless the water peak is sup-pressed. The CSI technique applied to 1day germinating mung bean seeds hasshown uniformly distributed oil, whichallowed the changes in the image with ger-mination to be attributed to the bulk waterfraction (Connelly et al., 1987). Oil andsucrose have been mapped in fresh maizekernels (Koizumi et al., 1995), germinatingbarley seeds (Ishida et al., 1990) and indeveloping pea seeds (Tse et al., 1996).

4.4.2.4. High-resolution multinuclear NMRspectroscopy

GENERAL REMARKS. High-resolution multi-nuclear NMR is used to detect ions andmetabolites of low molecular weight, theintracellular pH, the subcellular compart-mentation of compounds and the fluxthrough metabolic pathways (Ratcliffe,1994; Schneider, 1997; Roberts, 2000). Lowconcentration of the molecules of interest



and low receptivity of nuclei other than 1Hmake this approach rather insensitive. Thesensitivity increases with increasing fieldstrength. High-resolution NMR spectrome-ters are usually equipped with high-fieldsuperconducting magnets in the range4.7–14.1 T, corresponding to a 1H fre-quency of 200–600 MHz. The sensitivitycan be increased by multiple scanning andusually permits the detection of millimolarconcentrations of metabolites (Ratcliffe,1994). To increase the sensitivity further,the tissue volume within the detector hasto be maximized. Cell suspensions andexcised tissues are more suitable for suchexperiments than whole plants or seeds.

1H NMR. Different nuclei can be used for dif-ferent purposes. The high sensitivity makes1H attractive for metabolite detection.Nevertheless, the need to suppress the watersignal and the complexity of spectra limitthe possibilities for in vivo 1H NMR. Thesmall differences in chemical shift and con-siderable overlapping of broad signals in tis-sues make 1H spectra poorly resolved. Forexample, in germinating seeds only peaksfrom sugars and oil under conditions of par-tial water signal suppression can beresolved (Koizumi et al., 1995; Ishida et al.,1996). 1H NMR spectra of oil in dry seedscan be obtained because the signals fromother nuclei are broadened due to immobi-lization. However, the resolution of lines isnot good because of differences in magneticsusceptibility. The magic-angle samplespinning (MASS) technique eliminates linebroadening arising from differences in mag-netic susceptibility due to fast mechanicalrotation about an axis, making a magic angle(54°55�), and resulting in 1H spectra fromdry seeds with a good resolution (Rutar,1989). 1H NMR is widely used to analysetissue extracts for the presence of specificcompounds such as, for example, betaine inwild-type and transformed Arabidopsisthaliana seeds (Alia et al., 1998).

13C NMR. 13C NMR is more attractive forapplication in vivo for two reasons. First,the chemical shift scale of the 13C nucleusis more than an order of magnitude greater

than that of the 1H nucleus, which reducesoverlapping in the spectra. Secondly, thelow natural abundance of 13C opens possi-bilities for labelling the tissue and monitor-ing metabolic pathways. The biological useof NMR to study metabolism is described inSection 4.3.2. 13C NMR has also been usedto establish changes in soybean seeds dur-ing maturation and germination. The mois-ture content-dependent disappearance orappearance of narrow peaks associated withsugars in in vivo NMR spectra is indicativeof the presence of free water in these seeds(Ishida et al., 1987, 1988a). The sensitivityof natural abundance 13C NMR can beenhanced, by applying low-speed magic-angle spinning (Ni and Eads, 1992) or bythe detection of 13C by protons coupled tothe 13C nucleus (Heidenreich et al., 1998).13C labelling gives opportunities for probingdifferent metabolic pathways, such as lipidsynthesis in soybean ovules (Schaeffer etal., 1975) and the metabolism of dormancy-breaking chemicals in red rice (Footitt etal., 1995).

31P NMR. In vivo 31P NMR has many applica-tions because of the convenient magneticproperties of the 31P nucleus and the physi-ological importance of the information thatcan be deduced from the spectra. The mea-surement of cytoplasmic and vacuolar pH isone of the most important applications of invivo 31P NMR, which is based on the depen-dence on pH of the chemical shift of Pi.This, together with the slow exchange of Piacross the tonoplast, allows the origin of thePi signal – either cytoplasmic or vacuolar –to be determined and, consequently, thecytoplasmic and vacuolar pH. A number ofimportant phosphorylated metabolites canbe resolved in 31P spectra. For some of them(Pi, polyphosphates), information on thesubcellular distribution can also be obtainedbecause of the pH-dependent chemical shift.31P NMR has been applied to study the pHof intracellular compartments in germinat-ing seeds of Phacelia tanacetifolia (Espen etal., 1995). Changes in chemical shifts of thepH-dependent 31P signal from cytoplasmicand vacuolar inorganic phosphate correlatewith seed germination. 31P can also be used



to monitor phosphorus compounds andtheir changes during maturation and germi-nation of seeds, both in extracts and in vivo.Because of line broadening in in vivo exper-iments, a lower number of phosphorus com-pounds can be resolved (Ishida et al., 1987,1988a) in comparison with extracts (Ricardoand Santos, 1990). 31P spectra can be usedfor the identification of the appearance ordisappearance of vacuoles in seeds duringgermination and maturation (Ishida et al.,1990).

4.4.2.5. Structure and dynamics of cellularmembranes

GENERAL REMARKS. NMR provides a rapid,non-invasive method for investigating thestate of membranes in isolated cellularfractions and in living tissues. Theapproach in the study of membrane struc-ture and dynamics is solid-state NMR,because of the anisotropic nature of themembranes. The main nuclei used for thisstudy are 31P and 2H. Sometimes, labellingwith 13C has been used, although the lineshape is difficult to analyse.

31P NMR The chemical shift of the phospho-lipids depends on the orientation of thephosphate groups with respect to the mag-netic field. In the case of unrestrictedmotion, all directions are averaged and thespectrum is isotropic and contains the nar-row symmetrical 31P NMR line (Cullis andde Kruijff, 1979). In some cases, peaks fromdifferent phospholipids can be resolved(Smith, 1985). In the case of restrictedmobility of phospholipids in membranes,the spectrum is anisotropic. The shape ofthe anisotropic 31P NMR spectrum dependson the type and rate of motion of the phos-pholipids. Thus, 31P NMR spectra are sensi-tive to the physical state of thephospholipids. From the spectra, the orderparameter can be calculated (Smith, 1985).There are a few examples of the successfulapplication of 31P NMR in the field of desic-cation tolerance. Lee et al. (1986, 1989)studied the interaction of trehalose with thephospholipid, dipalmitoylphosphatidyl-

choline (DPPC). It was shown that the headgroups are in a rigid state above and belowthe phase transition for both dry DPPC anda mixture of dry DPPC and trehalose.Tsvetkova et al. (1998) used 31P NMR in acomparative study of the interaction of glu-cose, trehalose and hydroxyethyl starchwith dry DPPC. The differential effect of car-bohydrates on the behaviour of head groupshas been related to the role of trehalose inmembrane protection upon drying.

Phospholipids arranged in bilayers or inan inverted hexagonal phase have differentline shapes (31P pattern) (Cullis and deKruijff, 1979). These differences betweenbilayer and hexagonal phase spectra arisefrom the fact that the lipids are restricted inmotion to the plane of the membrane in thelamellar state. In the case of the hexagonalphase, a rapid motion about the cylinderaxis averages the chemical shift anisotropy.These differences in 31P pattern can be usedto detect the presence of either phase.

For many years researchers have beeninterested in the membrane transition upondrying from the bilayer into the hexagonalphase (Simon, 1974). In an attempt todetect this membrane transition, Priestleyand de Kruijff (1982) applied 31P NMR toseveral dry biological systems. The in vivospectra were complicated by the superposi-tion of the signals from phospholipids andphosphorus-containing compounds. Pollenof Typha latifolia was the most suitable forspectra analysis. At 5.2% MC, the lineshape of the spectrum was broad and notsuitable for analysis. At MC � 8.8%, onlyisotropic signals from phosphorus low-weight molecules could be identified, but,at 10.9% MC, a clear peak from phospho-lipids organized in bilayers became evi-dent. Thus, no evidence was obtained forthe presence of a hexagonal phase in thepollen on drying to 10.9% MC.

2H NMR. The relatively small quadrupolemoment of deuterium makes it an idealprobe of membrane lipids (Smith, 1985).Fatty acids labelled with 2H at differentpositions must be synthesized. The 2HNMR spectrum of membranes containsthree clearly separated lines (‘rabbit ears’),



and the separation relates to the ordering ofthe 2H-labelled segment. Quadrupole split-ting, overall pattern and relaxation timesare usually used to characterize 2H spectra.Spin–lattice relaxation is sensitive to rela-tively rapid motions, whereas spin–spinrelaxation is sensitive to slow motions(Smith, 1985). This technique can be usedto study membrane phase transitions, theinfluence of acyl chain saturation on mem-brane fluidity and changes in membranefluidity.

2H NMR was applied by Lee et al. (1986,1989) in a study on the effect of interactionof trehalose with dry DPPC on the behav-iour of acyl chains. 2H quadrupole spectraof dry DPPC labelled at the 7th positionshowed that the disorder of lipid acylchains is much greater in the case of inter-action of DPPC with trehalose above thephase transition than in hydrated or dryDPPC without trehalose. The new type ofliquid-crystalline phase observed in the drymixture of trehalose and DPPC is believedto play a main role in maintaining mem-brane stability in dehydrating organisms.

13C NMR. 13C-labelled phospholipids can beused to study the particular dynamics ofmembranes in the interfacial region. Lee etal. (1989) used 13C-labelled sn-2-carbonylof DPPC to study the influence of the inter-action of dry DPPC with trehalose on inter-facial behaviour. No changes in 13C NMRpowder spectra were observed during thephase transition of a dry mixture ofDPPC/trehalose, whereas hydrated DPPCexhibited pronounced changes during thephase transition.

4.4.3. Fourier transform infrared (FTIR)spectroscopy

4.4.3.1. General description of infraredspectroscopy

Infrared (IR) spectroscopy deals with thetransition between vibrational energy lev-els that permanently exist in a system, incontrast to NMR and EPR where the transi-tion occurs between energy levels thatarise in a system only in an external mag-

netic field. IR spectroscopy is sensitive tovibrations that modulate a molecule’sdipole moment. The range of frequenciesis around 1012–1014 Hz or 400–4000 cm�1.The main problem of IR spectroscopy ishigh water absorption in the IR region.D2O substitution or dry films are oftenused. In plotting IR spectra, the intensityof absorption (A) against wave number(1/) is used. The main characteristics ofthe absorption band are wave number ofthe maximum absorption (Amax), the widthof the band determined at half of theheight of Amax, the optical density at Amaxand the shape of the band. Every band canbe assigned to a certain chemical groupand a certain type of vibration. In the caseof simple molecules, IR spectra consist ofnarrow lines. In the case of macromole-cules, the spectrum is characterized by rel-atively broad bands because of theoverlapping of a great number of individ-ual lines corresponding to different typesof bonds and different conformations.

4.4.3.2. Biological applications

With the introduction of FTIR spectrome-ters in the 1970s, in vivo studies becamepossible, which was not the case with thegrating IR spectrometers because of theirlow energy throughput. FTIR spectroscopycan be used for the analysis of certain com-pounds, or to study the interaction betweenmolecules. In dry organisms, the techniqueis particularly useful because of the‘absence’ of water. The absorption of waterusually obscures other absorption bandsand thus complicates the interpretation ofspectra. A considerable advantage of in vivoFTIR spectroscopy is that it permits theanalysis of macromolecules in their naturalenvironment as opposed to in a solvent. Adisadvantage is that information is obtainedon the average vibrational absorption of allmolecular groups contributing to the IR-absorption band under study.

For analysis of small samples or the loca-tion of certain compounds in specific tis-sues, an IR microscope fixed to the opticalbench can be used. Improvement in sensi-tivity has been reached by the application of



liquid nitrogen-cooled MCT (mercury/cad-mium/telluride) detectors, which allowpollen, microorganisms or slices of seeds tobe studied. Peak positions or presence ofshoulders in the spectra can be analysed bycomputer-assisted derivative analysis (Susiand Byler, 1983) and deconvolution (Bylerand Susi, 1986) procedures, respectively.Depending on transmittance and scatteringcharacteristics of a sample, a transmission,reflection or attenuated total reflection(ATR) mode can be used.

An example of the in vivo analysis of cer-tain compounds in seeds is scanning in thetransmission mode along a slice of tissue.Thus, it has been confirmed that the aleu-rone layer is enriched in proteins and theendosperm in starch. In the case of dehy-drating organisms, the change in molecularinteractions or conformation is of interest.The occurrence of an absorption bandaround 2850 cm�1 originating from the sym-metric stretching vibration of CH2 can safelybe attributed to acyl chains, either from oilor from membranes. If the organism is lowin oil, it is possible to follow, in vivo, thedecrease in C–H vibrational freedom in theacyl chains of membranes with dehydration(Cameron et al., 1983; Crowe et al., 1989;Hoekstra et al., 1992). Restriction of vibra-tional freedom by molecular interaction(van der Waals interactions in the case of gelphase formation) leads to shifts of theabsorption peaks to lower wave number andsharpening of these peaks. If the sampleholder is temperature-controlled, it is possi-ble to determine the gel-to-liquid crystaltransition temperature of these membranesfrom shifts in the absorption maxima withtemperature. The same information can beobtained from shifts in other absorptionbands, e.g. the asymmetric CH2 stretcharound 2920 cm�1 and the C�O stretch ofthe ester bond of the acyl chains around1740 cm�1 (Sowa et al., 1991). Although thegeneral melting behaviour of oil in seedscan also be analysed by other techniques(e.g. differential scanning calorimetry), thatof membranes is difficult with other meth-ods because of the small amount involved.

In vivo FTIR spectroscopy has beensuccessfully applied in the study of pro-

tein secondary structure with dehydration(Wolkers and Hoekstra, 1995, 1997;Golovina et al., 1997c; Wolkers et al.,1998a,b). Conformational changes of pro-teins can be derived from peak positionsin the amide I (1600–1700 cm�1) and II(around 1550 cm�1) regions (Byler andSusi, 1986; Surewicz and Mantsch, 1988).The amide I band mainly arises from theC�O stretching vibration of the peptidegroups, and the amide II band from the N–H bending vibration of the proteinbackbone (Susi et al., 1967). The C�Ostretching frequency is very sensitive tochanges in the nature of the hydrogenbonds arising from the different types ofsecondary structure. This causes a charac-teristic set of IR-absorption bands for eachtype of secondary structure (Susi et al.,1967). Curve fitting of the different bandsallows, to a certain extent, the amounts of�-helix, random coil, turn and �-sheetstructures to be established (Surewicz etal., 1993). In some model enzyme sys-tems, a highly characteristic low wavenumber band (around 1625 cm�1 in theamide I region (the intermolecularextended �-sheets) is indicative of the for-mation of large protein aggregates withdrying (Prestrelski et al., 1993). Theseaggregates have also been found in vivo onheat denaturation. The stability of pro-teins against heat denaturation can be fol-lowed by scanning over a range oftemperatures (Wolkers and Hoekstra,1997; Wolkers et al., 1998a). In the situa-tion where the absorption band of water(HOH scissoring vibration band at1650 cm�1) interferes with the proper esti-mation of the different protein secondarystructures, H2O can be replaced by D2O,which causes a downward shift in wavenumber. The accessibility of the proteinsfor D2O can help identify the protein sec-ondary structure.

Recently, it was established that theglassy state can be studied in vivo byinspection of the OH-stretch at around3300 cm�1 (Wolkers et al., 1998c, 1999).The interaction of sugars with proteins orwith polar head groups has been verifiedin dry model systems in the 3300 cm�1



(Wolkers et al., 1998d) and 1240 cm�1

regions (Crowe et al., 1996), respectively.Such interaction upon desiccation has notbeen established with certainty in vivo dueto the possible absorption of other molecu-lar groups in these regions. Although invivo FTIR spectroscopy has disadvantagesin that it is an averaging technique andthat it is difficult to establish with cer-tainty from which molecules the spectraoriginate, it has the considerable advan-tage that molecules are studied in theirnative environment. The disadvantagescan be partly alleviated by parallel in vitroexperiments, also employing other meth-ods of analysis.

4.5. Additional Techniques to StudyBiochemical and Biophysical Aspects of

Desiccation Tolerance

4.5.1. Differential scanning calorimetry(DSC)

DSC is applied to the study of thermalevents associated with lipid and waterphase/state transition. In plant anhydro-biotes, it is used for two main purposes:(i) to determine the calorimetric proper-ties of water present in the system; and(ii) to construct a state–phase diagram inwhich the glass transition temperature(Tg) and the ice formation/melting temper-ature are plotted as a function of moisturecontent (Vertucci, 1990; Leprince andVertucci, 1995; Buitink et al., 1996). Thecalorimetric behaviour of the glass transi-tion can be characterized although DSCdoes not give direct access to the physicaland biological properties of glasses.Sometimes, the heat released during theglass transition is below the sensitivity ofthe equipment. For example, in seedspecies such as rice and tobacco, Tg can-not be detected by DSC (O. Leprince and J.Buitink, unpublished data). Furthermore,in oily seeds such as neem and Impatiens,the lipid melting transitions often maskthe thermal events associated with water,

thereby limiting the range of moisturecontent that can be studied (Buitink et al.,1996; Sacandé et al., 2000). However, thefuture of DSC in studying anhydrobiosisis questionable since no major differencein the calorimetric properties of water wasfound between desiccation-tolerant andsensitive organisms (Sun et al., 1994;Buitink et al., 1996; Fig. 10.2, Chapter 10).

4.5.2. Electron microscopy

Owing to technical difficulties in studyingultrastructural characteristics of organ-elles in the dry state and upon rehydration,two promising microscopic techniques areworth mentioning because they can be con-sidered as non-invasive techniques: atomicforce microscopy (AFM) and low-tempera-ture scanning electron microscopy(LTSEM). AFM is particularly suitable forimaging, non-invasively, the surface topog-raphy of membranes at a nanometer scale.Furthermore, AFM can be used to obtaininformation on the mechanical propertiesof surfaces (Heinz and Hoh, 1999;Claessens et al., 2000). LTSEM overcomesproblems linked to aqueous fixation. Itallows a fast and direct observation offreeze–fractured specimens with great reso-lution without altering the sample watercontent. Application of LTSEM was foundto be powerful for studying ultrastructuraldamage resulting from imbibitional injuryin seeds (Leprince et al., 1998; Nijsse etal., 1998; Sacandé et al., 2001) and cellu-lar collapse in lichens (Scheidegger et al.,1995). In the near future, new technologi-cal developments (so-called semi-in-lens)will improve the resolution, which is cur-rently limited to 100 nm in most commer-cially available equipment. Non-invasivefixation (freeze-substitution) and a newnon-aqueous fixative for immunocyto-chemistry (acrolein) are becoming avail-able for transmission electron microscopystudies (Grote et al., 1999), allowingmicroscope observation without disturb-ing the sample water content.



4.6. Acknowledgements

The authors thank Dr F.A. Hoekstra forhis contribution to the section on spec-troscopy methods and for critically read-ing the manuscript. O.L. acknowledgesthe financial support of the Ministère de

l’Agriculture et de la Pêche, the Contratde Plan Etat-Région and INRA; E.A.G.gratefully acknowledges the financialsupport by a grant from the WageningenCentre for Food Sciences and by NATOcollaborative linkage grant # LST.CLG975082.


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pulsed NMR. Lipid Technology 10, 135–137.Weber, H., Rolletschek, H., Hein, U., Golombek, S., Gubatz, S. and Wobus, U. (2000) Antisense-inhibition

of ADP-glucose pyrophosphorylase in developing seeds of Vicia narbonensis moderately decreasesstarch but increases protein content and affects seed maturation. Plant Journal 24, 33–43.

Wehmeyer, N. and Vierling, E. (2000) The expression of small heat shock proteins in seeds respondsto discrete developmental signals and suggests a general protective role in desiccation tolerance.Plant Physiology 122, 1099–1108.

Wilson, D.O. and McDonald, M.B. (1986) A convenient volatile aldehyde assay for measuring soy-bean seed vigour. Seed Science and Technology 14, 259–268.

Wiseman, H. and Halliwell, B. (1996) Damage to DNA by reactive oxygen and nitrogen species: rolein inflammatory disease and progression to cancer. Biochemical Journal 313, 17–29.

Wolk, W.D., Patrick, F.D., Copeland, L.F. and Dilley, D.R. (1989) Dynamics of imbibition in Phaseolusvulgaris L in relation to initial seed moisture content. Plant Physiology 89, 805–810.

Wolkers, W.F. and Hoekstra, F.A. (1995) Aging of dry desiccation-tolerant pollen does not affect pro-tein secondary structure. Plant Physiology 109, 907–915.

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Wolkers, W.F., Alberda, M., Koornneef, M., Léon-Kloosterziel, K.M. and Hoekstra, F.A. (1998a)Properties of proteins and the glassy matrix in maturation-defective mutant seeds of Arabidopsisthaliana. The Plant Journal 16, 133–143.



Wolkers, W.F., Bochicchio, A., Selvaggi, G. and Hoekstra, F.A. (1998b) Fourier transform infraredmicrospectroscopy detects changes in protein secondary structure associated with desiccationtolerance in developing maize embryos. Plant Physiology 116, 1169–1177.

Wolkers, W.F., Oldenhof, H., Alberda, M. and Hoekstra, F.A. (1998c) A Fourier transform infraredmicrospectroscopy study of sugar glasses: application to anhydrobiotic higher plant cells.Biochimica et Biophysica Acta 1379, 83–96.

Wolkers, W.F., van Kilsdonk, M.G. and Hoekstra, F.A. (1998d) Dehydration-induced conformationalchanges of poly-L-lysine as influenced by drying rate and carbohydrates. Biochimica etBiophysica Acta 1425, 127–136.

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Part III

Biology of Dehydration



5 Desiccation Sensitivity in Orthodox andRecalcitrant Seeds in Relation to Development

Allison R. Kermode1 and Bill E. Finch-Savage21Department of Biological Sciences, Simon Fraser University, Burnaby, BC,

V5A 1S6, Canada; 2Horticulture Research International,Wellesbourne,Warwick CV35 9EF, UK

5.1. Introduction 1505.2. Development and Acquisition of Desiccation Tolerance 151

5.2.1. Changes in water status during development of orthodox seeds 151

5.2.2. Acquisition of desiccation tolerance during development of orthodox seeds 152

5.2.3. Loss of desiccation tolerance following germination of orthodox seeds 153

5.2.4. Effects of the rate and extent of desiccation on the acquisition of tolerance of orthodox seeds 153

5.2.5. Variation in desiccation tolerance across species 1555.2.5.1. Seed development in recalcitrant species 1575.2.5.2. Time-dependent effects of storage and drying rate 1595.2.5.3. Desiccation tolerance differs between seed tissues 160

5.2.6. Mechanisms underlying the acquisition of desiccation tolerance: recent findings and speculations 1615.2.6.1. The effects of premature desiccation during the

tolerant and intolerant stages of orthodox seed development 161

5.2.6.2. Cellular and metabolic changes during the transition to a desiccation-tolerant state 161

5.2.6.3. Desiccation-tolerance mechanisms in sensitive seeds 170

5.3. Conclusions 1745.4. References 175



5.1. Introduction

The development of most seeds can bedivided conveniently into three confluentstages (Fig. 5.1). During histodifferentiation,the single-celled zygote undergoes exten-sive mitotic division, and the resultant cellsdifferentiate to form the basic body plan ofthe embryo (the axis and cotyledons); con-currently, there is the formation of thetriploid endosperm or haploid megagameto-phyte. Thereafter, cell division ceases dur-ing the seed expansion stage and there iscell expansion and the deposition ofreserves (normally proteins along withlipids or carbohydrates), primarily in thestorage tissues (i.e. cotyledons, endospermor megagametophyte). Finally, the develop-ment of most seeds is terminated by somedegree of drying (maturation drying), whichresults in a gradual reduction in metabo-lism as water is lost from seed tissues and

the embryo passes into a metabolicallyinactive or quiescent state.

The majority of seeds are referred to as‘orthodox’, in which desiccation occurs asa pre-programmed and final stage in theirdevelopment (Fig. 5.1). Seeds of the ortho-dox type and other desiccation-tolerantstructures such as spores and pollen areunique in the degree of water loss toler-ated; as much as 90–95% of the originalwater is removed during their develop-ment. In this dehydrated state, the seed cansurvive the vagaries of the environmentand, unless dormant, will resume full meta-bolic activity, growth and developmentwhen conditions conducive to germinationare provided (Fig. 5.1). This chapter dis-cusses some of the mechanisms underlyingdesiccation tolerance of seeds and recentapproaches to elucidate the precise roles ofprotective molecules and repair processesat the cellular and subcellular levels.

150 A.R. Kermode and B.E. Finch-Savage

Cell division

Desiccation-intolerant

Reservebreakdown(Mature dry

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Development Germination growth

Histodifferentiation Maturation Desiccation Dry

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Fig. 5.1. Some events associated with seed development, germination and growth. (From Kermode, 1995.)

(Expansion)

,


An important approach to elucidatingthe basis of desiccation tolerance in seedsis comparative analyses between seeds thatdiffer in their capacity to withstand waterloss, i.e. seeds of orthodox and recalcitrantplant species. ‘Orthodox’ seeds can bestored for long periods under conventionalconditions, i.e. in the dry state and at lowtemperature. Recalcitrant seeds, on theother hand, do not undergo maturationdrying, nor are they capable of withstand-ing water loss of the magnitude of thatexperienced by orthodox seeds. The seedsare shed at relatively high moisture con-tents and are highly susceptible to desicca-tion injury; in order to remain viable, theymust not undergo any substantial changein moisture. They are not storable underconditions suitable for orthodox seeds and,even when stored under moist conditions,their viability is frequently brief and onlyrarely exceeds a few months (reviewed inChin and Roberts, 1980; Bewley and Black,1994; Smith and Berjak, 1995; Vertucci andFarrant, 1995; Berjak and Pammenter,1997; Pammenter and Berjak, 1999). Thusthe terms ‘orthodox’ and ‘recalcitrant’ havebeen used to describe the storage behaviourof seeds. A category intermediate betweenorthodox and recalcitrant is now recog-nized (e.g. coffee) in which seeds survivedesiccation but become damaged duringdry storage at low temperatures (0°C and�20°C) (Ellis et al., 1990, 1991a). It isimportant to note, however, that the situa-tion is more complex and there is a gradualcontinuum of desiccation tolerance acrossorthodox and recalcitrant species.

The question arises as to whether thedesiccation sensitivity of recalcitrant seedsis at least partially the result of an insuffi-cient accumulation of protective proteins,or whether other factors (including a lackof protective sugars) are more important.Since desiccation tolerance is arguably aquantitative feature (Vertucci and Farrant,1995), the amount of protective proteins, orthe rate at which the proteins accumulate,may determine the level of tolerance.

Other features that may be part of thebasis of desiccation sensitivity include aninability to repair desiccation-induced dam-

age upon subsequent rehydration and aninappropriate proportion or distribution offreezable and non-freezable (bound) waterwithin the seed (Berjak et al., 1992;reviewed in Bewley and Oliver, 1992;Vertucci and Farrant, 1995). Recent researchin this area will be discussed briefly but seealso Chapters 6–8 of this volume.

5.2. Development and Acquisition ofDesiccation Tolerance

5.2.1. Changes in water status duringdevelopment of orthodox seeds

The three major phases of seed develop-ment characteristic of orthodox seeds(namely histodifferentiation, expansionand maturation drying; Fig. 5.1) are markedby distinctive changes in fresh weight, dryweight and water content (Fig. 5.2). Duringhistodifferentiation and early cell expan-sion, there is a rapid increase in wholeseed fresh weight and water content.Generally, a period of rapid dry-weightgain follows (when whole seed freshweight is relatively stable); this takes placeduring the later part of the seed expansionphase of development. Most seeds losewater during this phase as reserves aredeposited primarily within storage tissues,displacing water from the cells. Thisdecline in water content slows as the seedapproaches its maximum dry weight. Then,as the seed undergoes maturation dryingand approaches quiescence, there is aperiod of fresh weight loss accompanied bya rapid decline in whole seed water con-tent (Kermode, 1990; Fig. 5.2).

Little is known about the mechanism androute of water loss from seeds. Some studiessuggest the existence of a passive mecha-nism whereby water is lost primarily byevaporation from the surface of surroundingseed structures (Nechiporenko andRybalova, 1983; Lee and Atkey, 1984;Goncharova et al., 1985). Another suggestionis that water moves from the seed to the par-ent plant by a metabolically active process,i.e. the plant actually ‘pumps’ the water fromthe seed (Meredith and Jenkins, 1975).

Desiccation Sensitivity in Relation to Seed Development 151


In soybean and castor bean, the desicca-tion period is most probably initiated bythe severing of the vascular supply to theseed (funiculus detachment) and senes-cence of the pod or capsule (Greenwoodand Bewley, 1982; Murray and Nooden,1986). This would suggest that relocationof water from the seed to the parent plantis not the means by which water lossoccurs. Similarly, pectic substances in thelumina of xylem elements of the rachis ofwheat and barley (laid down during thefinal stages of grain maturation) may leadto the progressive dehydration of the ear bycutting off its water supply (Cochrane,1985).

5.2.2. Acquisition of desiccation toleranceduring development of orthodox seeds

Seeds of orthodox plant species cannot tol-erate drying at all stages of their develop-ment. During very early development, seedsare generally intolerant of drying, but theylater undergo a transition to a desiccation-tolerant state at a particular time (reviewedin Kermode, 1990, 1995). In many cases,

drying of seeds at a desiccation-tolerantstage of their development promotes germi-nation upon subsequent rehydration. Air-dried grains of wheat not only germinate atan earlier stage of development than non-dried grains, but at later stages may alsogerminate at a faster rate than their non-dried counterparts (Mitchell et al., 1980;Symons et al., 1983). Seeds of Phaseolusvulgaris (French bean) undergo a transitionto a desiccation-tolerant state around 26DAP (days after pollination) approximatelyhalfway through development (Dasgupta etal., 1982). Seeds at 26–32 DAP can beinduced to germinate to increasing extentsif first dried over silica gel, whereas thosedried at 22 DAP fail to germinate whensubsequently rehydrated and they eventu-ally deteriorate. The 22 DAP seeds do notrecover their full cellular and metabolicintegrity following the premature dryingtreatment. At later times of development,however, the seeds acquire a tolerance ofdesiccation and also germinability isinduced.

A similar situation exists for the castorbean seed (Ricinus communis) (Kermodeand Bewley, 1985). Here, germinability is


Gra

ms

HistodifferentiationExpansion

(reserve deposition)Maturation

drying

fw

dw

WC

I II III

Days of development

Fig. 5.2. A general scheme of changes in whole seed fresh weight (fw), dry weight (dw) and water content(WC) during the histodifferentiation, expansion and maturation drying phases of development of orthodoxseeds. Three major periods are noted: I, rapid fresh-weight gain; II, rapid dry-weight gain; III, fresh-weightloss. (From Kermode and Bewley, 1986.)


not achieved until 50–55 DAP, whereas pre-mature drying will promote the germina-tion ability of seeds as young as 25 DAP. Atearlier stages of development, drying notonly fails to induce germination ability, butalso kills the seed. For both P. vulgaris andR. communis, the seeds acquire a toleranceof desiccation and an ability to be potenti-ated to germinate by this treatment around25 days after development commences.

The transition to a desiccation-tolerantstate approximately midway throughdevelopment is also characteristic of otherseeds, e.g. soybean, maize, barley,Agrostemma githago (Adams and Rinne,1981; deKlerk, 1984; Bartels et al., 1988;Bochicchio et al., 1988). Tolerance of desic-cation is gained over only a few days ofdevelopment (e.g. between 20 and 25 DAPin R. communis); it is achieved well beforethe completion of major developmentalevents such as reserve deposition and thecommencement of normal maturation dry-ing (Kermode and Bewley, 1985; Kermodeet al., 1986) (Figs 5.1 and 5.3).

5.2.3. Loss of desiccation tolerance followinggermination of orthodox seeds

During germination, seeds initially remaintolerant of reimposed desiccation, but atsome stage after axis elongation this abilityis lost (Fig. 5.1). Germinating soybeanseeds are tolerant of drying during theearly stages, up to 6 h after commencingimbibition, but they become increasinglyintolerant after this time. Thus, desiccationat 36 h after the start of imbibition kills theseed (Senaratna and McKersie, 1983). Theplasma membrane appears to be a majorsite of damage in seeds during the desicca-tion intolerant stage of germination, asindicated by ultrastructural studies(Crevecoeur et al., 1976) and by theincreased solute and electrolyte leakageupon subsequent rehydration (Senaratnaand McKersie, 1983). There appears to be adifferential sensitivity of different seed tis-sues with respect to the loss of desiccationtolerance. For example, axes of soybeanrapidly lose their tolerance to desiccation

(brought about by air-drying to 10% watercontent) during the course of germination,while the cotyledons remain tolerant for aconsiderably longer period (Senaratna andMcKersie, 1983).

As will be discussed below, changes thatoccur on dehydration of the most desiccation-sensitive seeds (e.g. those of themangrove, Avicennia marina) can be verysimilar to changes brought about by desic-cation of orthodox seeds during the intoler-ant stage following germination (Farrant etal., 1986). Some recalcitrant seeds initiategermination-related metabolism shortlyafter shedding (reviewed in Vertucci andFarrant, 1995) and, in A. marina, 10–15days before shedding (Farrant et al., 1993b).As germination events progress, the seedsbecome increasingly sensitive to drying andattempting to store these seeds is akin tostorage of germinated, orthodox seeds(Farrant et al., 1986, 1988). There is noclear-cut event delineating the end of seeddevelopment and the start of germination;during both phases, recalcitrant seedsappear to remain metabolically active,although the axes may undergo a very briefperiod of relative quiescence.

5.2.4. Effects of the rate and extent ofdesiccation on the acquisition of tolerance of

orthodox seeds

The rate at which drying is imposed duringearly development is critical for the subse-quent expression of germinability, andthus, when it is stated that a seed acquiresa tolerance of desiccation at a particularstage during its development, it is neces-sary to define the rate of water loss towhich it is subjected (see Chapters 2 and3). Whole seeds of several legumes (Adamset al., 1983; Ellis et al., 1987) and R. com-munis (Kermode and Bewley, 1985) areunable to withstand rapidly imposed dry-ing (over silica gel or under regimes similarto ambient laboratory conditions) at earlystages (i.e. during most of development,prior to maturation drying) and exhibit nogerminability upon subsequent rehydra-tion. This contrasts with seeds at the same




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stage of development dried slowly over sat-urated salt solutions or air-dried whileenclosed in the pod, where full germinabil-ity is evident. Tolerance of rapid dryinggenerally occurs only at or near the com-pletion of reserve deposition (as indicatedby the attainment of maximum dry weight)just after the onset of natural drying(Rogerson and Matthews, 1977; Kermodeand Bewley, 1985; Ellis et al., 1987),although there are exceptions (see subse-quent discussion).

Gradual water loss may allow protectivechanges to occur and hence increase theseed’s resistance to disruption by dehydra-tion. Rapid drying presumably would notallow such protective changes to take placeand may cause considerable disruption tocellular membranes and internal structures(see Section 5.2.6). As a result, the seedrequires time for metabolic readjustment(i.e. repair) following rapid drying. Thiscannot take place during drying itselfbecause the seed reaches a critical dry (andquiescent) state before the repair processescan be initiated. Such repair is alsoimpeded upon imbibition because of a too-rapid influx of water, which cannot beaccommodated by the weakened or dam-aged structural components of the cell. Infact, rapid rates of drying may predisposeseeds to imbibitional injury, as indicatedby increased rates of solute leakage, asymptom of cellular membrane disruption.However, slowing the rate of hydrationmay prevent a loss of germinability ofrapidly dried seeds by allowing time forrepair to occur. Germinability of soybeanseeds (following an accelerated ageingtreatment) is increased from 10% to 90%by controlling the rate of imbibition(Tilden and West, 1985). Since seedsbecome capable of surviving rapid waterloss during later development, they mustacquire a greater cellular and metabolicresistance to this event or have anenhanced ability to effect repair during theearly stages of imbibition.

The capacity to withstand rapid or slowdesiccation during the tolerant phase oforthodox seed development varies betweenspecies. An intolerance of rapid desiccation

occurs during the first 55 days of develop-ment of R. communis seeds, although theseseeds can tolerate slow drying at stages asearly as 25 DAP (Kermode and Bewley,1985). Seeds (and isolated embryos) of somemembers of the Gramineae, on the otherhand, can survive and germinate following adrastic drying treatment (which reducestheir water content to around 5%) at rela-tively early stages of development (Bartels etal., 1988; Bochicchio et al., 1988). The rea-sons for the variation between species in therate of water loss tolerated during theirdevelopment are not known.

5.2.5. Variation in desiccation toleranceacross species

(see also Chapter 8)

A wide range of species growing in differ-ent habitats produce seeds that cannot sur-vive drying to the low moisture contentthat enables prolonged storage of orthodoxseeds. These species are spread widelythrough the plant kingdom and updatedlists continue to be produced (Chin andRoberts, 1980; Hofmann and Steiner, 1989;Hong et al., 1996). Since Roberts (1973)introduced the terms orthodox and recalci-trant, it has become clear that the degree towhich seeds can survive desiccation variesgreatly both within and between non-orthodox species. The moisture contents towhich seeds of different species can sur-vive dehydration ranges from just less thanthat of vegetative tissues to almost com-plete tolerance. A recent study has shown acontinuous scale of desiccation toleranceacross 64 orthodox and recalcitrantspecies, with critical water contents forsurvival ranging from 0.1 to > 1.2 g g�1 dryweight (Sun, 1999). Even in geneticallyrelated species there is wide variation indesiccation tolerance at shedding, forexample, within the genera Shorea, Hopeaand Dipterocarpus (Tompsett, 1987), Citrusand Quercus (Sun, 1999) and Coffea(Dussert et al., 1999). Species within thegenus Acer (Hong and Ellis, 1990; Dickie etal., 1991; Fig. 5.4) produce orthodox andrecalcitrant seeds.



The restrictive categorization of seedsintroduced by Roberts (1973), particularlywith the introduction of an intermediatecategory (Ellis et al., 1990, 1991a,b),remains useful in so far as it fulfils its origi-nal purpose to describe storage behaviour.However, it does not accurately reflectknowledge of seed response to desiccation.

Consideration of variation in the desicca-tion tolerance of seeds across species as acontinuum of behaviour in response to dry-ing is arguably more realistic (Farrant etal., 1988; Berjak and Pammenter, 1997;Pammenter and Berjak, 1999). Such varia-tion, while tedious to categorize, providesan opportunity to increase our understand-


See

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Seed development

100

80

60

40

20

Germinationbefore drying

Germinationafter drying

August September October November

Norway maple

Sycamore

Shedding

Seed development

Fig. 5.4. Seed development on adjacent trees of orthodox Acer platanoides (Norway maple) and recalcitrantAcer pseudoplatanus (sycamore). Adapted from data in Hong and Ellis, 1990 and Dickie et al., 1991.


ing of the basis of desiccation tolerance,which has only just begun to be exploited.

A number of studies have shown thatdesiccation-sensitive seeds do not passthrough a fully desiccation-tolerant phaseduring their development, but tolerancetends to increase to a maximum near thetime of shedding as moisture contentdeclines (Figs 5.4 and 5.5; reviewed byFinch-Savage, 1996; Berjak andPammenter, 1997). Thus, the extent ofseed maturity at harvest is one of a num-ber of factors, discussed below, that deter-mines the degree of desiccation toleranceobserved in sensitive seeds (e.g. as deter-mined by critical water content experi-ments). The increase in desiccationtolerance as water content declines duringdevelopment (Fig. 5.5) and the apparentcontinuous range of critical moisture con-tents observed across species suggests thatdesiccation tolerance is a quantitative fea-ture. However, it is more accurate toexpress the degree of desiccation tolerancein terms of water potential as this reflectsthe amount of water available to the cyto-plasm (see Chapter 2). When data are pre-sented in this way, there is convincingevidence that tolerance appears to beacquired in discrete water potential stepsduring development (Farrant and Walters,1998), and the tolerance of species may begrouped according to these steps (Walters,1999; see Chapter 9), though in other stud-ies this was thought unlikely (Sun, 1999).It is argued that these critical water poten-tials, which have different water activitiesand associated metabolic processes(reviewed by Vertucci and Farrant, 1995;see also Chapters 2 and 9), may be relatedto specific desiccation stresses and dis-crete patterns of gene expression (Walters,1999). Thus, critical water potentials maybe determined by specific tolerance mech-anisms or by sufficient accumulation ofdesiccation protectants.

Within a species, seed tolerance of des-iccation can vary according to provenanceeven to the extent that one species, neem(Azadirachta indica), has been describedas both orthodox and recalcitrant (Berjakand Pammenter, 1997). However, it is not

known what proportion of this variation isgenetic in origin or due to the environ-ments in which seed development, storageor drying occurred. Temperature of dryingcan alter tolerance to desiccation (Ellis etal., 1990, 1991a; Berjak et al., 1994). Rateof drying and the extent of storage beforedrying are also important time-dependentfactors that could alter the extent of metab-olism-induced damage accumulated dur-ing desiccation (Pammenter and Berjak,1999). Such damage would alter the seed’sinherent capacity for desiccation toleranceand may obscure discrete critical waterpotentials for survival. A further compli-cating factor is that the onset of metabo-lism leading to the completion ofgermination is often observed in recalci-trant seeds following harvest or naturalshedding (Farrant et al., 1988; Pammenterand Berjak, 1999), and this is known toprogressively increase sensitivity in toler-ant species (Section 5.2.3).

5.2.5.1. Seed development in recalcitrantspecies

With the exception of A. marina, the phys-iology of seed development following ini-tial histodifferentiation has strongsimilarities across the recalcitrant speciesso far studied in detail (reviewed by Finch-Savage, 1996; Berjak and Pammenter,1997). This general pattern of recalcitrantseed development is also similar to that oforthodox seeds before they reach maxi-mum dry weight (mass maturity) andrapidly lose water following vascular sepa-ration (Finch-Savage, 1996; Farrant et al.,1997). For example, when adjacent trees ofthe sympatric species Acer pseudoplatanus(recalcitrant) and Acer platanoides (ortho-dox) are compared, there is a strong tempo-ral correlation in developmental eventssuch as the accumulation of seed reservesand the development of germinability (Fig.5.4). However, there are a number of com-mon characteristics of recalcitrant seeddevelopment that contrast with those oforthodox seeds and which result in seedsadapted for rapid germination and estab-lishment. In general, recalcitrant seeds at



shedding have had almost no net loss ofwater; they can still be accumulating dryweight, they retain active metabolism,remain desiccation-sensitive and have norequirement for desiccation to stimulatesubsequent germination. Despite theseapparent adaptations for rapid germina-tion, a few temperate recalcitrant speciesare dormant at shedding (Aesculus hip-pocastanum, Tompsett and Pritchard,

1993; A. pseudoplatanus, Hong and Ellis,1990). In contrast, viviparous germinationis a common event in other tropical recal-citrant species such as Telfairia occiden-talis (Akoroda, 1986) and A. marina(Farrant et al., 1993b).

During development, tolerance to desic-cation increases throughout reserve accu-mulation as percentage moisture contentdecreases in most recalcitrant seeds as it


Seed development

Reserve accumulation(a)

(b)

70

60

50

40

30

20

35

30

25

20

Moi

stur

e co

nten

t at 5

0% v

iabi

lity

(%)

40 45 50 55 60 65 70 75 80 85

45 46 47 48 49 50 51 52 53 54 55

Moisture content at harvest (%)

Fig. 5.5. The relationship between moisture content at harvest and desicccation tolerance (moisture contentat 50% viability) (a) during seed development in Quercus robur in 1989, and (b) following shedding in 1989(� ), 1990 (� ), 1991 (�), early in 1993 (� ) and late in 1993 (∆ ). The linear regression in (a) and (b) isfitted to 1989 data (r 2 = 0.937, d.f. 5). (From Finch-Savage and Blake, 1994.)


does in orthodox species (Hong and Ellis,1990; Finch-Savage, 1996; Berjak andPammenter, 1997; Farrant et al., 1997).These changes are concomitant with areduction in vacuolar volume, the appear-ance of a semi-viscous state and otherchanges associated with desiccation toler-ance (Farrant and Walters, 1998). However,in recalcitrant seeds there appears to be noclear end point to development. For exam-ple, seeds of Quercus robur are shed at dif-ferent moisture contents in different yearson the same tree and those shed with thelowest moisture content are most tolerantto desiccation (Fig. 5.5; Finch-Savage andBlake, 1994). There is a linear relationshipbetween moisture content at harvest (pre-mature and at shedding) and the moisturecontent at which 50% of seeds remainviable during drying. This contrasts withorthodox species, where desiccation toler-ance continues to increase after the acqui-sition of maximum seed dry weight, duringmaturation drying, which results in a qui-escent seed (Sanhewe et al., 1996). In Q.robur, development is indeterminate, butconsistent in several respects with that oforthodox seeds shed early before massmaturity and therefore before full desicca-tion tolerance is acquired (Finch-Savageand Blake, 1994). It is therefore tempting tosuggest that the level of desiccation toler-ance may depend upon how far seeds of aspecies progress through development,possibly an evolutionary consequence ofthe environmental and selection pressuresthat were exerted on them in the past.These ideas are taken further in a compari-son of development in orthodox P. vulgarisseeds, and the recalcitrant seeds of A. hip-pocastanum and A. marina (Farrant et al.,1997). One possibility in temperate cli-mates is that the onset of winter truncatesseed development so that full tolerancedoes not develop. However, seeds of Q.robur produced on trees grown outside thetemperate climate zone in the apparentlynon-limiting environment and seasonlength of South Africa also failed to pro-duce desiccation-tolerant seeds (Finch-Savage and Blake, 1994). In addition,experimental manipulations of seed devel-

opment in recalcitrant species have not sig-nificantly improved their desiccation toler-ance, suggesting inherent limitations to thedevelopment of full tolerance.

A. marina will tolerate little drying andcan be considered to be at the extreme sen-sitive end of the tolerance continuumacross species; developmental age has lit-tle influence on the desiccation sensitivityof seeds (Farrant et al., 1993b). In someother species, maximum desiccation toler-ance is reached at a point before shedding(e.g. A. pseudoplatanus, Hong and Ellis,1990) and tolerance may then subse-quently decline (e.g. Litchi chinensis,Clausena lansium and Coffea arabica)(Ellis et al., 1991a; Fu et al., 1994). Thisdecrease in tolerance may be due to theinitiation of germination (Farrant et al.,1988; Hong and Ellis, 1992). A gradualdecrease in tolerance is then shown as ger-mination proceeds in desiccation-sensitiveseeds (Farrant et al., 1988) as it does inorthodox seeds (Hong and Ellis, 1992).

5.2.5.2. Time-dependent effects of storageand drying rate

Many recalcitrant seeds are characteristi-cally large and consequently dry slowly.But even when the seeds are of similar sizeto comparable orthodox seeds, such as inA. pseudoplatanus (recalcitrant) and A.platanoides (orthodox), the recalcitrantAcer takes 12 times as long to reach 20%moisture content under the same dryingconditions (Greggains et al., 2000a). A fur-ther characteristic of recalcitrant seeds isthat even in the absence of desiccation theydeteriorate rapidly and are therefore short-lived. Differences in the reported criticalwater contents for recalcitrant seeds can begreatly influenced by these factors, and thethreshold water potentials for a number ofother physiological processes can be highlydependent on the postharvest history of theseed (Tompsett and Pritchard, 1998). Acommon observation is that if seeds arestored before drying they become moredesiccation-sensitive (Finch-Savage et al.,1996). This may be because of damageaccumulated during storage (Greggains et



al., 2000b) or because storage has allowedthe initiation of germination (Farrant et al.,1985; Berjak et al., 1989). However, insome other species, particularly in seedsshed early, development may continue sothere is a considerable delay before theinitiation of germination (Berjak andPammenter, 1997).

Drying rate can affect the apparent toler-ance of whole seeds (Farrant et al., 1985;Pritchard, 1991; Pammenter et al., 1998,1999; Chapter 3), although this is notalways the case (Finch-Savage, 1992). Inembryonic axes, as discussed below, rapiddrying consistently improves their survivalto lower moisture contents, perhapsbecause there is less time for damage toaccumulate during drying. However,Pammenter and Berjak (1999) pointed outthat rapid drying does not confer improveddesiccation tolerance because axes thathave been rapidly dried to lower moisturecontents do not survive long under ambi-ent conditions.

5.2.5.3. Desiccation tolerance differsbetween seed tissues

In general, when isolated, the embryonicaxis of desiccation-sensitive species ismore tolerant than when it is dried in thewhole seed (Berjak et al., 1990; Pammenteret al., 1991; Leprince et al., 1999), whichmay result from its more rapid dryingwhen excised than when in situ (Berjak etal., 1990). It may also relate to a signifi-cantly smaller proportion of ‘matrix-bound’(Finch-Savage, 1992; Finch-Savage et al.,1993) or non-freezable water (water that isbound or structure-associated; Berjak et al.,1992) in the axis compared to storage tis-sues. Desiccation tolerance of Landolphiakirkii axes is also affected by developmen-tal status and, in contrast to the wholeseed, immature axes are more tolerant thanmature ones (Berjak et al., 1992) as a resultof a lower content of non-freezable water inthe immature axes. Despite their greaterdesiccation tolerance, the immature axes,unlike mature axes, do not survive expo-sure to very low temperatures and aretherefore unsuitable for cryopreservation

(Vertucci et al., 1991). It appears from thisand similar published work that desicca-tion sensitivity in recalcitrant seeds andexcised embryonic axes can occur at a min-imum of two levels, which are influencedby the rate of drying:

1. The removal of freezable water is toler-ated and minimum survivable moisturecontent coincides with the quantity of non-freezable (matrix-bound) water in the tis-sue. Farrant et al. (1988) suggested thatrecalcitrant seeds, unlike orthodox seeds,require this bound water for the mainte-nance of membrane integrity. 2. Seed viability is lost as freezable (free)water is removed.

The former situation usually occurs inrapidly dried embryonic axes (Berjak etal., 1992), but has also been reported inrelatively desiccation-tolerant recalcitrantseeds (Finch-Savage, 1992; Finch-Savageet al., 1993). The second situation occursin more sensitive recalcitrant species. Athird level of sensitivity, which is unaf-fected by drying rate, is thought to occurin the most sensitive seed: mechanicaldamage resulting from a reduction in cellvolume in the early stages of drying(Pammenter and Berjak, 1999). In allcases, solute leakage precedes viabilityloss during desiccation, suggesting thatsignificant membrane damage hasoccurred. These differences in criticalmoisture levels are consistent with theconcept of discrete water potential stepsassociated with desiccation tolerance(Farrant and Walters, 1998; Walters, 1999)and may be related to the different desic-cation stresses encountered. In specieswhere loss of viability appears to coincidewith removal of non-freezable water(Berjak et al., 1992; Finch-Savage, 1992),it may be that these seeds lack mecha-nisms required to stabilize membranes aswater is removed. Where viability is lostas freezable water is removed, othermechanisms may be limiting, such as theprovision of adequate protection againstfree-radical attack. The provision of puta-tive protective mechanisms in seeds isreviewed in the following section.



5.2.6. Mechanisms underlying theacquisition of desiccation tolerance: recent

findings and speculations

5.2.6.1. The effects of premature desiccationduring the tolerant and intolerant stages of

orthodox seed development

Premature desiccation during the earlydevelopmental stages of P. vulgaris (up to22 DAP, i.e. during the desiccation-sensi-tive stage) drastically reduces the metabolicand cellular integrity of the axis upon sub-sequent rehydration. Particularly evident isa loss in the capacity to recover polyribo-some levels and to resume protein synthe-sis (Dasgupta et al., 1982). Considerabledamage is inflicted upon cellular organelles(including protein bodies and mitochon-dria) and upon the nuclear membrane. Incontrast, such severe perturbations do notoccur following desiccation at a tolerantstage, e.g. at 32 DAP. Moreover, the limiteddamage that is sustained during drying atthis stage is rapidly reversed followingrehydration; cells regain their normalappearance within a very short time.

Studies on the effects of desiccationduring the sensitive stages of seed develop-ment (or germination) suffer the limitationof not distinguishing between the causes ofdesiccation intolerance and changes duringthe death of cells as a consequence ofundergoing desiccation. Nevertheless, afew of these studies (particularly those thathave compared the effects of drying at thesensitive and tolerant stages) have pro-vided some useful information on the cel-lular sites and/or metabolic processes thatare most susceptible to damage during des-iccation/rehydration, and hence requireprotection for retention of viability (seeChapters 9 and 12). As implied earlier, theintegrity of membranes in seeds is of cru-cial importance to the maintenance of via-bility; any undue disruption of themembrane systems during drying is likelyto be of immediate consequence once theseed imbibes. It is probable that somechanges in membrane structure are pro-voked as a consequence of desicccation(even during the tolerant stages of develop-

ment), but the physical nature of suchchanges is enigmatic. While drasticchanges to membranes have been observedupon their drying in vitro (Crowe et al.,1992), the evidence suggests that mem-branes (in the desiccation-tolerant state)appear to be protected against certainmajor alterations (e.g. transformation of thebilayer arrangement to hexagonal-typearrangements). The importance of a preser-vation of basic membrane composition dur-ing drying is obvious, for cells wouldsurely perish without the prompt re-estab-lishment of functioning membranes uponrehydration when they are challenged by aswiftly changing hydration environment.

Fourier transform infrared microspec-troscopy has been useful for elucidatingsome of the changes to membranes andother cellular constituents (e.g. proteins) fol-lowing desiccation at the tolerant and sensi-tive stages of seed development (Wolkers etal., 1998b, 1999; see Chapter 4). Isolatedimmature maize embryos acquire a toler-ance to rapid drying between 22 and 25DAP, but can tolerate slow drying from 18DAP onwards. Rapid drying at the tolerantstages is associated with lower membranepermeability upon rehydration in contrastto embryos rapidly dried at a sensitive stage,in which there is an almost complete loss ofmembrane integrity. In addition, there is agreater proportion of �-helical protein struc-tures in embryos rapidly dried at a tolerantversus an intolerant stage (Wolkers et al.,1998b). The proportion of �-helical proteinstructures increases in the axes of embryosduring slow drying of 20 and 25 DAP seeds(as compared with that within fresh devel-oping seeds at these stages), and this factorcoincides with the acquisition of additionaltolerance of desiccation.

5.2.6.2. Cellular and metabolic changesduring the transition to a desiccation-tolerant

state

DEHYDRINS AND LATE EMBRYOGENESIS ABUNDANT

PROTEINS ARE PRODUCED AS PART OF THE DEVELOP-MENTAL PROGRAMME. As noted earlier,orthodox seeds are not capable of with-



standing desiccation at all stages duringtheir development, but their potentialacquisition of tolerance is usually sub-stantially earlier than the onset of the nat-ural drying event itself. A highlyabundant set of hydrophilic proteinsexhibiting temporal regulation duringseed development (i.e. the late embryoge-nesis abundant (LEA) proteins firstdescribed in cotton) has been implicatedin desiccation tolerance (Dure, 1993; seealso Chapters 1, 10 and 11). The genesencoding these proteins arise as highlycoordinately regulated sets, which on thisbasis comprise two distinct classes in cot-ton (Hughes and Galau, 1989); the mRNAsthat correspond to the two classes peakjust prior to, or during, desiccation(Hughes and Galau, 1989). LEA proteinsynthesis constitutes a large proportion ofthe translational activity of the cottonembryo during late maturation (up to25%), regulated at the level of transcrip-tion, i.e. by the abundance of lea mRNAs(Hughes and Galau, 1987). In mature cot-ton embryos they comprise about 2% ofthe total soluble protein (Dure, 1993) orabout 30% of the non-storage protein moi-ety (Hughes and Galau, 1987).

Since their description in cotton, mes-sages homologous to the lea cDNAs of cot-ton (representing at least five conservedfamilies of corresponding proteins) havebeen found in abundance in mature dryembryos and storage organs of manydiverse plant species includingArabidopsis thaliana, several crop speciesand gymnosperms. Protein families relatedto some of the LEA proteins are inducedduring drying of xerophytic species, e.g.the desiccation-tolerant resurrection plant(Craterostigma plantagineum), which iscapable of surviving in the desiccated statefor long periods and resumes full physio-logical activity within several hours ofrehydration (Bartels et al., 1990;Piatkowski et al., 1990; reviewed inIngram and Bartels, 1996; see Chapter 11).The desiccation-related proteins accumu-late in leaves; some are also present withinroots and in seeds. Proteins sharing fea-tures with plant LEA proteins (e.g. a high

glycine content and a high hydrophilicityindex) also accumulate in Escherichia coliand in the yeast Saccharomyces cerevisiaeas an adaptive response to hyperosmoticconditions; the authors suggest that mostLEA proteins are part of a more wide-spread group that they term ‘hydrophilins’(Garay-Arroyo et al., 2000).

A subset of the LEA proteins (includingthe LEA D-11 family and some denotedRAB (responsive to abscisic acid (ABA)) inrice) have been termed dehydrins; theyexhibit some common features in theirstructure that may be important for theirputative protective function (Close, 1996).Dehydrin genes exhibit a flexible expres-sion repertoire, being responsive to bothdevelopmental and environmental cues(reviewed by Thomas et al., 1991).Transcription of these genes is alsoinduced in virtually all seedling tissuessubjected to water stress (i.e. non-lethaldesiccation). Thus, the protective role ofdehydrins in the survival of water loss ispurported to be dual: during maturationdrying of the developing seed and follow-ing germination/growth of the mature seed(i.e. in seedlings or plant vegetative tissuesundergoing mild water stress) (Fig. 5.3).Precocious appearance of the proteins andtheir mRNAs can be induced in culturedimmature embryos by exogenous ABAtreatment. It has been hypothesized that,during normal development, high levels ofABA induce the accumulation of thesepolypeptides and hence prepare theembryo for desiccation or possible cellulardisruption upon subsequent rehydration(reviewed by Kermode, 1990, 1995; Bray,1991, 1993; Bewley and Oliver, 1992;Chandler and Robertson, 1994; Ingram andBartels, 1996).

REGULATION OF DEHYDRIN AND LEA GENE EXPRESSION

BY ABA. Since ABA has been implicated as amediator of stress responses, especiallywhere water stress is concerned, its poten-tial role as the primary regulator of leagenes in vegetative tissues has been inves-tigated (reviewed by Bray, 1991; Chandlerand Robertson, 1994; Kermode, 1995;Ingram and Bartels, 1996; Plant and Bray,1999). In some cases (but not all) ABA



application stimulates the accumulation ofthe mRNAs in the absence of water stress,and there is some evidence that endoge-nous ABA plays a regulatory role in theirexpression in seedling tissues (Fig. 5.3).For example, there is excellent correlation(e.g. in barley and maize) between theamounts of mRNA and ABA in shoots,roots and aleurone layers from either well-watered, dehydrated or dehydrated/rehy-drated seedlings (Chandler et al., 1988;Gomez et al., 1988). Other stresses thatlead to increased endogenous ABA (e.g.salt, cold and wounding) are often alsocapable of eliciting expression of thesegenes. The most convincing evidence forthe role of ABA in dehydrin gene expres-sion comes from studies of ABA-deficientmutants of maize (Pla et al., 1989, 1991).When exposed to dehydration stress,seedlings homozygous for mutations lead-ing to vivipary (e.g. vp2 and vp5) fail toelevate ABA levels and show a correspond-ing inability to produce dehydrins. Similarresults have been found in an ABA-defi-cient mutant of tomato (Cohen and Bray,1990; reviewed by Bray, 1991).

What is the evidence that ABA plays acentral regulatory role in the expression oflea genes within the developing seed?Generally, the mRNAs encoding LEA pro-teins are detected in embryos around mid-development; highest levels of expressionoccur either at incipient desiccation orduring maturation drying itself (Gomez etal., 1988; Mundy and Chua, 1988; Close etal., 1989). The mRNAs are preserved inthe mature dry seed but are rapidlydegraded upon imbibition, although, insome cases, certain proteins persist follow-ing imbibition (Han et al., 1996).Precocious appearance of the proteins andtheir mRNAs can be induced in culturedimmature embryos by exogenous ABA.Thus, high levels of ABA during mid-development are thought to induce theaccumulation of these polypeptides andhence prepare the embryo for desiccationor possible cellular disruption upon subse-quent rehydration (Fig. 5.3).

A comparative analysis of wild-type,ABA-deficient and ABA-insensitive mutants

of maize has been undertaken in order toelucidate the possible regulatory role ofABA and to address whether discrete paral-lel ABA and stress response pathways existin developing maize embryos (Finkelstein,1993). However, substantially differentresults have been obtained depending onthe type of LEA protein under study andmore systematic and detailed investigationis needed. Several 23- to 25-kDa proteinscorresponding to RAB17 are expressed nor-mally in the ABA-deficient mutants ofmaize (vp-2 and vp-5) in contrast to thedependency on applied ABA for theirexpression in vegetative tissues of themutant seedlings (Pla et al., 1989). Likewise,the regulation of the Rab28 gene (a homo-logue of the cotton lea D34 gene) in excisedyoung embryos of the ABA-deficient vp-2mutant closely resembles that found in non-mutant excised young embryos (Pla et al.,1991). In contrast, embryos of the ABA-insensitive mutant of maize (vp-1) do notaccumulate Rab28 transcripts to significantamounts during development; surprisingly,induction of Rab28 mRNA can be achievedin these young vp-1 embryos by ABA treat-ment (Pla et al., 1991). Expression of themaize Em gene (a group 1 lea gene) may bedependent on both the presence of ABAwithin embryos and its perception via afunctional Vp-1 gene product; it is barelydetectable in the ABA-deficient mutantembryos and is undetectable in the ABA-insensitive vp-1 embryos. Nevertheless, vp-1embryos do exhibit a response to both ABAand osmotica at the molecular level, sincethey accumulate specific gene products (22-and 30-kDa polypeptides) differentiallyupon imposition of osmotic stress or exoge-nous ABA (Butler and Cumming, 1993).

The Vp-1 gene is thought to encode anovel type of transcription activator, whichplays a role in the expression of ABA-responsive genes during seed development(e.g. maize globulin and Em genes), similarto the Abi-3 gene product in Arabidopsisand other species (reviewed by Giraudat etal., 1994; Hattori et al., 1995; McCarty,1995). The promoter elements of the riceOsem gene (an Em-type gene) required forregulation by VP-1 have been identified



(Hattori et al., 1995). These includeTACGTGTC (an ABA-responsive element orABRE), a small sequence located just down-stream of the ABRE and a quantitiative ele-ment (the sph box/RY repeat), conserved inmany seed-specific gene promoters.

Accumulation of group 3 LEA proteinsin maturing maize embryos may be depen-dent upon ABA but appears to have nospecific requirement for the Vp-1 geneproduct (Thomann et al., 1992).

Interestingly, when an ABA-deficient,viviparous mutant of maize (vp-5) ismanipulated either genetically or viabiosynthesis inhibitors to induce gib-berellin (GA) deficiency during early seeddevelopment, vivipary is suppressed indeveloping kernels and the seeds acquiredesiccation tolerance and storage longevity(White et al., 2000). Major accumulation ofGA1 and GA3 occurs in wild-type maizekernels, just prior to a peak in ABA contentduring development. It is speculated thatthese GAs induce a developmental pro-gramme that leads to vivipary in theabsence of normal amounts of ABA, andthat a reduction of GAs re-establishes anABA/GA ratio appropriate for suppressionof germination and induction of matura-tion. Induction of GA deficiency does notsuppress vivipary in vp-1 mutant kernels,suggesting that VP-1 acts downstream ofboth GA and ABA in programming seeddevelopment (White et al., 2000).

Two ABA-deficient mutants of sun-flower have been isolated – nd-1, an albino,non-dormant and lethal mutant exhibitinga very low ABA content and no accumula-tion of ABA in response to stress, and w-1,a wilty mutant, with reduced ABA accu-mulation during embryo and plantletdevelopment and drought stress (Giordaniet al., 1999). The w-1 mutant exhibits areduction of dehydrin transcripts in theearly stages of embryo development ascompared with wild-type embryos, indicat-ing that ABA affects dehydrin accumula-tion; however, the amount of dehydrintranscripts appears to be independent ofABA content during late embryogenesis.Accumulation of dehydrin transcriptsoccurs in the leaflets and cotyledons of nd-

1 plantlets subjected to drought stress.Thus, there may be two regulation path-ways that mediate dehydrin transcriptaccumulation in seeds and stressed vegeta-tive tissues – an ABA-dependent pathwayand an ABA-independent pathway;together, these pathways may have cumula-tive effects (Giordani et al., 1999).

Ectopic expression of the Abi-3 geneproduct (Giraudat et al., 1992) allows theABA-mediated activation of lea genes invegetative tissues of A. thaliana (Parcy etal., 1994). Seed viability is not altered inABA-deficient (aba) and ABA-insensitive(abi-3) mutants of A. thaliana, yet seeds ofdouble mutants exhibiting these two traitsdo not undergo desiccation on the parentplant, are intolerant of artificial desiccationand fail to produce some of the late abun-dant proteins (Koornneef et al., 1989;Meurs et al., 1992). These double-mutantseeds accumulate only low amounts of themajor storage proteins and are deficient inseveral low-molecular-weight polypep-tides, both soluble and bound, some ofwhich are heat-soluble. During develop-ment (14–20 DAP), the low amounts of var-ious maturation-specific proteins aredegraded and proteins characteristic of ger-mination are induced, in the absence ofgermination. Here, the seed developmentalprogramme is not completed, and there is apremature (yet incomplete) switching to agermination programme in the absence ofsubstances presumed to be protectiveagainst desiccation. Seeds become desicca-tion-tolerant when the plants are wateredwith an ABA analogue (LAB 173711) or byincubating isolated immature seeds (11–15DAP) with ABA and sucrose. Whereassucrose may protect desiccation-sensitivestructures from damage, ABA inhibits pre-cocious germination and may be requiredfor, or is accompanied by, completion ofthe seed developmental programme andassociated acquisition of desiccation toler-ance (Meurs et al., 1992). In another study,expression of a specific lea gene (a group 1,D19/Em homologue) was found to beslightly reduced in seeds of theArabidopsis aba mutant, but was reducedby approximately tenfold in the abi-3



mutant; expression in the double mutantwas not studied (Finkelstein, 1993). A dif-ferent Arabidopsis abi-3 mutant (abi-3-3,isolated by screening for mutants that ger-minate in the presence of the GA biosyn-thetic inhibitor, Uniconazol) showedabnormal seed development, remaininggreen until maturity, had dramaticallyreduced amounts of storage proteins, wasdesiccation-sensitive, and lacked dor-mancy, indicative of a possible role for theAbi-3-3 gene in the control of the synthesisof seed storage proteins and desiccationprotectants (Nambara et al., 1992). ABI5, amember of the family of basic leucine zip-per transcription factors, regulates a subsetof lea genes during seed development andin vegetative tissues in the presence ofABA (Finkelstein and Lynch, 2000).

Further studies to clarify the role ofABA and other components of the signaltransduction pathway leading to lea geneexpression and other late maturationevents in developing seeds will be awaitedwith interest. Recessive mutants ofArabidopis with lesions at the Fusca3(fus3) and Leafy Cotyledon (lec1) gene locilead to various abnomalities during mid-embryogenesis and late embryogenesis,including loss of dormancy and failure toacquire desiccation tolerance (Kirik et al.,1998). FUS3 and LEC1 modulate the abun-dance of ABI3 protein in seeds and syner-gistic interactions between the threeproteins (ABI3, FUS3 and LEC1) arethought to control various key events,including accumulation of chlorophyll andanthocyanins, sensitivity to ABA andexpression of individual members of the12S storage protein gene family (Parcy etal., 1997). Interestingly, part of FUS3 (acontinuous stretch of 100 amino acids)shows significant similarity to the B3domain of the ABI3 and VP-1 proteins(Luerssen et al., 1998), a domain whichinteracts with the RY cis promoter motif ofseveral seed proteins. Thus, both FUS3 andABI3 may be essential components of a reg-ulatory network acting in concert throughthe RY-promoter element to control geneexpression during late embryogenesis andseed development (Reidt et al., 2000).

OTHER PROTECTIVE PROTEINS IMPLICATED IN DESICCA-TION TOLERANCE. Specific small heat-shockproteins (HSPs) of the cytosolic classes (Iand II) accumulate in seeds of several plantspecies. These proteins appear to be homo-geneously distributed in all tissues of theseed and a role in the acquisition of desic-cation tolerance has been suggested (Cocaet al., 1994; Wehmeyer et al., 1996; seeChapters 1 and 10). In Arabidopsis andother species, class I small HSPs are firstdetected during mid-maturation andbecome most abundant in dry seeds(Wehmeyer et al., 1996; Carranco et al.,1999). In some seeds (e.g. Arabidopsis), theproteins decline rapidly during germination(Wehmeyer et al., 1996); in others (e.g. sun-flower), they persist (Coca et al., 1994). TheAbi-3 gene product may activate expressionof genes encoding specific small HSPs dur-ing seed development. Transcriptional acti-vator mutants of Arabidopsis (abi-3-6,fus3-3 and lec1-2) that are desiccation-sen-sitive have undetectable amounts of HSP17.4(abi-3-6) or highly reduced amounts of theprotein (fus3-3 and lec1-2), i.e. less than 2%of that in wild-type seeds (Wehmeyer andVierling, 2000). Interestingly, a chimericgene consisting of the small HSP gene pro-moter linked to �-glucuronidase (GUS)shows strong expression in mutant seedsthat are heat-stressed, indicating that thegenes are under distinct developmentaland stress regulation.

Polypeptides produced in sunflowerseeds (e.g. HSP17.6 and HSP17.9, belong-ing to different families of cytoplasmicsmall HSPs) are indistinguishable fromlow-molecular-weight HSPs expressed invegetative tissues in response to waterdeficit, but they are different from homolo-gous proteins expressed in response tothermal stress (Coca et al., 1994; Carrancoet al., 1997, 1999). Proteins immunologi-cally related to two sunflower small HSPsare detected in unstressed vegetative tis-sues of the desiccation-tolerant resurrec-tion plant C. plantagineum and areinduced to higher levels in these tissuesby water stress and heat shock. In desicca-tion-sensitive Craterostigma callus tissue,there are no detectable small HSP-related



polypeptides, but their expression, and theconcurrent acquisition of desiccation tol-erance is induced by exogenous ABA(Alamillo et al., 1995).

Small HSPs, immunologically related toa 20-kDa HSP from desiccation-sensitivechestnut (Castanea sativa) seeds have beendetected in orthodox and recalcitrant seedsof 13 woody species; hence additional pro-teins or mechanisms are likely to beinvolved in desiccation tolerance (Colladaet al., 1997) (see later discussion).

Major intrinsic proteins (MIPs) are afamily of channel proteins that are mainlyrepresented by aquaporins in plants. Theyare generally divided into TIPs (tonoplastintrinsic proteins) and PIPs (plasma mem-brane intrinsic proteins) according to theirsubcellular localization (reviewed byMaurel et al., 1997). The vacuolar mem-brane protein, �-TIP (a water-channel pro-tein), accumulates during seed maturationin the parenchyma cells of seed storageorgans. Synthesis of this integral membraneprotein does not appear to be related (in aquantitative manner) to storage proteindeposition and a role in seed desiccation,cytoplasmic osmoregulation and/or seedrehydration has been suggested (Johnson etal., 1989). The water-channel activity of theprotein can be regulated by phosphoryla-tion and the protein assembles as a 60 Å �60 Å square in which each subunit isformed by a heart-shaped ring comprised of�-helices. This structure is remarkably sim-ilar to that of mammalian PIPs, suggestingthat the molecular design of functionallyanalogous and genetically homologousaquaporins is maintained between the plantand animal kingdoms (Daniels et al., 1999).

In the desiccation-tolerant resurrectionplant C. plantagineum, homologues to PIPsand TIPs are regulated by dehydration andABA, with members of a subset of PIPs(PIPa) being regulated by ABA-dependentand ABA-independent pathways (Mariauxet al., 1998).

In many seeds, the acquisition of desic-cation tolerance during the seed expansionstage of development occurs well beforethe completion of reserve deposition.

However, it is possible that the attainmentof a critical level of reserves is requiredbefore the seed can withstand desiccation(Kermode, 1997). Highly vacuolated cells(hence containing little reserve material)may undergo severe mechanical disrup-tion during water loss, and tearing orshearing of membranes (or other cellularcomponents) could lead to irreversiblechanges in their internal morphology. Thepresence of a critical level of cellularreserves would limit such changes (Table5.1). The quantity of reserves may alsomerit consideration in relation to the lossof tolerance during seed germination. Asnoted earlier, while soybean seed axesrapidly lose their tolerance to desiccationduring the course of germination, thecotyledons remain tolerant for a consider-ably longer period (Senaratna andMcKersie, 1983). The major breakdown ofreserves within the cotyledons is a post-germinative event; however, catabolism ofreserves within the axes occurs relativelyearly (i.e. during germination) to provide asource of nutrients. The decline ofreserves below a critical level within theaxes may contribute to a loss of desicca-tion tolerance within this germinating tis-sue. Interestingly, certain seed storageproteins are suggested to play a moredirect role in desiccation tolerance. Onemember of the vicilin superfamily in pea(psp54) is expressed during seed desicca-tion and is not detected prior to this stage;the mRNA encoding the protein declinessoon after imbibition, but can be detectedin vegetative tissues in response to water-deficit-related stresses and ABA (Castilloet al., 2000). A lower-molecular-weightprotein (p1), which corresponds to the C-terminal third of p54, shares some proper-ties with dehydrins and is suggested toprotect chromatin structure during desic-cation (Castillo et al., 2000). Seed storageglobulins of spermatophytes are thought tohave evolved from a group of ancient single-domain proteins of prokaryotes and fungi functional in cellular desicca-tion/hydration processes (Baumlein et al.,1995; Shutov et al., 1998).




Tab

le 5

.1.

Pos

sibl

e co

mpo

nent

s of

des

icat

ion

tole

ranc

e in

see

ds a

nd th

eir

prot

ectiv

e ac

tion.

a(B

ased

on

Ker

mod

e (1

990)

. With

per

mis

sion

from

CR

C P

ress

, Inc

.)

Site

or

proc

ess

affe

cted

Pro

tect

ive

com

pone

ntP

rote

ctiv

e ac

tion

Pos

sibl

e m

ode

of p

rote

ctio

n

Mem

bran

esC

arbo

hydr

ates

: suc

rose

Pre

vent

cha

nges

in s

elec

tive

perm

eabi

lity

Hyd

roxy

l gro

ups

of s

ucro

se r

epla

ce w

ater

on

plus

raf

finos

e an

d/or

due

to la

tera

l pha

se s

epar

atio

n of

hydr

ophi

lic (

pola

r) e

nd g

roup

s of

mem

bran

e

stac

hyos

eph

osph

olip

ids

in th

e bi

laye

r an

d th

e ph

ase

phos

phol

ipid

s; o

ligos

acch

arid

e (r

affin

ose

and/

or

tran

sitio

n fr

om li

quid

cry

stal

line

to g

elst

achy

ose)

inhi

bits

suc

rose

cry

stal

lizat

ion

durin

gdr

ying

, pre

vent

ing

loss

of i

ts p

rote

ctiv

e po

tent

ial

Lipi

d-so

lubl

e an

tioxi

dant

sA

s ab

ove;

pre

vent

de-

este

rifica

tion

ofS

cave

ngin

g ac

tivity

incr

ease

s re

sist

ance

to

(e.g

. toc

ophe

rols

)m

embr

ane

phos

phol

ipid

and

free

fatty

free

-rad

ical

-med

iate

d de

sicc

atio

n in

jury

acid

acc

umul

atio

nS

truc

ture

/met

abol

ism

Res

erve

s: c

arbo

hydr

ates

,P

reve

nt ‘w

hole

sca

le’m

echa

nica

lC

ritic

al le

vel o

f res

erve

s in

vac

uole

s/st

orag

e bo

dies

lipid

s, p

rote

ins

disr

uptio

n of

cel

lula

r co

mpo

nent

sco

nfer

s m

echa

nica

l str

engt

h to

who

le c

ell

Pre

vent

loss

of t

ight

ly b

ound

(‘v

ital’)

wat

erW

ater

-bin

ding

cap

acity

of c

ells

enh

ance

d w

ith

nece

ssar

y fo

r st

ruct

ural

and

func

tiona

lin

crea

sed

num

ber

of s

orpt

ion

site

sin

tegr

ity o

f bio

mol

ecul

esH

ydro

phili

c, d

enat

urat

ion-

As

abov

eW

ater

-bin

ding

cap

acity

of c

ells

enh

ance

d w

ith

resi

stan

t pro

tein

s (e

.g. L

EA

s,in

crea

sed

num

ber

and

stre

ngth

of s

orpt

ion

site

s;

othe

r de

sicc

atio

n-in

duci

ble

nativ

e co

nfor

mat

ion

of p

rote

ctiv

e m

olec

ules

mai

ntai

ned

poly

pept

ides

)th

roug

hout

dry

ing;

bin

d io

ns a

nd th

ereb

y co

unte

ract

dam

agin

g ef

fect

s of

incr

easi

ng io

nic

stre

ngth

s of

cy

toso

l dur

ing

dryi

ng‘R

epai

r’pr

otei

ns, p

rote

ases

,R

apid

re-

esta

blis

hmen

t of s

truc

tura

lE

ffici

ent r

epai

r of

mem

bran

es a

nd o

ther

cel

lula

rub

iqui

tin a

nd e

xten

sion

and

met

abol

ic in

tegr

ity fo

llow

ing

com

pone

nts

rest

ores

nor

mal

func

tioni

ng; a

idpr

otei

n, H

SP

/mol

ecul

arim

bibi

tion

prot

eins

in r

ecov

erin

g th

eir

nativ

e co

nfor

mat

ion;

chap

eron

es, s

ome

LEA

sde

grad

atio

n of

dam

aged

or

dena

ture

d pr

otei

ns

a Ref

er to

rev

iew

by

Ker

mod

e (1

990)

and

Ingr

am a

nd B

arte

ls (

1996

) an

d re

fere

nces

ther

ein.


ROLE OF SUGARS (see also Chapters 1, 10 and11). It is likely that the underlying basisof desiccation tolerance is diverse and isnot simply restricted to the synthesis ofspecific proteins. The ability to withstanddesiccation may also depend uponincreased amounts (or a heightened capac-ity to synthesize) molecules which stabi-lize membranes. Carbohydrates such astrehalose (a non-reducing disaccharide ofglucose) are effective in preserving thestructural and functional integrity of mem-branes in vitro at low water contents(reviewed by Crowe et al., 1992). Dryingand rehydration of the model membranesarcoplasmic reticulum usually results inthe fusion of vesicles and loss of the abilityto transport calcium. However, when disac-charides such as trehalose are present inconcentrations equivalent to those in desic-cation-tolerant organisms, functional vesi-cles are preserved. Membrane fusionduring desiccation is thought to be pre-vented as a result of the sugars’ hydroxylgroups interacting (i.e. forming hydrogenbonds) with the polar head groups of phos-pholipids and functional groups of pro-teins (Crowe et al., 1992). Thus, the sugarsare thought to alter physical properties ofdry membranes so that they resemble thoseof fully hydrated biomolecules (Crowe etal., 1992) (Table 5.1).

The occurrence of trehalose in high con-centrations in anhydrobiotic organismssuch as yeast and nematodes (up to 20% oftheir dry weight) suggests that this sub-stance may be involved in their desiccationtolerance (Crowe et al., 1992). A role forsucrose and raffinose (carbohydrates foundin much greater abundance in seed tissuesthan trehalose) in the preservation of mem-branes during drying has been suggested byLeopold and Vertucci (1986) and severalothers. Orthodox seeds accumulate consid-erable amounts of soluble proteins and sug-ars throughout maturation, and thesecollectively may be important in the acqui-sition of a desiccation-tolerant state (Amutiand Pollard, 1977). Stachyose accumulatesin immature soybean seeds subjected toslow drying, but does not increase signifi-cantly when seeds are maintained at high

humidity (Blackman et al., 1992). However,an increase in the amount of raffinose isnot correlated with the acquisition of des-iccation tolerance of wheat embryos (Blacket al., 1999). Accumulation of fagopyritolB1 (a galactopinitol) in buckwheat seeds istemporally associated with the acquisitionof desiccation tolerance during develop-ment (Horbowicz et al., 1998). This majorsoluble carbohydrate, which comprises40% of the carbohydrate of the maturebuckwheat embryo, declines with the lossof desiccation tolerance following germina-tion. Temperature conditions during seeddevelopment that have a favourable effect onvigour and storability of buckwheat seedsresult in seeds having a lower sucrose-to-fagopyritol ratio as compared with thosethat develop under non-optimal tempera-ture conditions (Horbowicz et al., 1998).

Although trehalose is not abundant invascular plants, it has been identified as amajor carbohydrate in more than 70species of desiccation-tolerant lower plants(reviewed by Muller et al., 1995). There arerecent reports of trehalose in relativelyhigh amount in two desiccation-tolerantangiosperms (reviewed by Muller et al.,1995; see references therein). One isMyrothammus flabellifolia, a dicotyledo-nous plant living in arid, rocky regions insouthern Africa, the leaves of which con-tain about 3% trehalose on a dry weightbasis. The other is the grass Sporobolusstapfianus, in which trehalose comprises2–5% of the total soluble carbohydrates.Interestingly, even though most higherplants contain low amounts of trehalose,high activities of trehalase, an enzymewhich degrades trehalose, have been found(reviewed by Muller et al., 1995). A.thaliana possesses genes for at least one ofthe enzymes required for trehalose synthe-sis, trehalose-6-phosphate phosphatase(Vogel et al., 1998).

As noted above, the protective effect ofsugars probably extends to preventing irre-versible changes to proteins. For example,phosphofructokinase is a tetramericenzyme, which is irreversibly denaturedduring desiccation, dissociating into inac-tive dimers. However, the disaccharides



sucrose, maltose and trehalose stabilize theactivity of the enzyme (in vitro) during dry-ing (Carpenter et al., 1987). Although theevidence from these experiments carriedout in vitro is convincing, the role of sugars(in vivo) in protecting cells during waterdeficit and during desiccation of orthodoxseeds remains to be elucidated.

One way sugars may protect the cellduring severe desiccation is by glass forma-tion (see Chapter 10); in the presence ofsugars a supersaturated liquid is producedwith the mechanical properties of a solid(Koster, 1991). Only sugar mixtures equiva-lent in concentration and composition tothose of desiccation-tolerant embryos areable to form glass at ambient temperature(Koster, 1991) and this ability has beenassociated with retention of viability ofmaize embryos (Williams and Leopold,1989). Glass formation has been suggestedto prevent cellular collapse during desicca-tion and to promote a state of metabolicquiescence by restricting diffusion of sub-strates and products within cells (Koster,1991). Carrot somatic embryos, when pre-treated with ABA, are able to tolerate slowdrying, but are still intolerant of rapid dry-ing. This appears to be due in part to theextent of protein denaturation, which isgreater after rapid drying (Wolkers et al.,1999). In contrast to slowly dried embryos,which form a glassy state at room tempera-ture, no clearly defined glassy matrix isformed in rapidly dried embryos. The aver-age strength of hydrogen bonding is less inrapidly dried versus slowly dried embryos,which may be indicative of less extensive‘molecular packing’ in the former. Sucroseaccumulates following rapid drying ofembryos; following slow drying, the trisac-charide umbelliferose is accumulated atthe expense of sucrose. In phospholipidmodel systems, both carbohydrates are ableto form a stable glass with drying; theydepress the transition temperature of dryliposomal membranes to an equal extent aswell as preventing leakage from dry lipo-somes upon subsequent rehydration.Likewise, both exhibit an equal capacity toprotect a desiccation-sensitive protein.Thus, increased umbelliferose in slowly

dried embryos cannot account for theirenhanced viability as compared withrapidly dried embryos; however, enhancedsynthesis of LEA proteins embedded in theglassy matrix may be a contributing factor(Wolkers et al., 1999).

The proteins of maturation-defectivemutants of Arabidopsis (e.g. abi-3 and lecmutants) appear to be more susceptible todenaturation during heating (Wolkers etal., 1998a). Proteins in dry wild-type seedsdo not denature at temperatures up to150°C; those of dry desiccation-sensitiveseeds (lec1-1, lec1-3 and abi3-5) denatureat 68, 89 and 87°C, respectively. In con-trast, in desiccation-tolerant seeds (abi3-7and abi3-1), denaturation commencedabove 120 and 135°C, respectively. The dif-ferential sensitivity of the seed proteins ofthe mutants to denaturation has beenattributed in part to differences in molecu-lar packing density, which is higher in drydesiccation-tolerant seeds than in dry des-iccation-sensitive seeds (Wolkers et al.,1998a).

OTHER MECHANISMS UNDERLYING DESICCATION TOLER-ANCE. The loss of desiccation tolerance dur-ing germination of soybean seeds is notassociated with any compositional changesin fatty acids, but is correlated with adecline in the quantity of lipid-solubleantioxidants in the membrane (Senaratna etal., 1985a,b). These antioxidants may con-tribute to the desiccation tolerance of axesduring the early stages of germination bypreventing changes in membrane fluiditycaused by free-radical attack on phospho-lipids in response to drying (Senaratna etal., 1985a,b). The transition to desiccationsensitivity following germination of pea andcucumber seeds is accompanied by a desic-cation-induced imbalance of metabolism(i.e. increased emission of CO2 and fermen-tation products such as acetaldehyde) in theradicle, which precedes loss of membraneintegrity (Leprince et al., 2000). Imbalancedmetabolism is significantly reduced whensensitive axes are dried in 50% O2 insteadof air and it is suggested that a balancebetween down-regulated metabolism and O2availability is associated with desiccation



tolerance. Products resulting from imbal-anced metabolism (e.g. acetaldehyde) dis-turb the phase behaviour of phospholipidvesicles and thus may aggravate membranedamage induced by dehydration (Leprinceet al., 2000).

Within the developing seed, the thiol-requiring (1-cysteine) peroxiredoxin familyof antioxidants may protect tissues (e.g. theembryo and aleurone layer of cereals) fromreactive oxygen species during desiccationand early imbibition (Haslekas et al., 1998;Stacy et al., 1999). PER1, a protein belong-ing to this family, is maintained in imbibeddormant barley seeds, but declines in thenon-dormant seeds. In immature embryosand aleurone layers, the protein resides inthe nucleus and is most abundant withinthe nucleolus (Stacy et al., 1999). In con-trast, in mature imbibed dormant seeds, anequivalent amount of protein is present inthe cytosol. In Arabidopsis, the expressionof AtPer1, a gene encoding a protein withsimilarity to barley PER1, is reduced inseeds of the ABA-insensitive mutant, abi3-1, but is unaltered in an ABA-defi-cient mutant of Arabidopis (aba-1)(Haslekas et al., 1998).

Serotonin accumulation in walnutcotyledons is thought to protect seeds fromtoxic ammonia concentrations followingseed desiccation (Schroder et al., 1999).

In conclusion, the basis of desiccationtolerance of developing seeds is still apoorly understood phenomenon. Whatemerges from the evidence available at pre-sent is a complex process involving variousmetabolic and/or structural adjustments,which allow cells to undergo extensivewater loss with a minimum of damage(Table 5.1). However, while maturationdrying may inflict limited damage on cellsof orthodox seeds, the capacity to reversesuch changes (i.e. to effect repair) uponsubsequent rehydration is probably an inte-gral feature of desiccation tolerance(Bewley and Oliver, 1992; Ingram andBartels, 1996; O’Mahony and Oliver,1999a,b). An L-isoaspartyl protein methyl-transferase that accumulates in wheatseeds during the late stages of caryopsisdevelopment may repair damaged proteins

by facilitating the conversion of abnormalL-isoaspartyl residues to normal L-aspartylresidues (Mudgett and Clarke, 1994). Asummary of some of the possible compo-nents of desiccation tolerance in seeds ispresented in Table 5.1.

DIFFICULTIES IN ASSESSING THE ROLE OF PROTECTANTS

IN DESICCATION TOLERANCE. As indicatedabove, a wealth of information has beenderived from the study of desiccation-toler-ant systems, such as seeds and resurrectionplants, and from the various molecular andbiochemical analyses that have contributedto our understanding of gene and proteinfunction. In orthodox seeds, the metabolicchanges that occur either prior to or duringmaturation drying (including the accumu-lation of oligosaccharides, sugars and LEAproteins) may have functional significancein protecting them against the rigours ofdesiccation and/or subsequent rehydration.However, some of the changes in metabo-lism of orthodox seeds during the time ofacquisition of desiccation tolerance maynot directly contribute to the ability towithstand water loss, but rather may bepre-programmed changes that are part ofother seed developmental programmes ulti-mately important for seedling survival.What are other research strategies that maycontribute to our understanding of theunderlying basis of desiccation tolerance?One strategy is the transfer of genes encod-ing putative desiccation protectants intotransgenic host plants with the ultimategoal of testing protein function andenhancing stress tolerance (see Chapter11). Another approach is the comparativeanalysis of LEA- and dehydrin-related pro-teins and other putative desiccation protec-tants in recalcitrant versus orthodox seeds(discussed below). Both approaches havelimitations.

5.2.6.3. Desiccation-tolerance mechanismsin sensitive seeds

In the previous sections we have shownthat development of orthodox seeds fol-lows a largely predetermined sequence ofevents that leads to desiccation and then



shedding of the seed in a quiescent (andsometimes dormant) state. In evolutionaryterms, it is not known whether the abilityto develop full desiccation tolerance hasbeen lost in species with recalcitrant seeds,was never gained, or is just not fullyexpressed. The progress of evolution is alsolikely to have differed among taxa (vonTeichman and van Wyk, 1994; see Chapter8). Despite increasing interest in recalci-trant seeds, it is clear from recent reviews(Berjak and Pammenter, 1997; Pammenterand Berjak, 1999) that the cause of theirdesiccation sensitivity is still far fromunderstood. However, comparison of desiccation-sensitive seeds with tolerantorthodox seeds can clarify our understand-ing of desiccation-tolerance mechanisms.In the following section, the occurrence ofthese putative mechanisms in desiccation-sensitive seeds is reported.

ABSCISIC ACID AND PROTEINS. As noted in theprevious sections, ABA may play a role inthe regulation of dehydrin and lea geneexpression in orthodox seeds. In the recal-citrant seeds of Theobroma cacao (Pence,1991) and Q. robur (Finch-Savage et al.,1992; Finch-Savage and Blake, 1994), thereis a clear pattern of ABA accumulationduring seed expansion, similar to thatreported in orthodox species, such as P.vulgaris (Prevost and Le Page-Degivry,1985a,b). In both the axis and cotyledons,ABA increases to a maximum and thendecreases before shedding. However, thedecline in ABA concentration prior toshedding in recalcitrant seeds is limitedand consistent with a continuing role forABA in preventing precocious germination(Finch-Savage and Farrant, 1997).Dehydrin proteins accumulate during seeddevelopment, and in response to seed dry-ing, in a number of recalcitrant species(Finch-Savage et al., 1994; Gee et al., 1994;Farrant et al., 1996; Han et al., 1997;Greggains et al., 2000a). However, dehy-drin proteins are not detected in matureundried axes of a range of recalcitrant trop-ical wetland species (Farrant et al., 1996).These species would not normally beexposed to significant drying and are con-

sidered to be particularly sensitive to des-iccation. The pattern of ABA concentrationin seeds of the tropical wetland species, A.marina, during seed expansion differs fromthat of other recalcitrant species; low anddecreasing concentrations of ABA are pre-sent in the axis during reserve accumula-tion (Farrant et al., 1993a). Moreover, ABAconcentration does not increase with dry-ing and dehydrin proteins are not detected(Farrant et al., 1996). In general, the pres-ence of dehydrins in recalcitrant seeds isassociated with those species that havehigh ABA concentrations and are mostlikely to be exposed to moisture stress(Farrant et al., 1996). In addition, an earlierpeak in ABA concentration of recalcitrantQ. robur seeds is associated with greaterdesiccation tolerance at shedding (Finch-Savage and Farrant, 1997). However, in T.cacao embryos in vitro, ABA is associatedwith maturation events as it is in orthodoxseeds, but does not influence desiccationtolerance (Pence, 1992).

In orthodox cotton, lea mRNAs thathave protein homology with dehydrinsaccumulate relatively slowly during theperiod of cotyledon expansion, but thenincrease rapidly at the point of vascularseparation (Galau et al., 1991). Recalcitrantseeds are often shed at a time when dryweight is still increasing and may thereforelack the phase of rapid increase in leamRNAs that occurs at the end of orthodoxseed development. Desiccation sensitivitymay therefore be due in part to an inabilityto accumulate a sufficient quantity of dehy-drins or other LEA proteins.

Small HSPs have also been associatedwith desiccation tolerance in orthodoxseeds (see earlier discussion; DeRocher andVierling, 1994; Wehmeyer et al., 1996), andhave been shown, like dehydrins, to accu-mulate to significant levels in recalcitrantC. sativa seeds during development and tobe present in seeds of other recalcitrantspecies (Collada et al., 1997). As discussedearlier, the presence of dehydrin and smallHSPs in recalcitrant seeds supports theview that they alone are not sufficient toconfer desiccation tolerance. However,Farrant et al. (1996) suggested that the con-



verse might be true, i.e. their absence mayimply an inability to tolerate desiccation,and the absence of a specific protectiveprotein cannot be ruled out as the cause ofdesiccation sensitivity. For example, themembranes surrounding oil bodies of seedscontain integral proteins, called oleosins,which may maintain the integrity of theseorganelles during desiccation and subse-quent imbibition (Murphy et al., 1995;Leprince et al., 1998). Oleosins are pre-sent in the membranes of oil bodies indesiccation-tolerant seeds, but are absent, ortheir amount is diminished, in desiccation-sensitive seeds (Leprince et al., 1998).Thus, a lack of oleosins may be an impor-tant factor in the desiccation sensitivity ofoil-storing recalcitrant seeds.

SUGARS. Studies with recalcitrant seedsshow that there is no clear link betweenthe presence of sugars and the level of des-iccation tolerance in seeds. For example,large amounts of sugars including sucroseand stachyose accumulate during develop-ment in the highly desiccation-sensitiveseeds of A. marina (Farrant et al., 1993b).In the more tolerant Q. robur, sucrose andraffinose accumulate in the cotyledons andaxes during the later stages of reserve accu-mulation (Finch-Savage et al., 1993; Finch-Savage and Blake, 1994) and, in matureaxes of Quercus rubra, desiccation sensitiv-ity is not caused simply by the absence ofnon-reducing sugars (Sun et al., 1994). In amore comprehensive study, Steadman etal. (1996) determined the sugar composi-tion of a range of recalcitrant, intermediateand orthodox species and combined thiswith published data for additional species.They found no simple relationship betweenseed type and total sugar content or sucroselevel; however, the content of raffinose andstachyose was generally lower in recalci-trant than in orthodox seeds. Theseoligosaccharides were also found to belower in seeds of recalcitrant A. pseudopla-tanus than in seeds of the orthodox A. pla-tanoides (Greggains et al., 2000a). Ingeneral, there are large variations in thecontent of sugars between tissues of desic-cation-sensitive seeds, but at least one tis-

sue in recalcitrant seeds tends to have amuch lower oligosaccharide:sucrose ratiothan that generally present in orthodoxseeds (Steadman et al., 1996). This ratio istherefore a potential indicator of seedbehaviour; however, seeds of cocoa and A.marina are exceptions (Steadman et al.,1996). Pammenter and Berjak (1999)pointed out that the proposed mechanismsfor the involvement of sugars in desicca-tion tolerance operate at moisture contentsbelow those at which most recalcitrantseeds can survive. It is therefore perhapsnot surprising that there is no clear rela-tionship between the degree of desiccationtolerance in recalcitrant seeds and sugaraccumulation.

High monosaccharide levels have beenlinked with desiccation sensitivity and thepotential for damage resulting from theMaillard reaction (Koster and Leopold,1988). In the later stages of development inorthodox seeds, monosaccharide levels arereduced and this also occurs in some(Farrant and Walters, 1998), but not all,species with recalcitrant seeds (Farrant etal., 1992, 1993b; Finch-Savage et al., 1993).Monosaccharide levels were generally lowin most of the 18 species studied bySteadman et al. (1996), including the recal-citrant ones.

IS DESICCATION SENSITIVITY DUE TO RETENTION OF

METABOLIC ACTIVITY AT SHEDDING? Recalcitrantseeds, perhaps because they remain moist,maintain active metabolism throughoutdevelopment to the time of shedding. Forexample, respiration of A. marina seeds,after a small decline at the start of reserveaccumulation, remains relatively constantuntil abscission (Farrant et al., 1992). Highrespiration rates have also been recorded inthe seeds of other recalcitrant species atshedding (Farrant et al., 1992, 1997;Poulsen and Eriksen, 1992; Finch-Savageand Blake, 1994; Salmen Espindola et al.,1994; Leprince et al., 1999). The absence ofsubstantial developmental arrest as seedsapproach shedding is confirmed by ultra-structural and biochemical studies (Doddet al., 1989; Berjak et al. 1992; Farrant etal., 1992; Farrant and Walters, 1998).



Indeed, in A. marina the limited de-differ-entiation of subcellular componentstowards the end of development allowschanges indicative of germination to beginimmediately upon shedding (Farrant et al.,1992). Interestingly, in contrast to this andother recalcitrant species studied, respira-tion in the dormant seeds of A. pseudopla-tanus declines to a rate similar to that ofthe orthodox A. platanoides at shedding(Greggains et al., 2000a).

There are differences in the activities ofrespiratory enzymes between the recalci-trant Guilfoylia monostylis and the ortho-dox Erythrina caffra (Nkang and Chandler,1986). These differences may be indicativeof the seeds’ different germination strate-gies; the recalcitrant seeds maintained abalance of enzymes suitable for immediategermination, whereas, in the orthodoxseed, biosynthetic processes were drasti-cally reduced (Nkang and Chandler, 1986).In contrast to recalcitrant seeds, the meta-bolic activity of orthodox seeds is thoughtto decline in a programmed way before orduring the early stages of maturation dry-ing, so that seeds are shed in a quiescentstate (Rogerson and Matthews, 1977; Milleret al., 1983; Farrant et al., 1997). This orga-nized decline in metabolic activity, whichpresumably has a role in protection againstdesiccation damage (reviewed by Vertucciand Farrant 1995), does not occur inspecies with the most sensitive seeds andis not completed in those species withmore tolerant recalcitrant seeds (Berjak andPammenter, 1997; Farrant et al., 1997).Recent studies on seeds of a number ofmore tolerant temperate recalcitrantspecies suggest that, although high at shed-ding, respiration rates decline like those oforthodox seeds during desiccation (V.Greggains and W.E. Finch-Savage, unpub-lished data). However, in the more sensi-tive Araucaria angustifolia, respiration isonly reduced by levels of desiccation thatcause viability loss (Côme and Corbineau,1996). In temperate Castanea sativa seeds,disruption of the electron transport chainoccurs during drying (Leprince et al.,1999), suggesting that the decline in respi-ration due to drying is not programmed,

like that thought to occur in orthodoxspecies.

In most cases, the viability of recalci-trant seeds is lost during drying in regionthree of the five hydration levels summa-rized in Vertucci and Farrant (1995). In thisregion (c. �3 to �11 MPa), seeds are meta-bolically active, respiration is measurableand presumably membranes are stillhydrated. However, at this level of hydra-tion, it is thought that metabolism becomes‘unregulated’, repair processes becomeinoperative and catabolic activities con-tinue unabated, but the processes utilizingthe high-energy intermediates are impaired(Vertucci and Farrant, 1995). As Q. roburseeds are dried, increasing quantities ofseveral harmful volatiles are produced,including ethanol and acetaldehyde(Finch-Savage et al., 1993). These volatilesare indicative of ‘unregulated’ respirationand are a potential source of the free radi-cals that accumulate around the time ofviability loss in Q. robur (Hendry et al.,1992). These free radicals could alter thephysical/chemical properties of mem-branes, causing them to lose liquid/crys-talline structure (McKersie and Leshem,1994). Thus, it is reasonable to speculatethat membrane damage and viability lossduring drying of recalcitrant seeds mayresult from unregulated metabolismenhanced by inadequate protection by free-radical scavengers.

ANTIOXIDANT SYSTEMS. Lipid peroxidation andfree-radical activity have been associatedwith seed viability loss in several recalci-trant species during desiccation (Hendry etal., 1992; Chaitanya and Naithani, 1994;Finch-Savage et al., 1996; Li and Sun,1999). So far, it is difficult to tell whetherthe reported accumulation of free radicalsis a cause or a consequence of viabilityloss. In either case, adequate protectivesystems to limit free-radical damage arelikely to be essential for the maintenance ofviability (Côme and Corbineau, 1996) and arange of antioxidant systems is present inrecalcitrant seeds (Hendry et al., 1992;Chaitanya and Naithani, 1994; Li and Sun,1999; Tommasi et al., 1999). In Q. robur,



there appear to be different protective sys-tems in the embryonic axis and cotyledons(Hendry et al., 1992). In the axis, protec-tion occurs predominantly through theantioxidants ascorbic acid and �-toco-pherol, whereas in the cotyledons protec-tion is largely enzymatic, with relativelyhigh and increasing activities of superox-ide dismutase and glutathione reductase.Decreased levels of protection from lipidperoxidation during desiccation may con-tribute to the loss of seed viability (Hendryet al., 1992). Increased lipid peroxidationduring desiccation, which precedes viabil-ity loss in Shorea robusta (Chaitanya andNaithani, 1994) and T. cacao (Li and Sun, 1999) seeds, was associated withdecreased activities of free-radical-scaveng-ing enzymes. In contrast to these findings,in a comparison of orthodox and recalci-trant Acer species, it was concluded thatthe limitation to desiccation tolerance doesnot result from inadequate free-radicalscavenging (Greggains et al., 2000a).However, A. pseudoplatanus used in thestudy can be placed at the most tolerantend of the continuum of desiccation sensi-tivity among recalcitrant species. Its respi-ration rate at shedding is similar to that ofthe orthodox Acer species, and there is noevidence of increased lipid peroxidation asviability is lost.

OTHER FACTORS. Differences in cellular struc-ture could influence desiccation tolerance(reviewed by Ruhl, 1996) such that the ini-tial loss of water and consequent reductionin cell volume in very sensitive seedscauses mechanical damage (reviewed byBerjak and Pammenter, 1997; Pammenterand Berjak, 1999). For example, the seeds ofA. marina are highly vacuolated and thishas been connected to their level of desicca-tion sensitivity compared with other species(Farrant et al., 1997). However, Vertucci andFarrant (1995) showed that the evidence forthis is conflicting. Considerable disruptionof the cytoskeleton has also been observedduring drying of Q. robur axes, and it is notreassembled during rehydration (Mycock etal., 1999), which may contrast with theapparently organized intracellular de-

differentiation that occurs in orthodox seedsduring maturation drying (reviewed byVertucci and Farrant, 1995).

It is essential that there is effectivemaintenance of the integrity of DNA duringdesiccation and that any damage isrepaired on rehydration for seed viabilityto be maintained (see Chapter 12). In A.marina, DNA repair processes aremarkedly compromised after limited dry-ing and DNA replication does not fullyrecover after only 8% water loss (Boubriaket al., 2000). The arrest of cell cycle activ-ity at the stage where DNA per nucleus islowest may render embryos more resistantto stress conditions (Deltour, 1985).Desiccation tolerance may therefore berelated to the stage of cell cycle activity atwhich desiccation occurs (Bino et al.,1992). However, Sacandé et al. (1997) havepresented data suggesting this is not true,and further convincing evidence againstthe possibility comes from a comparison ofthe orthodox species A. platanoides andthe recalcitrant species A. pseudoplatanus(Finch-Savage et al., 1998). Both speciesproduce seeds with stable high levels of 4CDNA during the later stages of develop-ment, and both contain nuclei arrested atthe 2C and 4C levels at maturity.

So far there has been little emphasis onthe study of post-desiccation repair mecha-nisms in seeds and few studies have beenpublished on this topic in recalcitrantseeds; this may well be a limiting factor indesiccation-sensitive seed tissues.

5.3. Conclusions

Essential components of desiccation toler-ance of seeds include the accumulation ofprotective substances, which limit theamount of damage that otherwise would beinduced by water loss, and the ability torepair cellular components upon subse-quent rehydration. Sugars (disaccharides,such as sucrose, and oligosaccharides, suchas raffinose and stachyose) have been sug-gested to play a key protective role byaccumulating under water deficit condi-tions and functioning to replace water, thus



stabilizing membranes and other sensitivesystems. Another protective mechanismmay involve dehydrins (Table 5.1).

Desiccation tolerance is acquired duringdevelopment of orthodox seeds; tolerance tofull desiccation is generally lost after germi-nation. Recalcitrant seeds, unlike orthodoxseeds, are sensitive to desiccation whenshed from the parent plant, and thus pro-vide a system to study temporal and stress-induced changes in dehydrins and otherputative desiccation protectants. Althoughstudies on recalcitrant seeds provide indi-rect evidence in relation to elucidating theroles of putative desiccation protectants,more work needs to be done in this area.

It is currently an exciting time to under-take the challenges of understanding thebiochemical and genetic components of desiccation tolerance of seeds. Several novel approaches are now available, includ-ing the yeast one- and two-hybrid systems(Ingram and Bartels, 1996; Frank et al.,

1998) for examining protein–protein inter-actions and the use of differential displayreverse-transcription PCR (Rodriguez-Uribeet al., 2000), gene and enhancer trap tag-ging (Rojas-Pierce et al., 2000) and micro-arrays (Nevarez et al., 2000) to identifywater-deficit-regulated genes. Proteomicsapproaches could yield invaluable infor-mation concerning post-translational con-trols over desiccation-induced geneexpression. These and similar researchavenues may yield more decisive resultsthan the traditional approaches.

Finally, it is noteworthy that desicca-tion tolerance of seeds is a complex andmultifaceted property involving a multi-tude of genes whose expression ultimatelyleads to mechanisms of both cellular pro-tection, to sustain limited damage duringdrying itself, and cellular repair, toreverse any desiccation-induced changeswhen the appropriate hydrated conditionsare re-established.


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6 Pollen and Spores: Desiccation Tolerancein Pollen and the Spores of Lower Plants and

Fungi

Folkert A. HoekstraLaboratory of Plant Physiology, Department of Plant Sciences, University of

Wageningen, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands

6.1. Introduction 1866.2. Pollen 187

6.2.1. Desiccation tolerance 1876.2.2. Characteristics 1886.2.3. Longevity 1906.2.4. Imbibitional stress 1916.2.5. (Cryo)preservation 191

6.3. Fern Spores 1926.3.1. Desiccation tolerance 1926.3.2. Characteristics 1926.3.3. Longevity 1936.3.4. Cryopreservation 193

6.4. Moss Spores 1936.4.1. Desiccation tolerance, longevity and imbibitional stress 1946.4.2. Characteristics 1946.4.3. Cryopreservation 194

6.5. Spores of Horsetails, Lycopodia and Selaginella 1946.6. Fungal Spores 195

6.6.1. Desiccation tolerance 1956.6.2. Characteristics 1956.6.3. Longevity 1976.6.4. Imbibitional stress 1976.6.5. Cryopreservation 197

6.7. Conclusion 1976.8. Acknowledgement 1976.9. References 199


Desiccation - Chap 06 18/3/02 1:56 pm Page 185

6.1 Introduction

Pollen grains of seed plants and spores oflower plants and fungi have diverse devel-opmental histories. However, they are simi-lar in size, usually below 200 µm, whichpromotes their effective dispersal via the air.The propagules are exposed to often hostileenvironmental conditions, without the abil-ity to actively control their own hydrationstatus. Under similar environmental condi-tions, microscopic propagules dry out muchfaster than, for example, the much largerseeds. It is, therefore, not surprising thatpollen and spores are endowed with mecha-nisms that allow them to withstand a cer-tain degree of water loss and to survive theperiod from their release from the produc-tion site to the target site and, often, beyondthat. The seeds of about 15% of all plantspecies are supposed to have problems as aresult of water loss (Hong et al., 1996).

Drought tolerance is the term used toindicate tolerance of moderate dehydration,for example, not below the moisture con-tent (MC) at which bulk water has disap-peared (approx. 20–25%, on a fresh weightbasis, or 0.25–0.33 g H2O g�1 dry weight).Desiccation tolerance refers to further dehy-dration and is understood to include notonly the ability of cells to become air-drywithout loss of viability, but also to success-fully rehydrate. The period of anhydrobio-sis in between these two events is referredto as longevity or life span in the driedstate. This chapter focuses on the possibletolerance of spores of eukaryotic plants toreduced levels of hydration.

Pollen, the male gametophyte ofhigher plants, is designed to deliver itshaploid sperm cells to the ovules inorder to bring about fertilization. Thereis a specific and highly specialized targetfor the pollen to land on – the stigma inthe case of angiosperms, or a pollinationdroplet or pollen-collecting apparatus inthe case of gymnosperms. In contrast, thepropagules of the spore-forming plantsand fungi are less restricted as to theirinitial target site, which is usually thesoil or a host organism. This differencein specialization between pollen and the

other propagules is linked with consider-able differences in physiology.

Pollen is dispersed mostly through theair by wind, insects or vertebrates to meet afemale receptive structure, where it is pro-voked to germinate immediately after rehy-dration, sometimes depending onrecognition and/or the presence of self-incompatibility. The rapid start of pollentube growth might be linked with the com-petition of the grains for the availableovules (Mulcahy, 1979), as discussed inSection 6.2. Those pollen grains that fail toland on the proper site are lost, becausethey have very little subsequent chance tomeet the appropriate vehicle for transport.After dehiscence, rainwater is generallydetrimental to pollen, as it causes loss ofviability, bursting or germination at inap-propriate sites (Lidforss, 1896). Thisextreme sensitivity to rainwater is associ-ated with the absence of dormancy inpollen. Only in some gymnosperms has itbeen found that the pollen remains viableafter several hours of soaking of dry pollenin rainwater followed by redrying(Hoekstra, 1983).

Fungal, fern and moss spores are alsooften transported via the air after therelease from the mother plant upon slightdrying of the spore-bearing structures.They will dehydrate to some extent,depending on the niche in which the par-ent organism grows. In these propagules,dormancy mechanisms occur, which sup-press germ tube emergence in the presenceof sufficient water, when the environmen-tal conditions are unfavourable for sup-porting growth. This also implies theactivity of dormancy-breaking mechanismsat some time when the conditions improve.As a consequence of the difference in tar-get, pollen does not survive in the hydratedstate for a long time, whereas the otherpropagules usually do.

A considerable amount of physiological,biochemical and biophysical research inrelation to desiccation tolerance has beenperformed on pollen and yeast, but com-paratively little on the other propagules.For each particular propagule described inthis chapter, special emphasis is placed on

186 F.A. Hoekstra


how widely spread the phenomenon ofdesiccation tolerance is (as far as isknown), including possible problems withthe rehydration of the dried specimens. Intests for desiccation tolerance, improperrehydration may kill otherwise viable dryspecimens, leading to false negatives.Longevity in the dried state is surveyed,because the span may be indicative of thedepth of desiccation tolerance. The occur-rence of compounds generally associatedwith anhydrobiosis is listed, particularlysugars, compatible solutes and dehydrationproteins. The feasibility of (cryo)preserva-tion is highlighted as a practical guidelinefor those interested in preserving thepropagules.

6.2. Pollen

The male gametophytes of higher plantsdevelop in the anthers (Stanley andLinskens, 1974). When the flower opens,the anthers become exposed to the environ-ment and dehydration then triggers amechanism in the anthers for the release ofthe pollen. In the process, pollen alsodehydrates. Continuous rain may preventanthers from opening. Once pollen haspassed physiological maturity, ageing maystart within the anthers under such condi-tions. Ultimately, pollen with reduced via-bility and vigour is released (Hoekstra and

Bruinsma, 1975; Linskens et al., 1989), orpollen may form germ tubes inside theanthers (Pacini and Franchi, 1982). Whenviable pollen rehydrates on the appropriatefemale receptive structure, it germinates,producing a filiform structure (pollentube), which grows through the styletowards the ovules by a process much likea parasitic invasive action.

6.2.1. Desiccation tolerance

The pollens of a large number of plantspecies can withstand air-drying (Stanleyand Linskens, 1974; Towill, 1985; Hoekstra,1986, 1995). Studies by the author of thepollen literature have revealed that the pol-lens of plants belonging to some genera ofabout half of all plant families have beentested for their tolerance to various levels ofdehydration. While desiccation toleranceappears to be widespread among pollen ofmost plant families studied, there are a fewexceptions. It is expected that more suchrecalcitrant pollen types will be found invery humid climates and niches not yetinvestigated, particularly in the hot, humidtropics.

From the results of in vitro and in vivogermination experiments, it is clear that thepollen of genera in Araceae, Cucurbitaceae,Gramineae and Zingiberaceae experienceproblems during drying (see Table 6.1 for

Desiccation Tolerance in Pollen and Spores 187

Table 6.1. List of plant families and their genera in which desiccation-sensitive pollens have beenreported.

Family Genus Reference

Araceae Dieffenbachia Henny, 1980a,bAglaonema Henny, 1985

Cucurbitaceae Cucurbita Wang and Robinson, 1983; Gay et al., 1987Momordica Dubey and Gaur, 1989

Gramineae Avena Wallace and Karbassi, 1968Hordeum Anthony and Harlan, 1920; Firbas, 1922Saccharum Sartoris, 1942; Moore, 1976Secale Chaudhury and Shivanna, 1987; Shi and Tian, 1989aSorghum Sanchez and Smeltzer, 1965Triticum Firbas, 1922; Goss, 1968Zea Barnabas, 1985; Digonnet-Kerhoas and Gay, 1990Pennisetum Aken’ova and Chheda, 1970

Zingiberaceae Zingiber Adaniya and Higa, 1988; Adaniya, 2002


details). They can be kept alive only under100% relative humidity (RH)(Zingiberaceae) or at slightly lower RH,albeit for short periods of time. AlthoughGramineae pollens are generally sensitiveto air-drying, they usually have a consider-able tolerance to drought. Thus, pollen ofSecale cereale is tolerant to MCs as low as11.5% (fresh weight basis) (Shi and Tian,1989a), and that of Zea mays to 13% MC(Digonnet-Kerhoas and Gay, 1990). In thegenus Pennisetum, tolerance (Pennisetumtyphoides (Pennisetum americanum),Chaudhury and Shivanna, 1986, 1987;Hoekstra et al., 1989) as well as intoler-ance (Pennisetum purpureum, Aken’ovaand Chheda, 1970) to air-drying have beenobserved. Some orchid pollens rapidlylose viability on storage above silica gel,which cannot be restored by a day of pre-hydration in humid air prior to the in vitrotest (Pritchard and Prendergast, 1989).This may point to reduced desiccation tol-erance, but may also indicate that thedried pollen is sensitive to the humid airtreatment (see Section 6.2.4).

6.2.2. Characteristics

On dehydration, pollen changes in shapein different ways, dependent on thespecies. While indentation is observed insome pollens, e.g. those of Gramineae, inothers the major to minor axis ratio isincreased as the shape changes fromround into ellipsoid along special fur-rows, as in the pollens of Solanaceae andPapaveraceae (for low-temperature scan-ning electron micrographs, see Fig. 6.1).The pattern of size reduction with dehy-dration also depends on the distributionand number of germ pores.

Germination ability of pollen is assayedin vivo by the analysis of tube growth incompatible stigmas or styles, or in vitro onartificial germination media (see Hoekstra,1995, for a review). Those pollens that areadapted to plants having wet stigmatic sur-faces easily germinate in liquid or solidifiedgermination media. In contrast, pollen grainsfrom plants with dry stigmatic surfaces have

more subtle rehydration requirements andare often difficult to germinate on suchmedia (Bar-Shalom and Mattsson, 1977).

During the development of the maleangiosperm gametophyte, two nuclear divi-sions take place. The first one leads to onevegetative and one generative cell thatgives rise to two sperm cells in the subse-quent division. Depending on whetherdehiscence occurs after the first or the sec-ond mitosis, mature pollen is bicellular ortricellular. Bicellular pollen grains have toperform the second mitosis during tubegrowth. Thus, tricellular pollen is ontoge-netically advanced. This characteristicoccurs in about 30% of angiosperm familiesthat are considered as being evolutionarilyadvanced (Brewbaker, 1959, 1967; Sporne,1969). Typical families with tricellularpollen are the Asteraceae, Caryophyllaceae,Chenopodiaceae, Cruciferae, Gramineae,Juncaceae and Umbelliferae. Tricellularpollen tends to be associated with dry stig-mas and is not readily germinated in vitro(Heslop-Harrison and Shivanna, 1977).

The time from contact with the germi-nation medium or appropriate stigmaticsurface to emergence of a pollen tube isgenerally short. It ranges from a few min-utes for Gramineae and Compositae pol-lens to a few hours for some other pollens(Hoekstra and Bruinsma, 1978; Hoekstra,1986). Only in the case of gymnospermpollens does it take a few days beforeemergence occurs (e.g. Pettitt, 1985). Arapid start of tube emergence is generallyfollowed by extremely fast tube growth ofup to 1–2 cm h�1 (Hoekstra and Bruinsma,1978). It has been argued that recurrentcompetition for the fertilization of avail-able ovules has led to such short lag peri-ods and fast growth, which may havemade mature pollen extremely sensitive tostress. This problem may have been suc-cessfully evaded by the regular productionof fresh pollen during the floweringperiod. Some characteristics of fast-grow-ing pollens that are often tricellular are thehigh proportion of polyunsaturated fattyacids (linolenic acid) in the phospholipidsand neutral lipids, the high rates of metab-olism associated with well-developed

188 F.A. Hoekstra


mitochondria and, in the case of shortstyles, even the absence of protein synthe-sis during tube growth (see Hoekstra,1986, for a review). The high level ofpolyunsaturated acyl chains that are par-ticularly sensitive to peroxidation mightcontribute to the intrinsically short lifespan.

Mature pollen is generally endowedwith high contents of sucrose, ranging from7 to 23% of the pollen dry weight, butoligosaccharides have not been encoun-tered (Hoekstra et al., 1992). Sucrose mayplay an essential role in the acquired toler-ance of severe dehydration, as illustratedby the following examples. Developingpollen of Papaver dubium becomes func-

tional and desiccation-tolerant at approxi-mately 1–2 days prior to anthesis, coinci-dent with the degradation of starch and adoubling of the amount of sucrose.Precocious drying leads to loss of viabilityand damaged plasma membranes. Whenimmature pollen is liberated mechanicallyfrom the anthers and allowed to mature inhumid air, starch degrades and sucrosecontent nearly doubles, and the grainsbecome largely functional and dehydration-tolerant. Apparently, maturation during thelast 3 days of development is independentof the parent plant, and sucrose may playan essential role in the acquired desicca-tion tolerance (Hoekstra and van Roekel,1988). During the slow dehydration of


Fig. 6.1. Low-temperature scanning electron micrographs of partly dehydrated (A,C) and hydrated pollens(B,D). Maize pollen, (A) partly dehydrated and (B) hydrated (fresh); poppy (Papaver rhoeas) pollen, whichwas liberated from an anther in a partly dehydrated state (C) and upon rehydration in water (D). Upondehydration, maize pollen becomes indented, whereas poppy pollen becomes elongated. Bars are 10 µm.(Micrograph, courtesy of Adriaan van Aelst, Laboratory of Experimental Plant Morphology and Cell Biology,Wageningen University.)


fresh maize pollen, the sucrose contentincreases from 5 to 12% of the dry weight.When fresh pollen is dried in the cold(2°C) or at a high rate, the increase insucrose content is curtailed, and the toler-ance of dehydration is affected, from whichit has been concluded that survival ofdehydration is correlated with the presenceof sucrose (Hoekstra et al., 1989). Themode of action of sugars in dry plant mate-rial is discussed in detail in Chapter 10.

Free amino acids are abundant inmature pollen grains. On the basis of ananalysis of the pollen of a large number ofplants (about 200 species from 63 families),it has been shown that pollen differs fromother organs in having an unusually highcontent of free proline, exceeding 1.5% percrude weight in many species (Britikov andMusatova, 1964). A high content of freeproline has repeatedly been confirmed forother pollens, even up to 3% of the dryweight. Instead of proline, free arginineand glutamate may also occur. There areindications that the free proline content ofpollen is positively related to viability(Palfi and Köves, 1984; Palfi and Mihalik,1985; Lansac et al., 1996). It is remarkableto note that the desiccation-sensitivepollen of Cucurbita has low proline con-tents (Gulyas and Palfi, 1986). On the otherhand, dehydration-sensitive maize pollen,which can withstand dehydration toapproximately 13% MC, does contain con-siderable amounts of proline (Linskens andPfahler, 1973; Palfi and Köves, 1984). Theaccumulation of proline is preceded by apeak in free abscisic acid (ABA) (Lipp,1991; Chibi et al., 1995). Whereas the ABAcontent decreases towards maturity (addedABA inhibits germination), the prolinecontent further increases to a maximum,coinciding with the stage of anther desicca-tion (Zhang et al., 1985). Proline added tothe germination medium makes pollenmore resistant to heat (Zhang and Croes,1983), which suggests that the highendogenous amounts in pollen may have arole in conferring resistance tounfavourable temperatures. The role of freeproline and some amino acids as compati-ble solutes, providing structural protection

to biopolymers under conditions whenthere is still bulk water present duringdehydration, is discussed in Chapter 10.

As in seeds, heat-stable proteins withlate embryogenesis abundant (LEA)(Wolkers et al., 2001) and dehydrin (Wanget al., 1996) characteristics have been iso-lated from pollen. There are further indica-tions that, as a result of osmotic stress,dehydration and ABA, a number of pro-teins are produced in pollen with homolo-gies to stress proteins in seeds. Thepossible mode of action of these proteins isdiscussed in Chapter 10.

6.2.3. Longevity

Longevity of pollen varies considerablyamong species and is dependent on watercontent and temperature (see Hoekstra,1986, for a review). Tricellular pollen tendsto be shorter-lived than bicellular pollen.However, short storage life and extremelyrapid tube emergence and growth have alsobeen found in bicellular pollen, e.g. in Balsaminaceae, Cucurbitaceae andCommelinaceae. The average survival peri-ods at 20–25°C and equilibrium RHs ofapproximately 40% range from a few hoursin desiccation-sensitive pollens to severalmonths in the most stable, desiccation-tol-erant pollens (Hoekstra, 1995). Only forgymnosperm pollens have longevities ofover 1 year under these conditions beenreported. The survival of pollen in storageconforms to a cumulative negative normaldistribution (van Bilsen and Hoekstra,1993; Buitink et al., 1998b). Based on thedata of Buitink et al. (1998b), an empiricalmodel for the storage behaviour of pollenhas been constructed, which can predictthe viability of a pollen lot over time at abroad range of different water contents andstorage temperatures (Hong et al., 1999a).There is a negative logarithmic relationbetween longevity and pollen MC and acurvilinear semi-logarithmic relationbetween longevity and temperature.

It has long been known that there is alower MC limit, below which further reduc-tion in MC does not increase pollen

190 F.A. Hoekstra


longevity, but, instead, decreases it (Pfundt,1910; Hoekstra, 1986). At high MC, pollenlongevity is not extended as in dormantspores and seeds, but the pollen germi-nates, or engages in inappropriate synthe-ses, such as callose deposition (Hoekstra,1986). As mentioned earlier, dormancymechanisms are absent in pollen. Anexception to the rule that fully hydratedpollen does not survive for a long time isorchid pollen that is enclosed in pollinia inlong-lasting flowers. On agar, survival timesof a few weeks at 2°C have been reported(Pritchard and Prendergast, 1989).

Recently, it has been established thatpollen is in a glassy state (see Chapter 10)at below approximately 10% MC at roomtemperature. In a glass, molecular mobilityis considerably reduced. It has been foundthat the molecular mobility of a small guest(spin-probe) molecule in the cytoplasm isinversely correlated with storage longevity(Buitink et al., 1998a). Thus, the slower themolecular mobility, the greater is the lifespan. Elevated water contents and tempera-tures increase molecular mobility and,thus, decrease the life span. For long-termsurvival it is therefore important that thepollen cytoplasm is in the glassy state.From the linear relationship between thelogarithms of molecular mobility andlongevity, Buitink et al. (2000a) have beenable to predict storage longevities undervarying conditions of temperature and MC.Thus, survival times at low temperatures,for which experimental determination ispractically impossible, can be estimated.This approach on the basis of molecularmobility probably gives more accurate esti-mates of survival times for sub-zero tem-peratures than does the empirical modelproposed by Hong et al. (1999a), which ismore conservative.

A possible cause of the critical lowerMC limit may be encompassed in the phe-nomenon that, during the removal of water,molecular mobility reaches a minimum,but increases again on further drying tovery low water contents (Buitink et al.,2000b). In addition, the MC at which thisminimum molecular mobility occursincreases with decreasing temperature, and

is predicted to be considerably above 10%MC at cryogenic temperatures. This may bethe reason for the successful cryogenicstorage of desiccation-sensitive Gramineaepollen, as mentioned below.

6.2.4. Imbibitional stress

Sensitivity of pollen to imbibitional stresshas been known for many years (seeHoekstra, 1986, for a review). The imbibi-tion of dry pollen in germination medium,particularly at chilling temperatures, canlead to loss of endogenous solutes anddelay of tube emergence, or even a com-plete failure of the grains to become turges-cent and form a pollen tube. The problemis widespread among pollens and can becircumvented by prehydration in humidair or by warm imbibition, or a combina-tion of both (Hoekstra, 1984; Hoekstra andvan der Wal, 1988). Some caution has to beexercised with prehydration in humid air,as this treatment may lead to a rapid loss ofviability in some cases. The extensive leak-age as a result of imbibitional stress isindicative of problems at the level of theplasma membrane. Few pollens have beenfound that are resistant to this stress. Theyare characterized by highly polyunsatu-rated acyl chains in their membranes(reviewed by Hoekstra and Golovina,1999), which may increase membrane flu-idity and thus more easily accommodatethe expanding protoplast on rehydration.

6.2.5. (Cryo)preservation

Desiccation-tolerant pollen that is air-dried to approximately 7% MC can bestored for extended periods of time atsub-zero temperatures. The experience ofthe author with air-dried cattail (Typhalatifolia) pollen is that a half-life of about25 years is possible at �18°C in a deep-freezer. However, for long-term storage ofmany decades or even longer, cryogenicstorage is essential. Some excellentreviews of this subject have been pub-lished (Binder et al., 1974; Stanley and



Linskens, 1974; Franklin, 1981; Shivannaand Johri, 1985; Towill, 1985).

The storage of desiccation-sensitivepollen is limited to several days or at mosta few weeks under conditions of high RHat 0–2°C (see Hoekstra, 1995). Desiccation-sensitive Gramineae pollen can usuallywithstand drying to 20–25% MC. This isthe MC at which bulk water has disap-peared and ice crystals do not rapidly format sub-zero temperatures. When pollen,carefully dried to just below this watercontent, is quenched in liquid nitrogen, aglassy state is rapidly formed, and viabilityis maintained because detrimental ice crys-tals cannot grow. In this way, successfulstorage at �196°C is possible with maizepollen (Barnabas and Rajki, 1976; Barnabaset al., 1988; Shi and Tian, 1989b; Barnabas,1994 (even for more than 10 years)) and ryepollen (Shi and Tian, 1989a). After pollina-tion with the thawed pollen, excellent seedset can be obtained.

It has been reported that storage in vac-uum ampoules at 5°C after vacuum-dryingallows maize pollen to survive for morethan 1 year (Jensen, 1964). Also freeze-dry-ing followed by storage at 0–5°C givessome survival up to 5 months (Nath andAnderson, 1975). Short lyophilization, fol-lowed by vacuum storage at �40°C pro-longs the life span of pollen of the grassDactylis glomerata for more than 2 years(Cauneau-Pigot, 1991). The beneficialeffect of the vacuum during dehydrationand storage might be associated with theexclusion of oxygen: oxidative stressmight be an important factor in the desic-cation sensitivity and storability ofGramineae pollen.

6.3. Fern Spores

Fern spores are formed by meiosis in spo-rangia, often underneath the leaves andabove ground. They are released whendehydration causes rupture of the sporan-gia. They are dispersed and can germinate,giving rise to prothalli, where eventuallyfertilization takes place followed by out-growth of the sporophytic plant.


Because the release of spores from the spo-rangia requires a certain degree of dehydra-tion, some tolerance to dehydration in thespores is likely. This has, indeed, beenestablished in the spores of a number offerns, e.g. Adiantum capillus-veneris(Uchida et al., 1998), Dryopteris filix-mas(Haas and Scheuerlein, 1991), Dryopterispaleacea (Scheuerlein et al., 1988; Hauptand Psaras, 1989), Anemia phyllitidis(Grill, 1988), Lygodium japonicum(Manabe et al., 1987) and Onoclea sensi-bilis (Raghavan, 1992), but precise informa-tion on longevity is scarce.


To detect spore survival by germinationtests is laborious and slow. Fern spores ger-minate in soil or on aseptic agar-containingor liquid nutrient media after a dormancy-releasing treatment. Criteria of viability areswelling of the cell, coat splitting, greeningand rhizoid formation, which usuallyrequire weeks after induction for theirexpression. A chlorophyll fluorescencetechnique has been applied, which has theadvantage that it can be used to quantifythe germination capacity of non-greenspores (D. paleacea) just 2 days afterphotoinduction (Scheuerlein et al., 1988).

There is ample evidence for the occur-rence of dormancy in fern spores. Sporegermination in hybrid Azolla increasesprogressively to attain a maximum after 5months of storage and declines after 7months to reach a minimum after 11months (Bhattacharyya and Kushari,1999). It is clear that fern spores do notsimply germinate when they come intocontact with water. They are stimulated byred light and reversibly inhibited by farred light. The red-light-induced germina-tion is irreversibly inhibited by ultraviolet(275 nm) and blue (440 nm) light (Sugaiand Furuya, 1985). The induction of sporegermination is mediated by phytochrome,and the effect of a red pulse irradiationcan be enhanced by nitrate added to the

192 F.A. Hoekstra


culture medium (Haas and Scheuerlein,1991). The application of gibberellic acid(GA3) also allows spores to germinate, butthis hormone strongly inhibits furthergametophyte development (Fernandez etal., 1997). The red-light-induced spore ger-mination is inhibited by AMO 1618, aninhibitor of gibberellin biosynthesis, fromwhich it has been suggested that red lightinduces the biosynthesis of gibberellin viathe phytochrome system and that gib-berellin induces spore germination(Kagawa and Sugai, 1991). Added ABAsignificantly inhibits spore germination(Singh et al., 1996).

Spores accumulate reserve lipids(Gemmrich, 1977) and storage proteins thatare genetically similar to seed storage pro-teins (Templeman et al., 1988). The spores ofOsmunda japonica contain large amounts offree proline and arginine (Wada et al., 1998).

6.3.3. Longevity

Storage of spores at 20°C in the air-driedstate and under hydrated conditions hasbeen compared for four species (Athyriumfilix-femina, Blechnum spicant, Polystichumsetiferum and Phyllitis scolopendrium)(Lindsay et al., 1992). The viability of theirhydrated, non-green spores remained un-changed after 2 years of storage, while thatof the air-dried spores decreased with time.In addition, the time required for germina-tion of the air-dried spores increased dur-ing storage. The chlorophyllous spores ofTodea barbara, which cannot be stored forlong periods by conventional methods, alsolost viability during storage much lessquickly when hydrated than when air-dried. From this, Lindsay et al. (1992) haveconcluded that wet storage of fern spores isfar more effective than dry storage.However, prior dehydration allows spores(Cyathea delgadii) to be stored at sub-zerotemperatures, which extends survivaltimes considerably in comparison withother storage methods (Simabukuro et al.,1998). Sporocarps of Azolla filiculoidesare able to survive storage in water for 3years and to germinate from mud samples

collected in the field (Janes, 1998). Fromthe scanty data in the literature, one cannotget an idea of how widespread the occur-rence of desiccation tolerance is amongfern spores.

Some fern species (e.g. A. filix-femina,Gymnocarpium dryopteris, Phegopterisconnectilis) form spore banks, which aresoil reservoirs of viable spores that remaindormant while buried, but germinate inlight if brought to the surface (Dyer, 1994).Fern spores remain stable in the soil formore than a year (Dyer and Lindsay, 1992).

6.3.4. Cryopreservation

It has been found that spores of Cyatheaspinulosa survive storage in liquid nitro-gen (�196°C) followed by slow thawing(Agrawal et al., 1993). In this case, thewater content of the spores was uncon-trolled. Prior dehydration to at least belowthe level of the non-frozen water content(approximately 0.25–0.33 g H2O g�1 dryweight) is expected to provide possibilitiesfor long-term survival under conditions ofcryogenic storage, if the spores survivedehydration to this low water content.

6.4. Moss Spores

Although the release of moss spores fromthe sporophyte capsules upon dryingwould suggest that desiccation tolerance inthese spores could be common, little isknown about how widely distributed thisphenomenon really is among species. Inthe soil, the unicellular haploid spore ger-minates to form a small green protonema.Later on, plants grow from these protone-mata. Beside the spores that arise from theinner tissue of the capsule by meiotic divi-sion, a variety of gametophytic cells andfragments, called ‘diaspores’, can be pro-duced from the gametophytic plant andprotonemata by different cellular separa-tion mechanisms (Duckett and Ligrone,1992). Here, diaspores will also be consid-ered in relation to their possible desicca-tion tolerance.



6.4.1. Desiccation tolerance, longevity andimbibitional stress

When comparing the survival of sporesfrom five species (Schistidium rivulare,Racomitrium aciculare, Dicranoweisiacrispula, Oligotrichum hercynicum andCeratodon purpureus) in water and afterdesiccation, Dalen and Soderstrom (1999)found that the spores generally survivedbetter in the dried state than in water.However, the survival times were limited,i.e. not exceeding 6 months. In these fivespecies, fragments (diaspores) have beenfound to survive equally well in water as inthe dried state. The (dia)spore bank ofbryophytes that are soil reservoirs of viablespores appears to play a role similar to thatof the soil seed bank in seed plants.

Redifferentiation of moss protonematainto spherical, thick-walled brood cells(brachycytes) is a widespread phenome-non, which occurs when protonemalcolonies are cultured for long periods oftime, allowed to dry out or are treated withABA. Brood cells in some species retainviability for long periods even in a desic-cated state and germinate rapidly in newmedium lacking ABA (Duckett et al., 1993;Schnepf and Reinhard, 1997). It is likelythat ABA is the natural compound thattriggers brood cell development andinduces tolerance to desiccation (Goode etal., 1993), including the synthesis ofextremely heat-resistant soluble proteins(Werner and Bopp, 1992). It has been con-cluded that ABA has the same function inbryophytes as in higher plants, acting as amediator in stress conditions (Bopp andWerner, 1993). Dry intact moss gameto-phytes are sensitive to imbibitional stress(Schonbeck and Bewley, 1981). Whetherdry spores and diaspores are similarly sen-sitive has not been studied.


Although no special studies have been con-ducted as to possible dormancy mecha-nisms in spores, the study of Miles andLongton (1992) suggests that dormancy is

present. These authors have found that onagar the germination of Archidium alterni-folium spores continues to increase over aperiod of several months. Percentage ger-mination was consistently less than 65% infreshly collected material, and increasedwith the age of the spores up to 4 years ofstorage. There is no information about themajor constituents of spores. For compari-son, sucrose is the main sugar in the driedgametophyte (Smirnoff, 1992).


Spores can be expected to survive drying,and also to survive well in the dried stateat sub-zero temperatures. However, cryo-preservation of (hydrated) cultures solvesproblems associated with long-term cul-ture, particularly when spores are short-lived or not produced. Improvements insurvival after cryopreservation have beenmade by preconditioning cultures in ABAand proline (Christianson, 1998). There isinformation that suggests that moss sporesburied in the Siberian permafrost have sur-vived 40,000 years, as they could still formprotonemata upon culture (www.science.nasa.gov/newhome/headlines/ast27jul99_1.htm).

6.5. Spores of Horsetails, Lycopodia andSelaginella

The chlorophyllous spores of the solehorsetail genus, Equisetum, survive desic-cation, yet do not live for more thanapproximately 2 weeks when desiccated at2% RH and 25°C (Lebkuecher, 1997).Under these conditions, disseminatedspores of Equisetum hyemale have anextremely short life span, possibly due tothe inability to recover losses of water oxi-dation and photosystem II core function.Storing ripe cones of E. hyemale at �70°Cextends the viability of the spores for morethan a year (Whittier, 1996).

Spores of Equisetum arvense, when cul-tured in Murashige and Skoog liquidmedium, germinate 2–3 days after sowing

194 F.A. Hoekstra


(Kuriyama and Maeda, 1999). Large amountsof free proline and arginine have beendetected in these spores (Wada et al., 1998).

The spores of the Lycopodiaceae, someof them chlorophyllous, germinate rela-tively slowly (months/years). Thereappears to be no information on desicca-tion tolerance of the spores, includingthose of Selaginella.

6.6. Fungal Spores

Spores of terrestrial fungi will be consid-ered here and not those of aquatic fungi. Atspore maturity, ejection mechanisms occurthat ensure the dispersal of spores into theair, but sometimes spores develop in anaqueous environment.


Desiccation tolerance has been establishedin the spores of a wide variety of fungi (seeTable 6.2). Particularly well studied are thedesiccation-tolerant spores of economicallyimportant fungi, including yeast. Amongthem are the entomopathogenic fungi of thegenera Beauveria, Metarhizium andPaecilomyces, the phytopathogenic fungi ofthe genera Alternaria, Helminthosporium,Pseudopezicula, Puccinia, Sclerotinia,Venturia, Uromyces and Ustilago, and thebioherbicidal fungus, Stagonospora. Themain issues in the research were the explo-ration of how long spores can survive undernatural conditions, which is important inrelation to plant pathogens, and the estab-lishment of culture conditions and dryingprotocols that give adequate shelf stabilityof spores in the case of commercial biologi-cal products for agricultural application.Desiccation-sensitive spores have beenfound as a spin-off in the search for toler-ance. For example, the conidia ofPenicillium bilaji appear to be desiccation-sensitive, because they could be kept aliveonly under conditions of 100% RH(Cunningham et al., 1990). However, using afluidized bed drier and drying system,Tadayyon et al. (1997) have managed to pro-

duce a powdered formulation of skimmedmilk/P. bilaji spore particles that could bekept alive under refrigeration for at least 3months. Drying techniques for spores andyeast cells that have been applied includeair-drying, freeze-drying, spray-drying andfluid bed-drying by contact-sorption.


Spores change their shape as a result ofdehydration, with size reductions to70–80% of the hydrated size. The criticalRH, above which spores are expanded andbelow which they are collapsed, rangesbetween 80 and 90% RH, as established fora number of urediniospores (Littlefield andSchimming, 1989).

Desiccation tolerance of the spores isenhanced when the fungi are grown in spe-cial media, e.g. enriched with glycerol, glu-cose and casamino acids, or when sporesare harvested at mature rather than earlystages of development. It has been foundthat the life span of Metarhiziumflavoviride conidia is greater after slowrather than rapid drying (Hong et al.,2000). This suggests that there is a certaintime required for protective mechanisms tobe expressed during the stress, which isalso observed in other anhydrobioticpropagules, e.g. seeds and pollen.

A compound that typically accumulatesin fungal spores is trehalose (see Feofilova,1992, for a review). Additional stresses,such as heat shock or stationary phase(stress because of crowding) (Pedreschi andAguilera, 1997; Pedreschi et al., 1997) andhigh osmolality (Hallsworth and Magan,1994; Eleutherio et al., 1997), are particu-larly effective at increasing the content oftrehalose, and enhancing viability and des-iccation tolerance, such as in yeast cells.Apart from its effect as an osmoprotectant,trehalose causes a depression of the gel-to-liquid crystalline transition temperature ofmembranes in dried yeast (Leslie et al.,1994; see Chapter 10), alleviates the effectsof ethanol stress (Mansure et al., 1994) andcan be involved in the formation of a glassystate in the dried organism (Wolkers et al.,



1998). Saccharomyces cerevisiae mutants,in which proline and the charged aminoacids such as glutamate, arginine and lysineare increased, have a marked cryoprotectiveactivity, nearly equivalent to that of glycerolor trehalose, both known as major cryopro-tectants in this yeast (Takagi et al., 1997).Other typical products are heat-shock pro-teins (HSPs), which, for example, in yeastare produced in the stationary phase of fer-mentation (Praekelt and Meacock, 1990;Sanchez et al., 1992). They are thought to becrucial for the naturally high thermo-toler-ance of these cell types and for their long-term viability at low temperatures. A smallHSP in yeast may function in the protectionof the plasma membrane against desiccationand ethanol-induced stress (Sales et al.,2000). Further, high superoxide dismutase(SOD) activity is essential for stationaryphase survival in yeast (Longo et al., 1996).

Evidence for the existence of dormancyin spores comes from the observation that

the percentage germination increases withspore age (Perry and Fleming, 1989; BenZe’ev et al., 1990; Gazey et al., 1993) or afterchemical treatment (Rivero and CerdaOlmedo, 1994). Usually, exposure of thespores to low temperatures for some time, orto heat, certain chemicals or light, increasesthe germination percentage. The presence ofa thick wall may play an important role inthe constitutive dormancy of some spores(Ulanowski and Ludlow, 1989). In thespores of ectomycorrhizal and several sapro-trophic fungi, fluorescein diacetate (FDA)staining has been found to be unreliable asan indicator of viability, but a good predic-tor of dormancy (Miller et al., 1993). As thespores age, the percentage of fluorescentspores increases. It is not known whetherthis increase is the result of a better penetra-tion of FDA through the spore wall, or of thesynthesis or activation of esterase capable ofcleaving the fluorogenic ester to form thefluorescent dye, fluorescein.

196 F.A. Hoekstra

Table 6.2. List of spore types of different fungal species in which desiccation tolerance has beenestablished.

Species Type of spore Reference

Alternaria porri Conidia Hong et al., 1997Aspergillus japonicus Conidia Gornova et al., 1992Beauveria bassiana Conidia Hallsworth and Magan, 1994; Pfirter et al., 1999Beauveria brongniarti Conidia Hong et al., 1997Colletotrichum gloeosporioides Conidia Cunningham et al., 1990Helminthosporium oryzae Conidia Hong et al., 1997Metarhizium anisopliae Conidia Hong et al., 1997Metarhizium flavoviride Conidia Moore et al., 1997; Hong et al., 2000Neosartorya fischeri Ascospores Beuchat, 1992Paecilomyces farinosus Conidia Hallsworth and Magan, 1994Paecilomyces fumosoroseus Blastospores Cliquet and Jackson, 1997; Jackson et al., 1997

Conidia Stephan and Zimmermann, 1998Phycomyces blakesleeanus Zygospores Rivero and Cerda Olmedo, 1994Pseudopezicula tracheiphila Ascospores Pearson et al., 1991Puccinia graminis Urediniospores Eversmeyer and Kramer, 1994Puccinia recondita Urediniospores Eversmeyer and Kramer, 1994Saccharomyces cerevisiae Cells van Steveninck and Ledeboer, 1974Saccharomyces uvarum Cells Eleutherio et al., 1997Sclerotinia sclerotiorum Ascospores Hong et al., 1997Sordaria macrospora Ascospores Read and Lord, 1991Stagonospora convolvuli Conidia Pfirter et al., 1999Talaromyces flavus Ascospores Beuchat, 1992Trichoderma harzianum Conidia Jin et al., 1996; Pedreschi and Aguilera, 1997Uromyces appendiculatus Urediniospores Hong et al., 1997Ustilago scitaminea Teliospores Hoy et al., 1993Venturia inaequalis Conidia Becker and Burr, 1994


6.6.3. Longevity

Longevity of spores varies considerablyamong species. Thus, the viability ofteliospores of Ustilago scitaminea in air-driedsoils does not begin to decrease until after 18weeks, but is lost after maintenance, free ofsoil, for 23 weeks at ambient RH (Hoy et al.,1993). Air-dried spores of Stagonospora con-volvuli remain viable at 3°C for 180 days(Pfirter et al., 1999). Particularly long-livedare the heat-resistant ascospores ofNeosartorya fischeri and Talaromyces flavus,which have been shown to survive dry stor-age in fruit powders (RH = 23%; 25°C) for upto 30 months (Beuchat, 1992). It has beenreported that some Talaromyces spores sur-vive in coating material of dry seeds for aslong as 17 years at room temperature(Nagtzaam and Bollen, 1994).

The survival of conidia conforms to acumulative negative normal distribution. Asin seeds and pollen, longevity of conidia isdetermined by MC and temperature, besideendogenous factors (Hong et al., 1997, 1998).These authors have constructed an empiricalmodel for the storage behaviour of conidia,which can predict the viability of a spore lotover time at a broad range of different watercontents and storage temperatures. A nega-tive logarithmic relation has been observedbetween longevity and conidia MC. The MCof the conidia is dependent on the sorptionproperties of the endogenous compoundsand the RH in which the conidia are equili-brated. The relation between longevity andequilibrium RH can be described by a nega-tive semi-logarithmic relation. There is alower MC limit, below which value furtherreduction in MC does not increase conidialongevity, and an upper MC limit, abovewhich longevity no longer decreases.Analysis of the effect of different tempera-tures has shown that the Q10 for the loss inconidia viability increases, the warmer thetemperature (Hong et al., 1999b). With fluc-tuating day/night temperatures, the warmertemperature mainly determines conidialongevity. Viability of ungerminated desicca-tion-tolerant conidia is not affected by expo-sure to wet and dry intervals, but germinatedconidia (germlings) are generally sensitive to

dry intervals (Becker and Burr, 1994). Sporescan also survive for considerable periods oftime when stored fully hydrated. Septorianodorum spores have been shown still to beinfectious after 24 months of storage in water(Wilkinson and Murphy, 1990).

6.6.4. Imbibitional stress

As in pollen, there is evidence in fungi andyeast that dried cells suffer from beingplunged into liquid medium, particularly atlow temperatures. For example, dry conidiaof M. flavoviride (4–5% MC) displayreduced viability when rapidly rehydratedin free water. Prehydration in an atmosphereof high humidity allows dry conidia toabsorb sufficient moisture to avoid imbibi-tional damage (Moore et al., 1997). A similaravoidance of damage has been demonstratedwhen dried yeast was allowed to imbibe atelevated temperatures of 39–42°C (vanSteveninck and Ledeboer, 1974). The mecha-nism of imbibitional damage is dealt with inChapters 10 and 12.


Spores of vesicular–arbuscular (VA) myocor-rhizal fungi are usually stored in soil justabove 0°C, but some of them may be storedfrozen. When cryoprotectants such asdimethylsulphoxide (DMSO), glycerol, man-nitol or sucrose are ineffective, incubation offreeze-sensitive spores for 2 days in0.75–1.0 M trehalose confers a measure offreeze-damage protection. However, it hasbeen found that slow drying of spores in situ(in soil) prior to freezing gives satisfactorysurvival of the VA spores (Douds andSchenck, 1990). In the case of full desiccationtolerance, sub-zero temperature storage of thedried spores will generally be successful.

6.7. Conclusion

In the introduction, it was suggested thatpollen and spores are likely to have atleast a certain level of tolerance to dehy-



dration. This expectation appears to begenerally correct.

Most pollens are tolerant to MCsbelow 10%. However, pollens of someplant families have an elevated lower MClimit (>10% MC), below which viabilityis lost. Such a reduced tolerance is com-parable with that of the so-called ‘inter-mediate’ seeds. It seems that realdesiccation sensitivity (recalcitrance),i.e. sensitivity already below 40% MC, asis frequently observed in seeds, isuncommon among pollens. This might beexplained by the usually larger size ofthe seeds: under the same humid condi-tions, the comparatively large seeds donot dry out as fast as the microscopicpollens. Thus, desiccation tolerance isless imperative for seeds that are dis-persed in humid climates.

The phenomenon of desiccation toler-ance has been extensively studied inpollen. Some of the compatible solutes,such as sucrose and proline, that are typi-cally associated with drought and desicca-tion tolerance are abundantly present. Theheat-stable dehydration proteins, knownfrom seeds, also occur in pollen.

A similar picture emerges for fungalspores and yeasts. However, the situationis less clear for fern and moss spores,mainly because considerably less work hasbeen done on this material. Desiccation-tolerant fern and moss spores have beenfound, nevertheless, and drought tolerancemight at least be common. Only for thespores of the fern Osmunda has analysisshown that free proline and arginineabound. Little is known about horsetail,Lycopodium and Selaginella spores,except that dehydrating horsetail sporeshave a very short life span and containfree amino acids. The problem withscreening for desiccation tolerance inthese propagules (not pollen and fungalspores) is that germ tube emergence iscomparatively slow, taking at least a weekand often much longer. Until rapid assaymethods for viability are developed, it willnot be attractive to study anhydrobiosis inthese spores. In contrast, germ tube emer-gence is generally a matter of hours in fun-gal spores and even minutes in pollen,

which makes them more attractive tostudy. The economic importance of pollenand fungal spores has also encouragedconsiderable research, particularly into thefundamental aspects of stress tolerance.The occurrence of imbibitional damage indry pollen and fungal spores/yeast, andprobably also in the other propagules, mayhave reduced the enthusiasm for the appli-cation of dry storage: the probably stillviable propagules may have been killed onrehydration. Considering the generallylimited longevity of the pollen/spores atroom temperature, sub-zero temperaturestorage has to be applied for successfullong-term storage.

The life span in the dry state at roomtemperature generally ranges from a fewdays to several months for both desicca-tion-tolerant pollen and spores. This is insharp contrast to plant seeds, which oftensurvive a number of years (see Priestley,1986, for a review). Pollen, for example,is not typically designed to overcomelong periods of adverse conditions, as iscommon for seeds. Although the cause ofthe difference in longevity betweenpollen and spores, on the one hand, andseeds, on the other hand, is a matter ofspeculation, it may be envisaged that thecomparatively large size of seeds givesthem an extra protection against molecu-lar oxygen and peroxidative degradation.The exceptionally long life span, as foundin some thick-walled fungal spores, mightdepend on a possible impermeability ofthese walls to oxygen.

The major difference between pollenand the other propagules is the lack of dor-mancy in the former. In hydrated pollen,metabolism seems unrestricted, which leadsto a rapid loss of viability. Thus, while sur-vival in the hydrated state is a commonstrategy in spores, it is not in pollen.

6.8. Acknowledgement

The author is indebted to Dr Elena A.Golovina from the Timiryazev Institute ofPlant Physiology, Russian Academy ofSciences, Moscow, for critically readingthe manuscript.

198 F.A. Hoekstra


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7 Vegetative Tissues: Bryophytes, VascularResurrection Plants and Vegetative Propagules

Michael C.F. Proctor1 and Valerie C. Pence21School of Biological Sciences, University of Exeter, Washington Singer Laboratories,

Perry Road, Exeter EX4 4QG, UK; 2CREW, Cincinnati Zoo and Botanical Garden,3400 Vine Street, Cincinnati, OH 45220, USA

7.1. Introduction 2077.2. Bryophytes 208

7.2.1. Desiccation time and recovery time: the typical pattern of desiccation response 210

7.2.2. The effect of intensity of desiccation 2117.2.3. Effects of temperature 2137.2.4. Events on rehydration 2137.2.5. Drying rate and drought hardening 2157.2.6. Constitutive and induced tolerance 2167.2.7. How long is needed for complete recovery? Processes and

criteria of recovery; long-term survival 2167.3. Vascular Plants 217

7.3.1. Ecological and morphological adaptations 2247.3.2. The effect of the intensity of desiccation 2257.3.3. Effect of the rate of desiccation 2267.3.4. Morphological and cytological changes that occur

with drying 2267.3.5. Rehydration and recovery 2277.3.6. Vegetative propagules: bulbils, corms, tubers and plant

fragments 2287.4. Concluding Comments 2287.5. References 230


7.1. Introduction

We take for granted the desiccation toler-ance of seeds and pollen, but see it as amatter for remark when we encounter des-

iccation tolerance in vegetative parts ofplants. However, desiccation tolerance is awidespread phenomenon (Bewley andKrochko, 1982; Stewart, 1989; Crowe et al.,1992; Oliver and Bewley, 1997). It is found

Dessication 07 18/3/02 1:56 pm Page 207

in many microorganisms, in animals suchas tardigrades, rotifers and nematodes andin larval cysts of crustacea of seasonalwater bodies, and in plants it is a frequentand characteristic feature of the vegetativecells of terrestrial algae, lichens andbryophytes. In vascular plants it is thenorm in spores, pollen and seeds, butuncommon in vegetative tissues.

Desiccation tolerance implies the abilityof an organism to dry to equilibrium withthe ambient air, and to recover and return tonormal metabolism on remoistening. It is aqualitatively different phenomenon fromdrought tolerance. Drought-tolerant plantscan maintain more or less normal metabo-lism in the presence of soil water potentialsthat may often be low enough to wilt theleaves, but are killed if the relative watercontent (RWC) of the tissues falls below~ 0.2–0.3, i.e. 20–30% of full turgor, corre-sponding to a tissue water potential of perhaps �5 to �10 MPa. Many desiccation-tolerant plants withstand drying to waterpotentials of �100 MPa or lower, at whichno liquid phase remains in the cells andmetabolism is at a standstill. Desiccation tol-erance must have evolved early in the colo-nization of land by microorganismsincluding cyanobacteria and simple algae.Amongst more complex photosynthetic landorganisms, the bryophytes are a numerousgroup, growing in diverse habitats from thetropics to the polar regions, in which adegree of desiccation tolerance seems to be afundamental part of their life strategy, and ispresumably primitive. Much the same maybe said of the symbiotic associationsbetween fungi and the photosynthetic organ-isms that we call lichens (Kershaw, 1985;Nash, 1996). On the other hand, the vascularplants that dominate the world’s flora gener-ally depend on maintenance of a high inter-nal water content by conduction of waterfrom the soil, with varying degrees of controlof water loss by the stomata (Raven, 1977).Desiccation-tolerant vascular plants are therare exception. They are relatively few innumber and taxonomically scattered andisolated – products of independent selectionfor intermittently or seasonally desiccatedhabitats, mostly warm-temperate to tropical.

There are probably some features andmechanisms common to all desiccation-tolerant cells, but there are also some majordifferences. In general, the more tolerantbryophytes are what have been character-ized as ‘fully desiccation-tolerant’ (Bewleyand Oliver, 1992; Oliver, 1996; Oliver andBewley, 1997; Oliver et al., 2000); their tol-erance is essentially constitutive and littleaffected by the rate of drying. By contrast,many desiccation-tolerant vascular plantsshow little tolerance if they are dried fast,but tolerance is induced in the course ofslow drying, which, because of their vascu-lar system and relatively large size, is gen-erally what happens in nature. Some of theless tolerant bryophytes probably behavesimilarly. These have been referred to as‘modified desiccation-tolerant’ plants(Oliver and Bewley, 1997). Desiccation-tol-erant plants vary greatly in the length andfrequency of the wet and dry periods towhich they are adapted. Small bryophytesof sun-exposed dry habitats may achieve apositive carbon balance from moist periodsof an hour or less and can benefit frombrief showers, or even dewfall followingclear nights. Vascular plants that retaintheir chlorophyll when desiccated maytypically require around a day for recovery.In poikilochlorophyllous species, whichlose their chlorophyll on drying, regreen-ing and return to normal metabolism takesseveral days and these plants generallyoccupy situations with relatively long alter-nating wet and dry periods.

7.2. Bryophytes (see also Chapter 1)

Bryophytes are the second largest group ofphotosynthetic land plants, with some25,000–30,000 species occupying diversehabitats from the tropics (Pócs, 1982;Richards, 1984) to polar regions (Longton,1988). By contrast with the vascular plants,they may be seen as having followed analternative strategy of adaptation to theirregular and intermittent availability ofwater on land. Conduction of water is typi-cally external and water is freely gainedand lost over much or all of the surface of

208 M.C.F. Proctor and V.C. Pence


the plant, and dry periods are passed in astate of suspended metabolism until wetconditions return. Clearly, bryophytes arein some respects limited by this mode oflife; they cannot compete with vascularplants in size or in productivity on fertilewater-retentive soils. On the other hand,they are freed from some constraints: theycan occupy hard substrates that are impene-trable to roots and so untenable to vascularplants, and they are well adapted to nutri-ent capture in N- and P-deficient situations.

The majority of bryophytes will with-stand at least modest levels of desiccation(to equilibrium with, for example �20 to�40 MPa) for at least a few days, but someare much more tolerant than that. Breuil-Sée (1993) observed regrowth followingremoistening of gametophytes of the liver-wort Riccia macrocarpa kept as driedherbarium specimens for over 23 years.Maheu (1922) observed regeneration of pro-tonema from leaves of ‘Barbula’ (= Tortula,Syntrichia) ruralis that had been dry for 14years. Keever (1957) found that most speci-mens of Grimmia laevigata were still viableafter 3 years’ dry storage, but only 20%remained viable after 10 years. Of taxo-nomic orders within the bryophytes, thosethat stand out as particularly desiccation-tolerant include the Andreaeales (Andreaea:‘granite mosses’), Encalyptales (Encalypta),Pottiales (Tortula, Syntrichia, Tortella, etc.),Orthotrichales (Orthotrichum, Ulota,Zygodon), Hedwigiales (Hedwigia) andGrimmiales (Grimmia, Racomitrium, etc.),but a number of other orders include atleast some highly tolerant species, e.g.Polytrichales (Polytrichum piliferum, etc.),Dicranales (Dicranoweisia cirrata,Cheilothela chloropus, etc.), Hypnales (e.g.Eurhynchium pulchellum, Scleropodiumtourretii, Hypnum spp., Leucodon sci-uroides, Anomodon viticulosus). Someother groups, such as the Hookerialesamong the mosses and many genera of leafyand thalloid liverworts, are relatively sensi-tive and characteristically confined to shel-tered humid places. Most bryophytes fallsomewhere between these two extremes.

The responses of bryophytes to desicca-tion are multifaceted, a fact of which the

early investigators were well aware(Irmscher, 1912; Höfler, 1946; Clausen,1952; Abel, 1956; Hosokawa and Kubota,1957), though appreciation of their com-plexity was constrained by limited tech-niques. The earlier studies generally usedplasmolysis as an indicator of cell survival.This necessarily gave a somewhat staticpicture, but it allowed exploration of theeffect of different times and intensities ofdesiccation, and demonstration of drought-hardening (Höfler, 1946; Abel, 1956);Höfler, Clausen, Abel, Hosokawa andKubota and others laid a foundation ofknowledge which is still valuable as back-ground for more analytical research. Ried(1960a,b), in his work on lichens, measuredphotosynthesis and respiration by classicalgas-analysis techniques, which, thoughlaborious, gave newly dynamic insightsinto the process and timing of recovery.Various authors in the 1970s and early1980s (Hinshiri and Proctor, 1971; Dilksand Proctor, 1974; Schonbeck and Bewley,1981a,b) measured gas exchange manomet-rically, but by the mid-1980s that had beenlargely superseded by infrared gas analysis(IRGA). This allowed continuous measure-ment and, as IRGA systems developed, pro-gressively better stability, sensitivity andtime resolution (Sesták et al., 1971; Dilksand Proctor, 1976; Kershaw, 1985; Lange,1988; Long and Hällgren, 1993; Tuba et al.,1996). Since the 1990s, chlorophyll fluores-cence has provided a powerful newmethod for rapid in vivo assessment ofsome aspects of photosynthetic function tocomplement gas-exchange measurements(Seel et al., 1992b; Deltoro et al., 1998a,b;Csintalan et al., 1999; Marschall andProctor, 1999).

Alongside this strand of research centredon gas exchange and carbon balance,Bewley, Oliver and their co-workers haveconcentrated particularly on effects of desic-cation and rehydration on protein synthesis.Their very extensive work, reviewed byBewley (1979), Bewley and Krochko (1982),Bewley and Oliver (1992), Oliver (1996) andOliver and Bewley (1997), leads naturallyinto modern molecular biological tech-niques (Wood et al., 1999) and to the possi-

Desiccation Tolerance of Vegetative Tissues 209


bility of genetic engineering for increaseddrought tolerance in arid-zone crops.

7.2.1. Desiccation time and recovery time:the typical pattern of desiccation response

Figure 7.1 shows the photosynthetic per-formance of a rehydrated moss, Anomodon

viticulosus (measured as net O2 evolution),as a function of desiccation time (at c. �94MPa) on one axis and rehydration time onthe other (Hinshiri and Proctor, 1971). Forperiods of up to about 10–15 days desicca-tion, recovery is quick and essentially com-plete within 3–4 h. This may be seen as aperiod of ‘pure’ desiccation tolerance.From this point to about 40–45 days’ desic-


20

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12

22

28

35

42

49

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67

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–500

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Fig. 7.1. The course of the net photosynthesis rate of Anomodon viticulosus following moistening after variousperiods of desiccation at 50% RH, 20°C. Curves are interpolated for net photosynthesis rates attained 2.5, 5,10, 15 and 20 h after moistening. The shaded area indicates the part of the graph over which net C assimilationis negative. Warburg manometer measurements of oxygen evolution or uptake; CO2 concentration maintainedby carbonate–bicarbonate buffer in centre wells; with a few exceptions points are means of two readings.Measurements up to 28 days were made at 20°C, the remainder at 25°C. Somewhat simplified and in partredrawn from Hinshiri and Proctor (1971). (Reproduced with permission from Proctor, 2001.)


cation, recovery becomes progressivelyslower and less complete. Evidently, one ormore progressive damaging processes takeeffect, which can at least to some extent berepaired given sufficient recovery time.After prolonged desiccation, remoisteningleads to long-persistent oxygen uptake,implying corresponding carbon loss. Thisprobably reflects major metabolic disrup-tion, from which ultimate recovery, if ittakes place at all, can only be slow.

7.2.2. The effect of intensity of desiccation

In addition to the factors considered in Fig. 7.1, recovery and long-term survivaldepend on the equilibrium water potentialto which the bryophyte has been desiccated.Dilks and Proctor (1974) gave recovery datafor several species following desiccation atdifferent water potentials, which showedthat some, like Plagiothecium undulatum,are more severely damaged the lower thewater potential to which they have equili-brated, while others, such as Racomitriumlanuginosum, show better long-term sur-vival at low than at high water potentials.Experiments using chlorophyll fluorescenceas a measure of plant performance show thesame pattern (Proctor, 2001; Fig. 7.2).Species of more-or-less shaded habitats,never exposed in the field to extreme desic-cation stress, were most quickly andseverely damaged at the lowest water poten-tial, at which species of dry, open, sun-exposed situations, such as R. lanuginosumand Tortula ruralis, showed best survival ofprolonged drying. Given a modest level ofdaylight illumination (comparable with ashady woodland floor) all the investigatedspecies survived well for several weeks at�3 MPa, corresponding to a cell RWC ofabout 50%. Over this period, the highly des-iccation-tolerant species, R. lanuginosumand T. ruralis, performed best at either thishighest water potential or at the lowest, andit was only after about 6 weeks that materialstored dry outperformed material kept lightlywilted. It was noteworthy that the more des-iccation-tolerant species were damaged mostrapidly in the range �9 to �22 MPa. In fact,


Fig. 7.2. The chlorophyll-fluorescence parameterFv /Fm (estimating the maximum quantum efficiencyof photosystem II), measured 20 min afterremoistening, following desiccation at a range ofwater potentials (�3 to �154 MPa). The curves aresmoothed from the original data (n = 3) by a singlepass through a binomial-average smoothing routine.Collecting sites: Hookeria lucens, Stoke Woods,Exeter, Devon, UK, August 1999; Rhytidiadelphusloreus, White Wood, Holne, Devon, UK, August1999; Tortula ruralis, Fülöpházá, KiskunságNational Park, Hungary, July 1998.

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even for the most desiccation-tolerantspecies, there is an optimum range of waterpotential for longest survival and most rapidrecovery, generally around �100 to �300MPa. After 24 h recovery from desiccationperiods of up to 60 days, the most tolerantspecies showed very little differencebetween �41, �113, �218 and �412 MPa,the chlorophyll-fluorescence parameterFv/Fm (a measure of the maximum quantumefficiency of photosystem II, widely used asa general indicator of plant stress) recover-ing within 24 h to normal unstressed levelsafter all of them (Fig. 7.3). The water contentof a bryophyte (as % dry weight (dw)) isclosely determined by the water potentialwith which it is in equilibrium (Dilks andProctor, 1979; Schonbeck and Bewley,1981a). Water contents of 5% dw or lessmay be reached by bryophytes in exposedplaces in the heat of the sun, correspondingto water potentials below �300 MPa.

7.2.3. Effects of temperature

When fully hydrated, bryophytes showgenerally similar temperature responses toC3 vascular plants. The lower limit formost species is probably set by loss ofwater as the cell contents equilibrate withthe water potential of extracellular ice(Kallio and Heinonen (1975) found positivenet photosynthesis in R. lanuginosum andDicranum elongatum down to �8°C); pos-sible chilling effects in warm-climatebryophytes at non-freezing temperaturesseem not to have been explored. The upperlethal limit for metabolically activebryophytes is usually in the range40–50°C. Bryophytes become much moretolerant of extremes of temperature as theydry out (Meyer and Santarius, 1998).Although there has been a general aware-ness that temperature must influence thesurvival of dry bryophytes, there has beenrather little systematic work on the subject(Irmscher, 1912; Lange, 1955; Nörr, 1974).Desiccation-tolerant bryophytes, when dry,can survive extremely low temperatures.Becquerel (1951) reported survival ofBarbula and Grimmia leaves after cooling

to less than 0.05 K in liquid helium, andBewley (1973) reported recovery of proteinsynthesis in T. ruralis shoots that had beencooled in the dry state to –196°C in liquidnitrogen. This is perhaps no matter for sur-prise. However, by analogy with seeds instorage, increased temperature would beexpected to shorten survival time, and thisis indeed so. Hearnshaw and Proctor (1982)measured mean survival of seven species(in terms of chlorophyll content after aperiod of moist recovery) at temperaturesfrom 20 to 100°C. Five of the bryophytesgave good straight-line relationships on an‘Arrhenius plot’ relating the logarithm ofhalf-survival time to the reciprocal ofabsolute temperature, as did data for viabil-ity of rice in storage (Roberts, 1975;Proctor, 1982). This is the pattern thatwould be expected for the temperatureresponse of chemical reactions in general(Morris, 1974). Two Racomitrium speciesgave non-linear plots, but for all thebryophytes survival times ranged continu-ously from minutes at 100°C to weeks ormonths at normal ambient temperatures.Ambient ‘shade’ temperatures are com-monly in the range 0–30°C and rarelyexceed 40°C, but dry bryophytes in the sunon hot days can reach 40–60°C, and it isprobably these high temperature episodesthat drive selection for the apparentlyextravagant levels of desiccation toleranceseen in, e.g. Tortula ruralis, Grimmia laevigata, R. lanuginosum and Andreaearothii.

7.2.4. Events on rehydration

The preceding sections have principally con-sidered responses along the desiccation-time axis of Fig. 7.1. There are also impor-tant questions to answer on the rewetting(recovery) axis. As the time-resolution ofmeasurement techniques has improved, ithas become increasingly apparent thatrecovery of normal photosynthetic functionon remoistening can be very rapid.Manometric and IRGA measurements havedemonstrated recovery to near-normal ratesof net photosynthesis within an hour or



two of remoistening in a number of species(Hinshiri and Proctor, 1971; Dilks andProctor, 1974, 1976; Fig. 7.4). IRGA datashow net photosynthesis in T. ruralis to benegative 15 min after remoistening, butalmost completely recovered after 30 min(Tuba et al., 1996). Measurements frommodulated chlorophyll fluorometers showthat photosystem II (PSII) can return tonear-normal quantum efficiency within afew minutes. Initial recovery of the ratioFv/Fm on remoistening shoots of T. ruralisin the dark after 3 days air dry (c. �100MPa; water content c. 10% dw) approxi-mated closely to an exponential curve witha half-recovery time of c. 20 s. The corre-sponding initial half-recovery time for thependulous African forest moss Pilotrichellaampullacea (after 20 h at �37 MPa) was c.40 s (M.C.F. Proctor, unpublished data).This rapid initial phase of recovery is fol-lowed by a much longer phase of slowrecovery, which may last an hour or two inthe most tolerant species and many hoursin the more sensitive. Recovery of(Fm�Fm�)/Fm� (�PSII, a measure of effectivequantum yield of PSII) in light is slowerthan recovery of Fv/Fm. Even so, in tolerantspecies recovery of photosynthetic electronflow (which of course may include a pho-torespiratory component as well as carbonfixation) can be largely complete within15–20 min (Csintalan et al., 1999).

It is clear that full recovery must involve adiversity of processes. First, membraneintegrity must be re-established. Probably allbryophytes (indeed, all desiccation-tolerant cells (Crowe et al., 1992)) showdetectable leakage of solutes immediately onremoistening (Brown and Buck, 1979;Bewley and Krochko, 1982) but in tolerantspecies this in only transient and most leaked


0 60 120 180

Net photosynthesis

Dark respiration

20

10

0

–10

–20

–30

–40

6

4

2

0

–2

–4

–6

0 30 60 90 120

Recovery time (min)

Tortula ruraliformis:8 days desiccation

(a)8

6

4

2

0

–2

–4

–6

–8

CO

2 up

take

(vp

m)

Grimmia pulvinata:14 days desiccation

0 60 120 180 240

(b)

Andreaea rothii:4 days desiccation

(c)

Fig. 7.4. Rate of net CO2 uptake (�) and darkrespiration (�) of some desiccation-tolerant mossesfollowing remoistening, after various periods air dry(at ~ �100 MPa), measured by infrared gas analysis(LCA-2, ADC, Hoddesdon, UK); PPFD 500 �molm�2 s�1; ~ 23°C. (a) Tortula ruralis ssp. ruraliformis,8 days; Braunton Burrows, Devon, UK; (b) Grimmiapulvinata, c. 14 days; stone parapet of bridge, ClystSt Mary, Devon, UK; (c) Andreaea rothii, 4 days;granite outcrop, Haytor, Devon, UK.


solutes are rapidly reabsorbed; little or no netleakage of inorganic ions is detectable after afew minutes (Deltoro et al., 1998a). Of themajor metabolic systems, photosynthesis,dark respiration and protein synthesis(Gwózdz et al., 1973; Oliver, 1991) are reiniti-ated very rapidly (within a minute or two) indesiccation-tolerant species such as R. lanug-inosum, T. ruralis and A. viticulosus. Muchor all of the process of the recovery of allthree systems seems likely to be essentiallyphysical, involving reinstatement of waterinto macromolecules and re-establishment ofspatial and conformational relationships. Theevidence indicates that recovery of the photo-systems is essentially independent of proteinsynthesis, but that some protein synthesis isneeded for full return to predesiccation ratesof photosynthetic carbon fixation (Proctorand Smirnoff, 2000; Proctor, 2001).

Respiration recommences immediatelyon remoistening; indeed, in lichens thereare indications of low levels of respiratoryactivity even at water potentials as low as�100 MPa (Cowan et al., 1979). The initialrate of respiration on remoistening isalways higher, and sometimes very muchhigher, than the normal steady rate of darkrespiration, perhaps reflecting some (proba-bly variable) combination of uncoupling ofrespiration from other metabolic systemsand the demands of repair processes. In theIRGA measurements of Dilks and Proctor(1976), dark respiration of A. viticulosusand Rhytidiadelphus loreus took 5–10 h toreturn to steady levels following a few days’desiccation. The data of Tuba et al. (1996)showed dark respiration in T. ruralis andthe dry-grassland lichens Cladonia convo-luta and Cladonia furcata, following a fewhours’ desiccation, falling rapidly within 2h to a rather steady level still greater thanthat measured before or during drying.

The recovery of protein synthesis hasbeen the subject of extensive research byJ.D. Bewley, M.J. Oliver and their co-workers, reviewed by Bewley (1979),Bewley and Krochko (1982), Bewley andOliver (1992), Oliver (1996), Oliver andBewley (1997), Oliver et al. (1998) and inChapters 1 and 11 of this book. Protein syn-thesis declines and ceases quickly under

water stress, but recommences within aminute or two of remoistening. In T. ruralisthe pattern of protein synthesis in the firsthours of rehydration is distinctly differentfrom that of hydrated controls, but no noveltranscripts are made in response to desicca-tion; the changes appear to be due to alter-ations in translational controls. Oliver(1991) showed that in T. ruralis the synthe-sis of 25 proteins ceased (or substantiallydecreased) and the synthesis of 74 proteinswas initiated (or substantially increased)during the first 2 h of rehydration. Henamed these protein groups ‘hydrins’ and‘rehydrins’, respectively. The synthesis ofall the hydrins returned to control levelswithin 2–4 h, but, while some rehydrinswere synthesized only transiently withinthe first hour or two of rehydration, otherswere still being synthesized at elevated lev-els 10–12 h later. Synthesis of all proteinshad returned to normal levels within 24 h.The exact nature and function of most ofthese hydrins and rehydrins is not yetknown, but some homologies with knownproteins are beginning to emerge from mol-ecular biological investigations (Wood etal., 1999; Chapters 1 and 11).

Growth of the bryophyte plant requiresnot only a positive carbon balance and theability to synthesize proteins, but also there-establishment of the cell cycle, andtranslocation of metabolites, mineral nutri-ents and growth regulators to the meristem-atic regions. Cell division and probably alsotranslocation depend on processes mediatedby microtubules, which are broken down ondesiccation and must be reconstituted as apart of the recovery process. A degree ofrepair of DNA will always be necessary, andthe requirement for this is likely to increasewith increasing desiccation time.

7.2.5. Drying rate and drought hardening

Höfler (1946) and Abel (1956) found that thedesiccation tolerance of bryophytes wasenhanced following a period of less severedrying. Abel surveyed the recovery of a widerange of moss species after 24–48 h desicca-tion at a range of humidities from 0.08 to



96% relative humidity (RH), with and with-out a previous light drying at 96% RH for 24h. The predrying treatment led to clearlygreater desiccation tolerance in the majorityof species, sometimes very strikingly so, as inBryum capillare, Bryum caespiticium,Mnium marginatum, Plagiomnium rostra-tum, Ceratodon purpureus, Fissidensadiantoides, Pohlia elongata, Timmia austri-aca and P. undulatum. In some species (gen-erally the most sensitive and the mosttolerant) predrying made little difference, asin Bryum pseudotriquetrum (bimum),Mielichhoferia elongata, Philonotis seriataand Rhynchostegium riparioides (Platy-hypnidium rusciforme) (sensitive), and T.ruralis, Encalypta streptocarpa (contorta),Grimmia pulvinata, Hedwigia ciliata (albi-cans), Pleurozium schreiberi andRhytidiadelphus spp. (tolerant). Schonbeckand Bewley (1981a) explored the effects ofslow and fast drying and rehydration on pho-tosynthesis by T. ruralis following 2 days’and 7 days’ desiccation at �21 or �208 MPa.Rate of drying made little difference to recov-ery after desiccation at �21 MPa, but recov-ery of photosynthesis was severely impairedafter rapid drying to �208 MPa. Damagingeffects of rapid drying to –600 MPa werelargely eliminated if the moss samples werefirst dried slowly to �21 MPa, and weregreatly reduced if the samples were equili-brated for 5 h in saturated air before remoist-ening. Oliver and Bewley (1997) suggestedthat the limited ‘hardening’ effect seen in T.ruralis may reflect sequestration of ‘recovery’mRNAs during slow drying. Krochko et al.(1978) found very large differences betweenfast- and slow-dried material of the relativelydesiccation-sensitive moss Cratoneuron fil-icinum. There is clearly much of interest inthe effects of different rates of drying andrehydration, which invites further investiga-tion and study of a wider range of species.

7.2.6. Constitutive and induced tolerance

Desiccation tolerance apears to be largelyconstitutive in the highly desiccation-tolerantbryophytes on which most work has beendone, such as T. ruralis (Oliver and Bewley,

1997; Oliver et al., 1998), R. lanuginosumand A. viticulosus. However, in many speciesof more mesic habitats, tolerance is inducedto varying degrees. The physiology of theinduction of enhanced tolerance in suchimportant groups as the Bryaceae, Mniaceaeand the common pleurocarpous families inthe Hypnobryales is almost entirely unex-plored. Abscisic acid (ABA)-induced toler-ance (involving synthesis of specific proteinsduring drying) has been demonstrated in pro-tonema of Funaria hygrometrica (Werner etal., 1991; Bopp and Werner, 1993; Schnepfand Reinhard, 1997), and short, thick-walleddesiccation-tolerant protonemal cells formunder the influence of desiccation or ABA inAloina aloides (Goode et al., 1994) andDiphyscium foliosum (Duckett, 1994).Beckett (1999) showed that partial dehydra-tion of the moss Atrichum androgynum for 3days increased resistance to desiccation-induced cation leakage, and that treatmentwith ABA produced the same effect. Inducedor seasonally switched desiccation toleranceis probably common in marchantialean liver-worts of seasonally dry habitats. In Lunulariacruciata, the switch from the desiccation-sen-sitive winter state to the tolerant summerstate appears to be mediated by lunularicacid (Schwabe and Nachmony-Bascomb,1963). Hellewege et al. (1994) showed thatABA induces desiccation tolerance in the liv-erwort Exormotheca holstii, and Hellewege etal. (1996) have shown that ABA can bringabout the transition from the aquatic to theland form of Riccia fluitans. The ability torespond to ABA with enhanced desiccationtolerance thus seems likely to be widespreadamong bryophytes, but how widely endoge-nous ABA occurs within the bryophytes isunknown. ABA is undetectable in T. ruralis,in which a high level of desiccation toleranceis constitutive (Oliver and Bewley, 1997).

7.2.7. How long is needed for completerecovery? Processes and criteria of recovery;

long-term survival

The recovery processes outlined in Section7.2.4 proceed on different time scales.What should we take as the criteria of over-



all ‘recovery’? There may be no hard andfast answer to this question. Very shortmoist periods will lead to net carbon loss.Moist periods long enough for a positivenet carbon balance may be insufficient forcell division and growth, but might per-haps allow significant DNA repair. This isconjectural, but indicates the kind of ques-tions on which research is needed. Thelimited available measurements indicatethat the moist periods experienced by des-iccation-tolerant bryophytes in the fieldvary greatly in length (Proctor, 1990;Proctor and Smith, 1995). Maintenance of apositive net carbon balance must be impor-tant, even on a rather short time scale,whereas a bryophyte may have an entirelyviable annual life cycle in which growth islargely confined to particular seasons,while for the rest of the year the plant isdoing no more than maintaining itsfoothold in the habitat. Whether ‘recovery’is seen as return to a normal rate of carbonfixation, or as requiring full restoration ofall metabolic systems to their optimummoist-period activity, is a question of context – indeed, there may be no single‘optimum’. Further, bryophyte shoots com-monly show progressive formation of newleaves and death of old ones; recovery fromdesiccation of the apical parts of the shootmay be accompanied by accelerated senes-cence and death of the older parts.

7.3. Vascular Plants (see also Chapter 1)

Although often observed in seeds, sporesand pollen, desiccation tolerance is theexception in vegetative tissues of vascularplants. The combination of vascular tissueand intercellular spaces with cuticle andstomata allows these species to maintain awater potential higher than that of theirabove-ground environment (homoiohydry),avoiding the need to tolerate large fluctua-tions in moisture availability. Nevertheless,in intermittently arid habitats some specieshave adapted to survive desiccation ratherthan avoid it. However, of the quarter of amillion or so species of vascular plants,only some 330 species, or < 0.15% of the

total, have been documented as being desiccation-tolerant in their vegetative parts(Table 7.1; Porembski and Barthlott, 2000).Because the rehydration of some of theseplants gives the appearance of a revitaliza-tion of apparently dead tissues, they areoften referred to as ‘resurrection plants’.

The first scientific report of a resurrectionspecies was made by Hooker (1837) in adescription of Selaginella lepidophylla fromthe southwestern USA and Mexico.Subsequent reports were made in the nextcentury, the earliest from field observationsmade in dry areas of central Asia and sub-Saharan Africa. The remarkable ability ofthese plants to exhibit the effects of extremedesiccation and yet to remain alive andrevive when rewetted was reported withinterest and often careful notes (Thoday,1921; Vassiljev, 1931; Hambler, 1961, 1964;Hornby and Hornby, 1964). Carex physodes,Myrothamnus flabellifolia and Craterostigmaplantagineum were among the first desicca-tion-tolerant angiosperms described, whileearly reports confirmed the phenomenon inother pteridophytes, such as Polypodiumpolypodioides, Notochlaena marantae,Selaginella njam-njamensis and Platyceriumstemaria (Pessin, 1924; Iljin, 1931; Hambler,1961, 1964).

Resurrection species have been docu-mented thus far in nine families of pterido-phytes and ten families of angiosperms(Table 7.1). They are conspicuously lackingin gymnosperms, though foliage ofWelwitschia mirabilis shows some degreeof tolerance (Gaff, 1972). In angiosperms,they occur among both monocotyledonsand dicotyledons. Although a higher pro-portion of pteridophyte taxa than seedplants are tolerant of desiccation,angiosperms often exhibit a higher degreeof tolerance than ferns (Gaff, 1977).

Desiccation tolerance shows a wide tax-onomic scatter, and appears to haveevolved independently a number of timesas an adaptation to extremes in water avail-ability. Some genera, such as Cheilanthes,Pellaea, Selaginella and Xerophyta, havelarge numbers of tolerant species whileothers, such as Boea, have only a singlespecies that is known to be tolerant. A




Table 7.1. Desiccation-tolerant (DT) vascular plants. With a few exceptions, names and authorities arethose used by the cited authors. Species of a genus recorded in a single publication from the samegeographical area are listed together; otherwise the arrangement is alphabetical within major taxonomiccategories.

Species Country Reference

PTERIDOPHYTESLYCOPSIDA (Clubmosses)Isoetaceae

Isoetes australis Williams Australia Gaff and Latz (1978) (Only the cormsare DT; other terrestrial Isoetes spp. ofseasonally desiccated habitats, e.g. insouthern Europe, are likely to behavesimilarly)

Selaginellaceae

Selaginella caffrorum (Milde) Hieron., South Africa Gaff (1977)S. digitata Spring, S. dregei (C. Presl)Hieron., S. echinata Baker, S. imbricata (Forsk.) Spring ex Decaisne, S. nivea Alston

Selaginella convoluta Spring, South America Gaff (1987)S. peruviana (Milde) Hieron.,S. sellowii Hieron.

Selaginella lepidophylla (Hook. and Southwestern USA, Hooker (1837), Gaff (1971), Eickmeier Grev.) Spring Mexico (1979), Iturriaga et al. (2000)

Selaginella njam-njamensis Hieron. West Africa Hambler (1961)

Selaginella pilifera A. Br. Southwestern US Eickmeier (1980)

Selaginella sartorii Hieron. Mexico Iturriaga et al. (2000)

PTEROPSIDA (Ferns)Adiantaceae (sensu lato)

Actiniopteris dimorpha Pic. Serm. South Africa Gaff (1977)

A. radiata (Sw.) Link South Africa, India Gaff (1977), Sharma and Purohit (1986)

Adiantum incisum Forsk. South Africa, India Gaff (1977), Sharma and Purohit (1986)

Cheilanthes albomarginata India Sharma and Purohit (1986)

Cheilanthes bonariensis (Wild) Proctor, Mexico Iturriaga et al. (2000)C. integerrima (Hook.) Mickel.,C. myriophylla Desv.

Cheilanthes buchtienii (Rosenst.) South America Gaff (1987)Capurro, C. glauca (Cav.) Mett.,C. marginata H.B.K.

Cheilanthes farinosa (Forsk.) Klf. South Africa Gaff (1977), Sharma and Purohit (1986)

Cheilanthes capensis (Thunb.) Desv., South Africa Gaff (1977)C. depauperata Bak., C. dinteri Brause, C. eckloniana (Kunze) Mett., C. hirta Sw., C. inaequalis (Kunze) Mett.,C. marlothii (Hieron) Mett., C. multifida (Sw.) Sw., C. parviloba Sw.



Table 7.1. Continued


Cheilanthes distans (R. Br.), Australia Gaff and Latz (1978)C. fragillima F. Muell., C. lasiophyllaPic.-Ser., C. paucijuga Benth., C. tenuifolia (Burm.f.) Sw., C. vellea(R. Br.) F. Muell.

Cheilanthes lendigera Swartz Wittrock (1891)

Cheilanthes pringlei Daven., Southwestern USA, Helvy (1963)C. wrightii Hooker Mexico

Cheilanthes sieberi Kunze Australia, Gaff and Latz (1978), Gaff and South Africa McGregor (1979)

Doryopteris concolor (Lag. and Risch.) South Africa Gaff (1971, 1977) Kahn

Doryopteris kitchingii (Bak.) Bonap. South Africa Gaff (1977)

Doryopteris pedata (L.) Fée, D. triphylla South America Gaff (1987)(Lam.) Christ

Notholaena marantae R.Br. Europe Iljin (1931)

Notholaena parryi D. C. Eat. North America Witham (1972), Nobel (1978)

Notholaena R. Br. sp. South America Porembski and Barthlott (2000)

Paraceterach muelleri (Hook.) Copel. Northeastern Gaff and Latz (1978)Australia

Pellaea atropurpurea (L.) Link Mexico Pickett and Manuel (1926)

Pellaea boivinii Hook., P. calomelanos South Africa Gaff (1977)(Sw.) Link, P. hastata (L.f.) Link,P. quadripinnata (Forsk.) Prantl., P. viridis (Forsk.) Prantl.

Pellaea falcata (R. Br.) Fée Australia Gaff and Latz (1978)

Pellaea glabella Mett. USA Pickett and Manuel (1926)

Pellaea longimucronata Hooker Southwestern USA, Helvy (1963)Mexico

Pellaea ovata (Desv.) Weatherby (commercial) Iturriaga et al. (2000)

Pellaea rotundifolia (Forsk.) Hk. (RBG Kew: Gaff and Latz (1978)origin unknown)

Pellaea sagittata (Cav.) Link f. var. Mexico Iturriaga et al. (2000)cordata (Cav.) Tyron.

Pellaea ternifolia (Cav.) Link South America Gaff (1987)

Aspleniaceae

Asplenium aethiopicum (Burm. f.) South Africa Gaff (1977)Bech., A. rutifolium (Berg.) Kunze var. bipinnatum (Forsk.) Schelpe, A. sandersoni H. K.

Asplenium bourgaei Mediterranean Greuter et al. (1983)

Asplenium pringlei Davenp. Wittrock (1891)

Continued




Asplenium ruta-muraria L. Western Kappen (1964)Europe

Asplenium septentrionale (L.) Hoffm. Europe Kappen (1964)

Asplenium trichomanes L. Europe Wittrock (1891), Kappen (1964)

Ceterach cordatum (Thunb.) Desv. South Africa Gaff (1977)

Ceterach officinarum Lam. et DC Southern and Oppenheimer and Halevy (1962), Western Europe, Schwab et al. (1989), M.C.F. ProctorMediterranean (unpublished data)

Pleurosorus rutifolius (R. Br.) Fée Western Australia Gaff and Latz (1978)

Woodsia ilvensis (L.) R.Br. Europe Wittrock (1891)

Davalliaceae

Arthropteris orientalis (Gmel.) Porth. South Africa Gaff (1977)

Grammitidaceae

Ctenopteris heterophylla (Labill.) Tindale New Zealand Gaff and Latz (1978)

Hymenophyllaceae

Hymenophyllum tunbridgense (L.) Southwestern Smith, H. wilsonii Hook. England M.C.F. Proctor (unpublished data)

Hymenophyllum sanguinolentum New Zealand J.G. Duckett and M.C. Proctor(Forst. F.) Swartz (unpublished data)

Polypodiaceae

Platycerium stemaria (P. Beauv.) Desv. West Africa Hambler (1961)

Polypodium cambricum L. Southwestern M.C.F. Proctor (unpublished data)England

Polypodium polypodioides (L.) North America Pessin (1924), Stuart (1968), Gaff Hitchcock (1977), Iturriaga et al. (2000)

Polypodium virginianum L. North America Reynolds and Bewley (1993)

Polypodium vulgare L. Wittrock (1891), Kappen (1964)

Schizaeaceae

Anemia tomentosa (Sav.) Swartz South America Gaff (1987)

Mohria caffrorum (L.) Desv. South Africa Gaff (1971, 1977)

Schizaea Sm. sp. East Africa, Porembski and Barthlott (2000)Seychelles

ANGIOSPERMS

MONOCOTYLEDONS

Cyperaceae

Afrotrilepis pilosa (Boeck.) J. Raynal West Africa Hambler (1961), Owoseye and Sanford(1972)

Carex physodes M. Bieb Central Asia Vassiljev (1931)





Coleochloa pallidior Nelmes South Africa Gaff and Ellis (1974)

Coleochloa setifera (Ridley) Gilly South Africa Gaff (1971), Gaff and Ellis (1974)

Cyperus bellis Kunth South Africa Gaff and Ellis (1974)

Fimbristylis dichotoma (L.) Vahl Australia Gaff and Latz (1978)

Fimbristylis Vahl Tropical Africa Porembski and Barthlott (2000)

Kyllinga alba Nees South Africa Gaff and Ellis (1974)

Mariscus capensis Schrad. South Africa Gaff and Ellis (1974)

Microdracoides squamosa Hua West Africa Porembski and Barthlott (2000)(monotypic)

Trilepis Nees South America Porembski and Barthlott (2000)

Liliaceae (Anthericaceae)

Borya inopinata Australia Forster and Thompson (1997)

Borya nitida Labill. Australia Gaff and Churchill (1976)

Borya septentrionalis F. Muell. Australia Gaff and Latz (1978)

Poaceae

Brachyachne patentiflora (Stent and South Africa Gaff and Ellis (1974)Rattray) C.E. Hubb.

Eragrostiella bifaria (Vahl) Bor. Australia Gaff and Latz (1978)

Eragrostiella brachyphylla (Stapf) Bor., India Gaff and Bole (1986)E. nardioides (Trin.) Bor.

Eragrostis hispida K. Schum., South Africa Gaff and Ellis (1974)E. nindensis Fic. and Hiern,E. paradoxa Launert

Eragrostis invalida Pilger West, East and Gaff (1986), Nugent and Gaff (1989)South Africa

Micraira adamsii Australia Gaff (1989)

Micraira spinifera Lazar, M. tenuis Lazar Australia Gaff and Sutaryono (1991)

Micraira subulifolia F. Muell. Australia Gaff and Latz (1978)

Microchloa caffra Nees, M. kunthii Desv. South Africa Gaff and Ellis (1974)

Microchloa indica (L.f.) O. Kunze South America, Gaff (1987), Iturriaga et al. (2000)Mexico

Oropetium capense Stapf South Africa Gaff (1971), Gaff and Ellis (1974)

Oropetium roxburghianum (Steudel) India Gaff and Bole (1986)S. Phillips, O. thomaeum Trin.

Poa bulbosa L. Europe Gaff and Latz (1978)

Sporobolus atrovirens (Kunth) Kunth Mexico Iturriaga et al. (2000)

Sporobolus elongatus R. Br. Australia Gaff and Sutaryono (1991)

Sporobolus festivus Hochst. South Africa Gaff and Ellis (1974), Kaiser et al.(1985)


Continued





Sporobolus fimbriatus Australia Gaff and Ellis (1974)

Sporobolus lampranthus Prig. South Africa Gaff and Ellis (1974)

Sporobolus pellucidus Hochst. East Africa Gaff (1986), Nugent and Gaff (1989)

Sporobolus stapfianus Gandoger South Africa Gaff and Ellis (1974), Kaiser et al.(1985), Sgherri et al. (1994)

Tripogon capillatus Jaub. et Spach, India Gaff and Bole (1986)T. filiformis (Stapf) Nees ex Steud., T. jacquemontii Stapf, T. lisboae Stapf, T. polyanthus Naik. et Patunkar

Tripogon curvatus Phillips and Launert Africa Gaff and Sutaryono (1991)

Tripogon lolioformis (F. Muell.) Australia Gaff and Latz (1978)C. E. Hubbard

Tripogon minimus (A. Rich.) Hochst. South Africa Gaff and Ellis (1974)ex Steud.

Tripogon spicatus (Nees) Ekman South America, Gaff (1987), Iturriaga et al. (2000)Mexico

Velloziaceae

Aylthonia blackii (L.B.Smith) Menezes South America Gaff (1987)

Barbacenia flava Martius ex Schultes f., South America Gaff (1987)B. longiflora Martius, B. riedeliana Goethart and Henrare, B. selloviiGoethart and Henrard

Barbaceniopsis boliviensis (Baker) South America Gaff (1987)L.B. Smith, B. humahuaguensis Noher

Nanuza plicata (Speng) L.B. Smith South America Rosetto and Dolder (1996)and Ayensu

Pleurostima Raff. South America Porembski and Barthlott (2000)

Vellozia Vand. (~ 124 spp., probably all South America Gaff (1987), Porembski and Barthlott desiccation-tolerant) (2000)

Xerophyta Juss. (~ 28 spp., probably all Sub-Saharan Gaff (1971, 1977), Owoseye and desiccation-tolerant) Africa, Madagascar Sanford (1972); Gaff and Hallam (1974),

Hallam and Gaff (1978), Tuba et al.(1993), Porembski and Barthlott (2000)

DICOTYLEDONS

Acanthaceae

Talbotia elegans Balfour South Africa Gaff and Hallam (1974), Hallam andGaff (1978)

Cactaceae

Blossfeldia liliputana South America Barthlott and Porembski (1996)

Gesneriaceae

Boea hygroscopica F. Muell. Australia Gaff and Latz (1978), Kaiser et al.(1985)





Haberlea rhodopensis Friv. Southeastern Bewley and Krochko (1982), Müller et Europe al. (1997)

Ramonda myconi Reichb. Southwestern Gaff and McGregor (1979), Schwab et (= R. pyrenaica Rich.) Europe al. (1989)

Ramonda nathaliae Panc. and Petrov. Southeastern Bewley and Krochko (1982), Müller et Europe al. (1997)

Ramonda serbica Panc. Southeastern Markovska et al. (1994)Europe

Streptocarpus Lindley spp. Africa Porembski and Barthlott (2000)

Labiatae

Satureja gilliesii (Benth.) Briq. South America Montenegro et al. (1979)

Myrothamnaceae

Myrothamnus flabellifolia Welw. South Africa Thoday (1921), Child (1960), Gaff(1971, 1977)

Myrothamnus moschata (Baillon) Madagascar Gaff (1977)Niedenzu

Scrophulariaceae

Chamaegigas intrepidus Dinter ex Heil South Africa Gaff (1971, 1977)

Craterostigma monroi S. Moore, South Africa Gaff (1977)C. nanum Engl.

Craterostigma plantagineum Hochst. South Africa Gaff (1971, 1977), Schwab et al. (1989)

Craterostigma wilmsii Engl. South Africa Gaff (1971, 1977)

Ilysanthes purpurascens Hutch., South Africa Gaff (1977)I. wilmsii Engl. and Diels

Limosella L. South Africa Porembski and Barthlott (2000)

Lindernia All. spp. Tropical Africa Porembski et al. (1997)

large number of desiccation-tolerantspecies have been documented from cen-tral and southern Africa, Australia andSouth America (Gaff, 1971, 1977, 1986,1987; Gaff and Ellis, 1974; Gaff andHallam, 1974; Gaff and Churchill, 1976;Gaff and Latz, 1978; Gaff and Giess, 1986).Although fewer have been described fromEurope, Asia and North America, new des-iccation-tolerant species continue to befound as dry habitats are further explored(Gaff and Bole, 1986; Iturriaga et al., 2000).

Because these plants exhibit such a dra-matic change in morphology during desic-cation and rehydration, revival of normalappearance after apparent death was, atfirst, sufficient to indicate survival of

plants, and early studies centred on mea-suring the rates of drying and the loss ofmoisture in leaves of plants observed in thefield or laboratory (Child, 1960; Hambler,1961; Hornby and Hornby, 1964).Regreening of species that lose chlorophyllduring desiccation, as well as the use ofvital stains, was soon added to confirm tis-sue viability after rehydrating (Gaff andOkon’O-Ogola, 1971; Gaff and Ellis, 1974;Gaff, 1977; Hallam and Gaff, 1978), andearly measurements of respiration and pho-tosynthesis progressed from simple gasexchange to IRGA (Stuart, 1968; Viewegand Ziegler, 1969; Eickmeier, 1979). Soluteleakage from desiccated tissue was alsoused as a measure of damage (Leopold et


al., 1982). By the 1980s, research exploringthe relationship of metabolism to the desic-cation phenomenon was well under way,with a few species being notable modelsaround which much of this research cen-tred (e.g. S. lepidophylla, C. plantagineum,M. flabellifolia, Chamaegigas intrepidus,Polypodium virginianum, Sporobolus stap-fianus, Boea hygroscopica). This has natu-rally led to molecular studies, particularlywith Craterostigma plantagineum (seereviews by Hartung et al., 1998; Scott,2000), and these lines of research are exam-ined in detail in Chapter 12.

7.3.1. Ecological and morphologicaladaptations

Resurrection species are often small, low-growing plants with short internodes andcompact growth that are found as pioneersin shallow soils or on rocky outcroppings,areas that can experience extreme variationsin moisture availability. When the rains docome, these plants must be able to rehy-drate, photosynthesize and grow before dry-ing occurs again. At some point, they mustalso remain active for a period long enoughto reproduce. Some species are adapted forseasonal changes in water availability.Others dry and are rewetted at more irregu-lar intervals. Chamaegigas intrepidus growsin southern Africa in situations where watercollects into ephemeral pools. This aquaticplant may be subjected to as many as 20desiccation/rehydration cycles annually inaddition to surviving through at least 8months of dry dormancy (Schiller et al.,1998, 1999). The ability to survive rapid andfrequent desiccation and to quickly re-estab-lish normal function is a necessity undersuch conditions.

As pioneers, resurrection species maybe restricted to areas that are uninhabitableby other vascular species. The sedgeAfrotrilepis pilosa forms monospecificstands, which persist without encroach-ment by less well-adapted species(Porembski et al., 1996). Plants such asMyrothamnus flabellifolia, however, pro-vide shelter and trap organic materials,

promoting soil development and the estab-lishment of other species (Child, 1960).

In addition to physiological desiccationtolerance, some species share water-absorbing or retaining characteristics withdesiccation-intolerant species. Xeromorphiccharacteristics in the liliaceous desiccation-tolerant plant Borya nitida and the grass S.stapfianus help reduce water loss, with thelatter retaining almost 80% RWC after aweek of desiccation (Gaff and Churchill,1976; Vecchia et al., 1998). In otherspecies, specialized structures aid inabsorbing or dispersing water. Scales onthe underside of the leaf of P. polypodi-oides appear to function in distributingwater over the surface, thereby aiding inabsorption (Pessin, 1924; Stuart, 1968),while dead leaf bases around the stem of A.pilosa help retain water and slow the rateof drying (Hambler, 1961). Velamen on theroots of some species (Afrotrilepis pilosa,Coleochloa setifera, Xerophyta pinnifolia)allows for rapid absorption when water isavailable (Porembski and Barthlott, 1995).Other resurrection plants, such asCraterostigma plantagineum, C. wilmsiiand C. nanum, possess few morphologicalcharacteristics which help to avoid or slowdesiccation, and it is not surprising thatthese show a greater physiological toler-ance for rapid desiccation than do morexeromorphic species (Sherwin, 1995;Farrant et al., 1999). It should also be notedthat some desiccation-tolerant species areable to grow under conditions of extremewater deficits as well as in areas wheresoils are richer and water more plentiful,and that morphologies, such as leaf shape,may differ in the different environments(Hornby and Hornby, 1964).

Although they are adapted to extremedesiccation, often in hot, dry environ-ments, not all resurrection plants are toler-ant of either high light levels or hightemperatures. Many resurrection speciesdo tolerate full sun, but others, especiallypteridophytes, typically require a shadedhabitat and may be damaged by excessivesunlight (Gaff, 1977; Lebkuecher andEickmeier, 1991). Similarly, some resurrec-tion species, again often pteridophytes, fre-



quently occur in thermally buffered micro-habitats, such as in rocky crevices or out-croppings. This environment providessome shelter from direct sunlight, reducingdaytime temperatures while holding heatduring the cooler nights and winter days.The optimal temperature for photosynthe-sis in the desert fern, Notholaena parryi,has been measured as several degreescooler than the mean air temperature of itsgeneral environment, suggesting a modify-ing effect of its microhabitat (Nobel, 1978).Moderating the extremes of temperatureand light can also be important for surviv-ing desiccation, since desiccation damagecan be enhanced by photodamage and hightemperatures (Eickmeier, 1986; Muslin andHomann, 1992; Chapter 9).

Although research on desiccation toler-ance in vascular species has focused pri-marily on the sporophyte, gametophytes ofsome pteridophytes also display desicca-tion tolerance. The extent of this phenome-non has not been well documented, butseveral reports indicate that it may be asurvival mechanism in at least some pteri-dophyte species (Mottier, 1914; Kappen,1965; Page, 1979; Quirk and Chambers,1981). Some success has been achieved inrecovering in vitro-grown fern gameto-phytes of Adiantum tenerum, Adiantumtrapeziforme, Cibotum glaucum, Davalliafejeensis and Drymaria quercifolia afterexposure to liquid nitrogen when thegametophytes were first air-dried, indicat-ing that the tissues were dry enough toavoid freezing injury (Pence, 2000).

7.3.2. The effect of intensity of desiccation

The vegetative tissues of most vascularplants can survive equilibration with RHsonly in the range of 85–98%; the NamibianWelwitschia mirabilis will stand dryingonly to a RWC of 56% (Gaff, 1972). Trulydesiccation-tolerant species, however, canequilibrate with much lower RHs, often< 10%, and still recover on remoistening.Drying tissues may lose from 70 to 95% oftheir original water content, generally overa period of several days (Pessin, 1924;

Child, 1960; Oppenheimer and Halevy,1962; Kappen, 1964; Stuart, 1968; Gaff andChurchill, 1976; Reynolds and Bewley,1993). While some research on the limits ofdesiccation tolerance has been donedirectly on field-dried tissues, in the labo-ratory tissues have been air-dried, equili-brated over solutions producing knownRHs or dried over desiccating agents, suchas sulphuric acid or silica gel, for morerapid drying. The humidity over silica geland other strong drying agents is oftenreported as ‘0% RH’, but this limit (corre-sponding to a water potential of �∞) isunattainable in practice.

Levels of desiccation tolerance differamong species. Gaff (1977) examined 37desiccation-tolerant ferns and angiospermsfor their RH tolerance levels and foundthat only 30% of the pteridophytes couldsurvive equilibration with an RH close to0%, while 76% of the angiosperm taxa didso, reflecting the tendency of pterido-phytes to inhabit somewhat protectedareas. The least tolerant resurrection fern,however, was still able to survive equili-bration with 30% RH, significantly lowerthan the RH tolerated by non-resurrectionplants. The RWCs of most dry resurrectionplants are in the range of 5–10% or less(Scott, 2000).

As with dry seeds, vegetative tissuesthat are sufficiently dry may remain viablefor relatively long periods of time,although moisture levels, temperature andspecies differences will affect longevity.Dried Afrotrilepis pilosa remained viablefor a year at room temperature, and alsosurvived storage overnight at �10°C,whereas undried tissues did not survivefreezing (Hambler, 1961). Dry tissues ofseven vascular species survived for at least3.5 years as field-dried leaves when theywere sealed in plastic or air-tight glass(Gaff, 1977), and two of these species,Xerophyta squarrosa and C. setifera,showed 100% survival even after 5 years.As with seeds, lower temperatures mightbe expected to prolong viability of dry tis-sues, and such tissues should be good can-didates for low-temperature storage orcryopreservation.



7.3.3. Effect of rate of desiccation

In contrast to bryophytes, vascular plantsare buffered to varying degrees from ambi-ent moisture levels by their larger size, thepresence of a cuticle and stomata, andtheir ability to access underground mois-ture. In addition, desiccation-tolerantspecies with xeromorphic characteristicscan slow water loss even further. As aresult, the desiccation-tolerance mecha-nisms in desiccation-tolerant vascular taxaappear to be, in large part, inducible ratherthan constitutive, as in many bryophytes,with tolerance developing over the courseof 12–24 h.

Natural drying of resurrection species inthe field generally occurs over a period ofdays or even weeks. In Craterostigma plan-tagineum signs of desiccation were visiblewithin 1 week of the last rain (Hornby andHornby, 1964). Gaff (1977) observed 11species in the field in southern Africa forthe first signs of water stress in the leavesand noted that, for ten of the species, dry-ing times ranged from 40 to 96 h. Theexception was Chamaegigas intrepidus, asmall, aquatic plant of ephemeral rockpools, which was air-dry within an hour.

Critical evaluation of the maximum tol-erated rates of drying support the observa-tion that natural drying of resurrectionspecies is generally slow. A number ofstudies have shown that these species donot survive rapid desiccation, althoughslower rates of drying will allow survivalto very low levels of moisture. For exam-ple, leaves of Borya nitida which wereair-dried could not survive below equili-bration with 85% RH or less. If, however,the leaves were first exposed to 96–98%RH for 2 days, they were then capable ofsurviving close to 0% RH (Gaff andChurchill, 1976). Studies with Selaginellalepidophylla, Boea hygroscopica and otherspecies have demonstrated similarresponses (Eickmeier, 1983; Sgherri et al.,1994). Slow drying can be replaced byABA in several systems, suggesting that itis closely involved with the induction ofprotective mechanisms in resurrectionspecies (Reynolds and Bewley, 1993).

7.3.4. Morphological and cytologicalchanges that occur with drying

Drying of resurrection plants brings aboutvarious morphological changes. Theseresult from the loss of water at the cellularlevel, but provide protection at the wholeplant level. The curling of leaves to formlong, threadlike structures and the curlingof older leaves and stems over youngerleaves and buds slow the rate of drying inyounger, growing tissues, as well as pro-tecting inner dried tissues from photodam-age (Pessin, 1924; Child, 1960; Gaff andChurchill, 1976; Gaff, 1977). Younger leaftissue, in general, appears more tolerant ofdesiccation (Gaff and Ellis, 1974; Norwoodet al., 1999).

Changes in shape at the whole plantlevel are accompanied by a dramatic reduc-tion in size, loss of turgor and a decrease incell volume. Some resurrection species mayshrink to less than 20% of their original leafarea upon drying. These species are able tomaintain connections between the plasmamembrane and the cell wall and to undergocontrolled and extensive folding of the cellwall during drying, allowing the collapse ofthe tissue without the fatal results seen innon-tolerant species (Hartung et al. 1998;Vicre et al., 1999).

Damage from light has been observed inS. lepidophylla and P. polypodioides whencurling of the leaves was manually inhib-ited during illuminated drying (Muslin andHomann, 1992; Lebkuecher and Eickmeier,1993), and it is thought that a number ofmorphological and physiological processesassociated with desiccation are adaptationsto minimize damage from light in the drytissues. In several species, hairs or othersubstances help reflect light from the abax-ial leaf surfaces, which are exposed duringdesiccation-induced curling (Nobel, 1978;Sherwin and Farrant, 1998), as well asfrom adaxial surfaces in species withleaves that do not curl (Vecchia et al.,1998). Pigments in many desiccation-tolerant plants also help to reduce photo-damage, and colour changes in dried leaveshave been reported for a number of species.In C. wilmsii, Xerophyta viscosa and



Myrothamnus flabellifolia, anthocyaninlevels increased significantly in dried tis-sues, helping to mask chlorophyll andreduce damage by free radicals (Sherwinand Farrant, 1998; Koonjul et al., 2000).Photoprotection by mechanisms associatedwith the production of zeaxanthin has alsobeen demonstrated (Casper et al., 1993;Eickmeier et al., 1993).

Changes in colour are accentuated insome species by the loss of chlorophyllduring desiccation. Resurrection plants canbe classified as either poikilochlorophyl-lous desiccation tolerants (PDTs), whichlose chlorophyll during drying, orhomoiochlorophyllous desiccation toler-ants (HDTs), which retain chlorophyllthroughout desiccation. Gaff and Hallam(1974) reported that most resurrectionpteridophytes and dicots were HDTs, whileabout half of the monocots surveyed werePDTs. PDTs avoid photodamage to theirphotosynthetic apparatus by dismantlingit, a trade-off against the greater timeneeded for the re-establishment of photo-synthetic activity. Both HDT and PDTspecies can withstand severe desiccation(Gaff, 1989), but HDTs are fully functionalwithin about 24 h of exposure to water,while PDTs may take 48–72 h or longer toregreen and re-establish photosyntheticapparatus and function (Gaff and Ellis,1974; Tuba et al., 1993; and see below).Although the cells shrink during dehydra-tion and the cell walls and membranesbecome convoluted, most of the organiza-tion of the chloroplasts and otherorganelles is maintained in HDT species(Platt et al., 1994; Thomson and Platt,1997).

In contrast, desiccation in PDT speciesleads to the degeneration of chloroplastmembranes and the loss of the grana,stroma and thylakoid structure, and theunprotected chlorophyll suffers photode-struction (Owoseye and Sanford, 1972; Gaffet al., 1976; Hallam and Gaff, 1978; Bartleyand Hallam, 1980; Bergstrom et al., 1982;Tuba et al., 1993; Vecchia et al., 1998). Inthese species, there is also loss of innermitochondrial membranes and cristae.Nuclear, plastid and tonoplast membranes

remain intact, although the vacuole mayfragment into many small vacuoles. Thesespecies are generally adapted to areas thatexperience fewer fluctuations in wateravailability and thus do not require a rapidrecovery response.

An inherent problem in cytological stud-ies of these desiccated tissues has been thataqueous fixatives can initiate changes asso-ciated with rehydration in desiccated tis-sues. Fixatives with high osmolality havebeen effective in some studies (Platt et al.,1998), but the use of freeze-substitutiontechniques has proved most efficient inmaintaining the structure of desiccatedcells for electron microscopy (Thomson andPlatt, 1997; Platt et al., 1998).

7.3.5. Rehydration and recovery

The ‘resurrection’ of desiccation-tolerantvascular species centres on the events ofrehydration and recovery from apparentlifelessness. Very little rehydration occursfrom dew, but rains of 10 mm or greatergenerally stimulate rehydration (Gaff,1977). Rapid water uptake may occurthrough the leaves, and in experimentalsystems this is often the method used forrehydrating desiccated tissues. C. plan-tagineum can recover 85% RWC within5–6 h when submerged or substantiallyindundated, and desiccation-tolerantgrasses regain most of their shape within4–5 h of rehydration (Gaff and Ellis, 1974),In nature, however, water uptake will alsooccur through the roots, and larger species,such as M. flabellifolia, which rely primarilyon rehydration from the roots, will takelonger to rehydrate and recover than speciestaking up water primarily through theleaves. During desiccation, the flow ofwater through the vascular system is dis-rupted, leaving gaps of air. Upon rehydra-tion, the xylem must be refilled bycapillary action and/or root pressure inorder to resume functioning (Sherwin etal., 1998).

Several factors can influence the rateand type of recovery. Species of Selaginellafrom drier habitats resume photosynthesis



more quickly than those from moister habi-tats (Eickmeier, 1980), suggesting a naturaladaptation to the less frequent availabilityof water in the drier areas. There may alsobe developmental differences in desicca-tion tolerance. Recovery is limited toyoung growing tissues in a number ofspecies, suggesting a greater desiccationtolerance than in older tissues (Gaff andChurchill, 1976; Sherwin and Farrant,1996). When C. plantagineum was rehy-drated through the roots, rather thanleaves, water uptake was slower and manyof the older leaves did not recover com-pletely (Bernacchia et al., 1996).

The time needed for the recovery offunction will depend on how much of thephotosynthetic apparatus was dismantledduring the drying process. Photosynthesisis re-established in HDT plants within afew hours to a day after rewatering(Eickmeier, 1979; Bernacchia et al., 1996).HDT species generally retain a portion ofthe thylakoid system, as well as chloro-phyll, during desiccation, and, within aday of rehydration, functioning grana andlamellae are re-formed. In contrast, PDTspecies lose membrane structure as well aschlorophyll when dried, and these must allbe re-formed when water is available(Hallam and Gaff, 1978; Markovska et al.,1995; Sherwin and Farrant, 1996).Chlorophyll synthesis begins within a fewhours of water availability, but it can takeup to several days to re-establish function(Tuba et al., 1993; Drazic et al., 1999). PDTspecies are generally adapted to seasonalchanges in water availability, while HDTspecies may experience more frequent fluc-tuations in moisture.

7.3.6. Vegetative propagules: bulbils, corms,tubers and plant fragments

Many normal homoiohydric vascularplants produce vegetative propagules,which often serve to carry the plantthrough unfavourable dry periods. Theseinclude bulbils formed either in the soil (asin many Oxalis species (Robb, 1963)) or inleaf axils above ground, often replacing

flowers (as in Ranunculus ficaria,Cardamine spp., Saxifraga spp. and Alliumspp. (Richens, 1947)). There are also vari-ous kinds of stem and root tubers, andviable shoot fragments, which establish asindependent plants. Desiccation tolerancehas been noted in tubers of Anemone coro-naria and Ranunculus asiaticus (Antipovand Romanyak, 1983). Some other tubersand at least some bulbs, bulbils and similarstructures are likely to be desiccation-toler-ant too, but there seems to have been littlesystematic work to determine which of themore persistent of these are truly desicca-tion-tolerant and which are simply highlyresistant to water loss.

7.4. Concluding Comments

Various facets of the desiccation responsesof vegetative tissues have parallels in seedbiology, though these should be seen asillustrative and a prompt to thought ratherthan as necessarily implying close physio-logical correspondence. The shutting-downof metabolism on drying and recovery onremoistening after moderate periods of des-iccation suggest parallels in the maturationand then in the imbibition and germinationof seeds. The gradual loss of viability onprolonged desiccation and the relation ofthis to intensity of desiccation and temper-ature have obvious (even if not complete)parallels in the well-researched field ofseed storage. Fundamental prerequisites fordesiccation tolerance are maintenance (orrapid recovery) of membrane integrity,preservation of macromolecules in a func-tional state and maintenance of spatialrelationships between functional compo-nents of the cell. Protective substancessuch as sucrose and dehydration proteinsprobably combine to allow vitrification ofthe cell contents on drying (Crowe et al.,1998; Buitink, 2000; Chapter 10) to providea living (and reversible) equivalent of goodfine-structural fixation for electronmicroscopy. Some other factors, such asenhanced activity of anti-oxidant systems(Dhindsa and Matowe, 1981; Seel et al.,1991, 1992a; Smirnoff, 1993), may be seen



as extensions of processes common to allliving cells (Foyer et al., 1994; Alscher etal., 1997).

This underlines the distinction betweeneffects related to ‘pure’ desiccation toler-ance, characteristic of (and inseparablefrom) a drying and rewetting event, and thecumulative damaging effects of longer-termdesiccation. The effects of cumulative desic-cation damage are certainly diverse andcomplex (see, for example, Chapter 9). Gaff(1980) distinguished a number of possiblesources of injury operating at different rates,of which the slower types may be eitherstress-parallel or stress-inverse. Injury athigh water potentials may arise from contin-ued activity of metabolic processes. Inosmotic stress experiments, measurable res-piration and photosynthesis were founddown to c. �10 MPa in Anomodan viticulo-sus, and between �10 and �20 MPa inHomalothecium lutescens (Dilks andProctor, 1979); and photosynthetic activityhas been detected in the lichenDendrographa minor down to almost �40MPa (Lange, 1988). In various lichens, itwas found that incorporation of tritium intosugar alcohols, amino acids and some TCA-cycle intermediates was taking place ratherfreely at �40 MPa, and still detectable at�100 MPa (Cowan et al., 1979). Manybryophytes, especially those of woodlandhabitats, are remarkably tolerant of beingkept at or near full turgor in the dark forperiods of weeks, but under these condi-tions Tortula ruralis dies within a few days.As Fig. 7.2 shows, given a low level of light,it survives well at �3 MPa, but much lesswell at water potentials between �9 and�37 MPa. As has been noted for resurrec-tion vascular plants, slow growth ofpathogens, especially fungi, may be a com-mon cause of deterioration in this range(Gaff, 1997). At lower water potentials, otherfactors are likely to be important. High-lightdamage to dry thalli of the forest lichenLobaria pulmonaria has been demonstrated(Gauslaa and Solhaug, 1999), and photondamage is likely to be significant under fieldconditions for other desiccation-tolerantorganisms too. Very slow stress-paralleleffects may be envisaged at very low water

potentials (Gaff, 1980), and there are indica-tions that molecular mobility and rates ofageing generally decrease with falling watercontent, but increase again at water contentsin equilibrium with air at less than c. 10%RH (Buitink et al., 2000). The results in Fig.7.3 are broadly consistent with the conclu-sion that ‘desiccation-tolerant tissues areoften least damaged at values of � of about20 to 40% RH or �1300 to �2200 bars’(�130 to �220 MPa) (Gaff, 1980).

Bewley (1979) introduced the concept of‘repair’ in the recovery of bryophytes fromdesiccation, primarily in relation to therestoration of membrane integrity, and heand Oliver (Oliver and Bewley, 1984, 1997;Bewley and Oliver, 1992; Oliver et al.,2000) have extended the concept and asso-ciated it particularly with the recoveryprocesses in bryophytes. However, as theypoint out, ‘repair’ must be an element inthe recovery of all desiccation-tolerantplants, most of all in the poikilochloro-phyllous vascular species. The broad con-cept of ‘repair’ needs to be consideredanalytically and quantitatively in terms ofthe wide range of processes that it mustembrace. Some systems, such as protein-synthesis mechanisms and the photosys-tems, survive a drying–rewetting eventessentially intact and are functional withinseconds or minutes of remoistening, butexperimental evidence shows that recoveryof some other systems must be slower, andfull return of cell function to a steady statemay typically take hours or days.Underlying processes of recovery span asimilar range of time scales. Reinstatementof water into macromolecules and re-estab-lishment of normal membrane integrity arelikely to be primarily physical, and fast,taking place within seconds or minutes.Other processes are slower, such as re-establishment of normal water relations invascular ‘resurrection plants’ and synthesisof the rehydrins (which themselves evi-dently represent a diversity of processesproceeding at varied rates (see Chapter12)). There are important aspects of recov-ery of cell function after desiccation, suchas reinitiation of the cell cycle, of whichwe know very little (Paolillo, 1984).



What determines the success of desicca-tion-tolerant plants in some habitats andtheir exclusion from others? In broadterms, desiccation-tolerant plants are char-acteristic of situations where availability ofwater is strongly intermittent, and eitherthe physical nature of the site or climatemakes a continuous closed cover of vascu-lar plants impossible. However, there is awide range of possibilities within theseconstraints. Lichens on exposed rock facessuch as Rhizocarpon geographicum (Ried,1960a,b) or the crustose and endolithiclichens in the Negev Desert studied byLange et al. (1970) represent a ‘low-inertia’extreme (Tuba et al., 1998). Species withthis pattern of adaptation extend from thewettest to quite arid climates. Their watercontent tracks closely the incidence of pre-cipitation – rain, dew or impacted mistdroplets – from the atmosphere, and theydry quickly when precipitation ceases.Survival in such situations demands rapidreturn to a positive carbon balance onremoistening after desiccation. Somemosses such as Tortula ruralis respond tochanging hydration almost equally quickly.For larger lichens and many bryophytes thetime scale is longer. Cushions of the com-mon wall-top moss Grimmia pulvinatastore substantial amounts of water follow-ing rain and may take a number of hours todry out (Proctor, 1990; Proctor and Smith,1995; Zotz et al., 2000), and recovery of

carbon fixation, though still fast, is measur-ably slower. For species of shady habitats,such as R. loreus, Mnium hornum orPolytrichum formosum, recovery is sloweragain. Homoiochlorophyllous desiccation-tolerant vascular plants are especially char-acteristic of situations where a thin soilcover allows normal vascular-plant waterrelations to function for much of the year,but the soil is desiccated for more or lessextended periods. The ‘high-inertia’extreme is represented by poikilochloro-phyllous desiccation-tolerant plants suchas the Xerophyta species of central andsouthern Africa, which, essentially, areadapted to the switch between a wet and adry season. Tropical inselbergs provide thehabitats for a large proportion of all desic-cation-tolerant vascular plants (Porembskiand Barthlott, 2000).

By contrast with all desiccation-tolerantplants, drought-tolerant plants require con-tinued access to soil water at a physiologi-cally tolerable water potential. Vasculartherophytes, such as desert ephemerals andtemperate winter annuals, are droughtevaders, and succulents are droughtavoiders. Both groups commonly grow inthe same habitats as desiccation-tolerantspecies, which arguably (Proctor, 2000)may be seen as drought evaders no lessthan the therophytes, substituting desicca-tion-tolerant vegetative tissues for desicca-tion-tolerant seeds.


7.5 References

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Alscher, R.G., Donahue, J.L. and Cramer, C.L. (1997) Reactive oxygen species and antioxidants, rela-tionships in green cells. Physiologia Plantarum 100, 224–233.

Antipov, N.I. and Romanyak, A.N. (1983) Poikilohydric vegetative organs of reproduction in someflowering plants. Zhurnal Obshchei Biologii 44, 446–450 (in Russian).

Bartley, M. and Hallam, D. (1980) Changes in the fine structure of the desiccation tolerant sedgeColeochloa setifera under water stress. Australian Journal of Botany 27, 531–546.

Beckett, R.P. (1999) Partial dehydration and ABA induce tolerance to desiccation-induced ion leak-age in the moss Atrichum androgynum. South African Journal of Botany 65, 1–6.

Becquerel, P. (1951) La suspension de la vie des algues, lichens, mousses au zero absolu et rôle de lasynérèse reversible pour l’existence de la flore polaire et des hautes altitudes. Compte Renduhébdomadaire des Séances de l’Académie des Sciences, Paris 232, 22.

Bergstrom, G., Schaller, M. and Eickmeier, W.G. (1982) Ultrastructural and biochemical bases of res-


urrection in the drought-tolerant vascular plant, Selaginella lepidophylla. Journal ofUltrastructural Research 78, 269–282.

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8 Systematic and Evolutionary Aspects ofDesiccation Tolerance in Seeds

John B. Dickie and Hugh W. PritchardSeed Conservation Department, Royal Botanic Gardens Kew, Wakehurst Place,

Ardingly, West Sussex RH17 6TN, UK

8.1. Introduction 2398.1.1. Reviews and compilations of seed desiccation tolerance 2408.1.2. Classification of seed storage responses 2418.1.3. The phylogenetic classification of plants 2428.1.4. Evolution of desiccation tolerance in land plants 243

8.2. Systematics and Evolution of Seed Desiccation Tolerance 2448.2.1. The complete dataset 2448.2.2. Gymnosperms 244

8.2.2.1. Araucariaceae 2468.2.3. Angiosperms 247

8.2.3.1. Fagaceae 2498.3. Seed Desiccation Tolerance and Ecology sensu lato 250

8.3.1. Are recalcitrant seeds bigger than orthodox seeds? 2508.3.2. Are desiccation-sensitive seeds morphologically or anatomically

distinct from tolerant ones? 2518.3.3. Are desiccation-sensitive seeds associated with particular

habitats? 2528.4. Future Directions 2538.5. Conclusion 2548.6. References 254


8.1. Introduction

The ability of seeds to tolerate desiccationis a trait of major adaptive importance totheir survival and dispersal role. Thatseeds of some species do not withstanddrying presents a challenge for ex situ con-servation. In this review, we explore the

comparative biology of seed desiccationtolerance, looking at broad patterns in theoccurrence of this functional trait through-out the spermatophytes and examining sys-tematic, ecological and other potentiallyinformative correlations. We intend tocomplement the in-depth mechanisticstudies on a number of species described


elsewhere in this volume by discussingseed desiccation tolerance in an evolution-ary context. In so doing, we aim to high-light gaps in knowledge and hint atpossible new research directions. At amore practical level, a better understandingof the evolution of seed desiccation toler-ance is likely to suggest approaches to exsitu conservation for those species withdesiccation-sensitive seeds.

Much of the consideration of desicca-tion tolerance in seeds focuses on the‘other side of the coin’, sensitivity to desic-cation, as this appears to be the exceptionin seeds and leads to a number of practicalproblems. Our account relies on a survey ofthe literature, as well as some new analysisof existing data. In relation to the former,two other recent reviews (Farnsworth,2000; Pammenter and Berjak, 2000) havealso dealt with the subject from a compara-tive and evolutionary aspect. For the latter,heavy reliance is placed here on the dataaccumulated in the Compendium ofInformation on Seed Storage Behaviour

(Hong et al., 1998b), hereafter referred to asthe Compendium.

8.1.1. Reviews and compilations of seeddesiccation tolerance

Significant compilations of recalcitrant seeddata are listed in Table 8.1. The earliest listof recalcitrant (desiccation-sensitive) seededspecies included 73 species from 37 genera(Salix and Swietenia at genus level only) and29 families (King and Roberts, 1979). Withina year, the list had been shortened, firstly to68 species/42 genera/29 families (King andRoberts, 1980) and then to 49 species/36genera (Juglans and Swietenia at genus levelonly) and 27 families (Roberts and King,1980). Major divergences between the listsrelated to, for example, the inclusion/exclu-sion of Citrus spp., of which at least somewere now considered to be orthodox, andCoffea spp., about which there was somedoubt. A later review identified 186 recalci-trant-seeded species across 124 genera, after

240 J.B. Dickie and H.W. Pritchard

Table 8.1. Compilations of recalcitrant-seeded species.

Number of species Number of genera Number of families Reference

73 37 29 King and Roberts (1979)(Salix and Swietenia at genus level only)

68 42 29 King and Roberts (1980)(Malus and Swietenia at genus level only)

49 36 27 Roberts and King (1980)(Juglans and Swietenia at genus level only)

186 124 64 Hofmann and Steiner (1989)(Anthurium, Malus, Mauritia, Oxalis, Roystonea, Sabal, Swietenia and Thrinax at genus level only)

NGa NG 45 von Teichman and van Wyk (1994)

195 143 75 Farnsworth (2000)514b 192c 65d Hong et al. (1998b)

a NG, full details not given.b Out of a total of 6919 species considered, i.e. 7.4%.c Out of 2146 listed in electronic version, i.e. 9%.d Out of 251 families for which there are seed storage data, i.e. 25.9%.NB: there are c. 462 Angiosperm Phylogeny Group (APG) families.


the exclusion of 19 reclassified species(Hofmann and Steiner, 1989). Research sincethen indicates that the seeds of numerousother species can now be considered not tobe recalcitrant as long as they are treatedcarefully, e.g. Corylus avellana (hazelnut),Zizania aquatica (Indian wild rice), Elaeisguineensis (oilpalm) and Azadirachta indica(neem). The difficulties associated with allo-cating seeds to the recalcitrant grouping canbe gauged by the fact that 15–28% of thespecies in the early lists were reported ashaving recalcitrant behaviour that had notyet been fully confirmed.

Similarly, there is discrepancy betweenthe more recent lists of species. For exam-ple, 17 species listed by Farnsworth (2000)as recalcitrant or viviparous are shown inthe Compendium (Hong et al., 1998b) tohave seeds which are probably orthodox.The species are: Amomyrtus luma, Agathisrobusta, Caltha palustris, Chenopodiumquinoa, Cordia alliodora, Cupressus macro-carpa, Dovyalis hebecarpa, Fagopyrumesculentum, Fagraea fragrans, Flacourtiaindica, Hedera helix, Michelia champaca,Muntingia calabura, Nyssa aquatica, Piperhispidum, Santalum album and Vochysiahonurensis.

The latest hard-copy version of theCompendium (Hong et al., 1998b) draws ondata for 6919 species, of which 514 (7.4%)from 65 families are recorded as beingrecalcitrant or likely to be recalcitrant. Thatversion is an update of an earlier versionwith very limited circulation, sponsored bythe International Plant Genetic ResourcesInstitute (IPGRI). Readers should note thatthe analysis described below was actuallycarried out on an electronic version of thedataset (see IPGRI website for download –www.ipgri.cgiar.org/), consisting of recordsfor 7146 species, and which forms the basisof part of the Royal Botanic Gardens Kew’sSeed Information Database see Tweddle etal., 2002 for information on Release 2.0.

8.1.2. Classification of seed storage responses

The Compendium recognizes three maincategories of storage response: orthodox,

intermediate and recalcitrant. Orthodoxseeds can be dried without damage, to lowmoisture contents, usually much lowerthan those they would normally achieve innature. They can be conserved ex situ forrelatively long periods (at least decades) inseed banks, and many of them, but not all,form persistent seed banks in the soil. Overa wide range of storage environments, theirlongevity increases with reductions in bothmoisture content and temperature, in aquantifiable and predictable way (Ellis andRoberts, 1980; Dickie et al., 1990).Recalcitrant, or desiccation-sensitive, seedsdo not survive drying to any large degree,although the critical moisture level for sur-vival varies among species, from about25% to 40% seed/embryo moisture content(fresh mass basis) for cacao and red oak,respectively (e.g. Leprince et al., 1998).Thus, they are not amenable to long-termstorage for conservation, nor are they likelyto form persistent soil seed banks. For thisreview, this category includes those seeds,of some aquatic species in particular,described as viviparous (Farnsworth,2000). Intermediate seeds are more tolerantof desiccation than recalcitrants, thoughthat tolerance is apparently more limitedthan is the case with orthodox seeds, andwhen dry they generally lose viability morerapidly at 0°C and �20°C than at warmertemperatures around 15°C (Ellis et al.,1990, 1991).

Assignment of species to these classes ofresponse is not always clear-cut and sev-eral ‘likely’ and ‘probable’ epithets areused in the Compendium. One of the maindifficulties relates to a lack of a unifiedapproach to measuring the level of desicca-tion (in)tolerance. Whilst most studies usemoisture content on a fresh mass andsometimes dry mass basis (e.g. Tompsett,1984a,b; Pritchard and Prendergast, 1986;Farrant et al., 1988), estimates of criticalwater potentials for desiccation stress havebeen used more recently (e.g. Roberts andEllis, 1989; Pritchard, 1991; Poulsen andEriksen, 1992; Pritchard and Manger, 1998;Sun, 1999; Walters, 1999). Although suchan approach removes the potentially con-founding effects of differing chemical com-

Systematic and Evolutionary Aspects of Desiccation Tolerance 241


position on the assessment of critical mois-ture contents for desiccation stress, espe-cially the effect of oil content on thesorption properties of seeds (Cromarty etal., 1984), critical water potentials forstress have been suggested for the onset,mid-point and end of the viability lossresponse in seed populations (Pritchard,1991; Tompsett and Pritchard, 1998;Dussert et al., 1999; Sun, 1999; Walters,1999). In addition, the physiological stateof the seeds prior to dehydration is influ-enced by developmental age and post-har-vest storage conditions and this can have aprofound effect on the level of desiccationtolerance (e.g. Probert and Longley, 1989;Finch-Savage, 1992; Tompsett andPritchard, 1993, 1998; Pammenter et al.,1998; reviewed by Pammenter and Berjak,1999). Finally, the methods of desiccation,rehydration and germination testing alsoimpact on our perception of desiccationstress (in)tolerance (Grout et al., 1983;Kovach and Bradford, 1992; Leprince et al.,1998; Sacandé, 2000).

The consequent variability in data, andinterpretation, has led to divided opinionon whether there is a continuum of seeddesiccation tolerances between species(Berjak and Pammenter, 1994; Dussert etal., 1999; Sun, 1999) or approximately fivediscrete levels of desiccation tolerance(Walters, 1999). In addition, variability inresponse has tended to a proliferation of‘classes’, for example ‘sub-orthodox’(Bonner, 1990; Dickie and Smith, 1995)and ‘minimally recalcitrant’ (Berjak et al.,1989; Dickie et al., 1991). However, anddespite the caveats about assigning speciesto three classes of storage, the practical sys-tem of classification used in theCompendium is retained here. Also, for reasons of simplicity, our analyses of theCompendium data rely totally on the com-pilers’ assignment of species to a particularseed storage category, and we include thosecases where a classification is likely orprobable, as well as those where theobserved responses are more definite.Readers are referred to the extensive bibli-ography in the Compendium (and in

the Seed Information Database atwww.rbgkew.org.uk/data/sid).

8.1.3. The phylogenetic classification ofplants

Since Darwin’s Origin of Species, the aim ofsuccessive generations of plant systematistshas been to produce a classification that isever more ‘natural’, reflecting as closely aspossible the phylogeny or evolutionarydescent of species and higher groupings(see, for example, Woodland (2000) or Juddet al. (1999) for an account of the history ofplant classification systems and especiallythe more modern ones, such as those ofCronquist (1981) or Dahlgren (1983)). Thedevelopment of cladistic methods based onparsimony, together with the recent rapidincrease in the availability of DNA sequencedata, has led to the emergence of robustphylogenetic classifications of seed plants.A radically new ordinal classification of theflowering plants has arisen from the cooper-ative work of the Angiosperm PhylogenyGroup (APG, 1998). This classification recognizes only monophyletic families andorders, most, but not all, of which fit intoinformal higher-level groups or clades.Concordance with other, more formal, clas-sification systems may be rather forced atordinal and higher levels; and family cir-cumscriptions are not exactly the same inall cases, e.g. the sinking of Aceraceae intoSapindaceae, and Chenopodiaceae intoAmaranthaceae. Like all classification sys-tems, the APG system is a working hypothe-sis, not fact. Nevertheless, we believe thatits powerful representation of likely evolu-tionary pathways and relationships pro-vides the best background for comparativeinvestigations of physiological or ecologicaltraits such as seed desiccation tolerance,and we use it in the analysis of the flower-ing plants in the Compendium datasetbelow. The classification we use for thegymnosperms is basically the arrangementused by Judd et al. (1999), referring also totwo more recent molecular treatments of thegroup (Bowe et al., 2000; Chaw et al., 2000).



8.1.4. Evolution of desiccation tolerance inland plants

Vegetative desiccation tolerance is a wide-spread but uncommon occurrence in plants(Oliver et al., 2000; Chapters 1, 7). Althoughthe algae, lichens and bryophytes containthe most desiccation-tolerant plants, about120 spp. of ferns, fern-allies andangiosperms exhibit vegetative desiccationtolerance. The facts of its early evolutionare not certain, but it probably coincidedwith the colonization of the land by primi-tive plants during the Ordovician period(from about 510 million years ago (mya)),although it is frequent in the spores andcysts of extant representatives of moreprimitive organisms. For instance, theconidia of Metarhizium flavoviride with-stand drying, and once dry they respondquantitatively to moisture content and tem-perature in the same way as orthodox seeds(Hong et al., 1998a). Oliver et al. (2000)argued that desiccation tolerance was theancestral state for early land plants (liver-worts, hornworts and mosses), but that thistrait was lost early in the evolution of tra-chaeophytes, possibly beginning in theSilurian (from 439 mya). It has been postu-lated that desiccation tolerance was inde-pendently evolved (or possibly re-evolved)in both Selaginella and ferns, and at leasteight times in the angiosperms. In the lastgroup, vegetative desiccation tolerance isfound in the order Hamamelidales and thefamilies Poaceae (grasses), Cyperaceae(sedges), Velloziaceae, Liliaceae, Labiatae,Gesneriaceae and Scrophulariaceae (Oliveret al., 2000). (Note that Hamamelidales isnot recognized as monophyletic by theAPG, and the species concerned,Myrothamnus flabellifolius, is not assignedto an order, being placed in the CoreEudicots clade.) As plants adapted furtherto an existence on land, structural andmorphological modifications permittedgreater control of plant water status(homoiohydry), together with an increasein size and growth rate, and vegetative des-iccation tolerance was lost. A stationaryexistence was countered by the evolutionof dispersal structures that facilitated the

greatest opportunity for spatial and tempo-ral movement, and having desiccation-tol-erant propagules would offer a distinctecological advantage in environments notconstantly moist, e.g. seasonally dry tem-perate and tropical habitats. Oliver et al.(2000) speculated that such propagulesmay have evolved as a modification of veg-etative desiccation tolerance, i.e. usingexisting genes.

As noted by Pammenter and Berjak(2000), little is actually known about theevolution of seed desiccation toleranceitself, though rather more has beeninferred. Inference has relied on the associ-ation of seed desiccation tolerance withvarious aspects of systematics, ecology(particularly habitat and plant habit) andseed and fruit structure, among others (e.g.Tompsett, 1994; Hong and Ellis, 1998;Hong et al., 1998b).

Systematic analyses, mainly based onseed structural features that are regarded asprimitive (see later), have led to the pro-posal that seed desiccation sensitivity isthe ancestral state, with tolerance evolvingearly, and several times independently (e.g.von Teichman and van Wyk, 1991, 1994;Pammenter and Berjak, 2000). Meanwhile,based on a review of 195 sensitive species,Farnsworth (2000) has suggested that themost parsimonious explanation of the cur-rent distribution of species’ seed desicca-tion sensitivity is by convergent loss oftolerance from tolerant ancestors. Oliver etal. (2000) inferred that this may have beenthe case in seeds. We are aware of theinherent risks of inferring evolutionary pat-terns by extrapolating from extant specieson purely phylogenetic grounds, and thusour interpretation of the evolution of seeddesiccation tolerance is placed in a widerecological and functional biology context.The factors or features concerned areclosely interrelated and it is often difficultto disentangle the relative importance ofone from another in explaining seedresponses to drying. However, from a prac-tical standpoint the ability to diagnose orpredict the response from such diverseinformation would be a valuable tool forseed conservation planning.



For convenience we have chosen to con-sider systematic aspects separately first, andthen to cover ecology in the broad sense,including both habitat and parent plant fea-tures, as well as seed and fruit structure,including size. We believe that analyses ofsuch associations will ultimately illuminatethe evolution of the extremely valuable traitof seed desiccation tolerance.

8.2. Systematics and Evolution of SeedDesiccation Tolerance

8.2.1. The complete dataset

Species covered in the Compendium prob-ably represent less than 2.5% of all seedplant species, and the issues of coverageand potential bias (e.g. under-representa-tion of tropical moist forest species) in thedataset are real. The low level of samplingof terminal taxa is of particular concern inlight of the incidence of variation of behav-iour within genera; for example, the fol-lowing genera contain species with bothorthodox and recalcitrant seeds: Acer (see also Hong and Ellis, 1990, 1992a;Dickie et al., 1991), Agathis, Araucaria,Calophyllum, Castanopsis, Citrus,Coprosma, Diospyros, Garcinia, Magnolia,Pittosporum, Spondias, Vitex. However,the Compendium is substantially largerthan any of the earlier compilations usedby other authors as the basis for review(e.g. Hofmann and Steiner, 1989; vonTeichman and van Wyk, 1994), and is morerigorous in its assignment of species tostorage category. For example, apparentlymainly on account of its preference formoist habitats, Caltha palustris was classi-fied as having recalcitrant seeds byHofmann and Steiner (1989); and this hasbeen perpetuated in subsequent reviews byvon Teichman and van Wyk (1994) and byFarnsworth (2000). Yet seeds of thisspecies have been successfully stored air-dry and frozen for a number of years in theRoyal Botanic Gardens Kew (nowMillennium) Seed Bank at Wakehurst Place– its seeds are clearly orthodox andrecorded as such in the Compendium.

The evidence so far available indicatesthat desiccation tolerance is the rule formature seeds of the overwhelming majorityof species. Overall, of the 7146 specieslisted in the Compendium (electronic ver-sion) the great majority (c. 90%) are ortho-dox, with c. 7% recalcitrant and 2%intermediate. This is in stark contrast tothe tolerance of vegetative desiccation byadult plants: the overwhelming majority ofspermatophytes are sensitive (Oliver et al.,2000), and resurrection plants are veryrare.

8.2.2. Gymnosperms

Among the gymnosperms, seed desiccationsensitivity is just as uncommon as it isoverall and as it is in angiosperms (seeSection 8.2.3), with the same relative pro-portions found among the species repre-sented (87% tolerant, 6% intolerant and4% limited tolerance). Of the gym-nosperms represented in the Compendiumthe majority are conifers (200 species), andof these 174 are recorded as orthodox.Though their extant members are few andsampling is limited, all the cycads, ginkgosand gnetophytes so far examined have des-iccation-tolerant (or at least not desicca-tion-sensitive) seeds. With the demise ofthe ‘anthophyte hypothesis’ (Bowe et al.,2000; Chaw et al., 2000; Qiu et al., 2000), itis not now so easy to suggest that the puta-tive sister group to the angiosperms (gneto-phytes), and hence a common ancestor,possessed only orthodox seeds. However,the ancestral status of seed desiccation toler-ance within the gymnosperms still has sig-nificant support, from the fact thatrecalcitrant behaviour has not been observedin the cycads or ginkgos. These are regardedas basal in the group (e.g. Bowe et al., 2000;Chaw et al., 2000). Indeed, so far, desiccationsensitivity appears to be restricted to twoderived conifer families, Podocarpaceae andAraucariaceae. In the Podocarpaceae onlyPodocarpus usambarensis is recorded aspossibly orthodox, of ten species covered,the remainder having no or limited toler-ance of desiccation (seven recalcitrant, two




intermediate). Within the Araucariaceae,seed storage behaviour is polymorphic,with orthodox, intermediate and recalci-trant behaviour shown by congeners inboth Agathis and Araucaria (Tompsett andKemp, 1996).

In the gymnosperm fossil record, whilewell-preserved gametophytic tissue issometimes contained in Late Palaeozoicsediments, embryos are rarely found. Post-zygotic development in the earliest gym-nosperms is thought to have been rapidand continuous, making it less likely thatembryos would be fossilized (Mapes et al.,1989). Thus, the appearance in the Permo-Carboniferous (c. 370–240 mya) strata ofwell-developed cotyledonary embryos inrelatively small seeds (6–7 mm long) hasbeen used to suggest the development of aquiescent phase in embryo growth, i.e. theembryos may have been dormant (Mapes etal., 1989). Thus, by Permo-Carboniferoustimes, conifer seeds may have alreadyevolved two functionally important traits –dormancy and, possibly, desiccation toler-ance. Readers should note that Mapes et al.(1989) used the word ‘dormancy’, as doesFarnsworth (2000), in its broad sense, todenote the capability for embryonic devel-opmental arrest, no matter how long sus-tained or controlled, in contrast to‘vivipary’. Most seed biologists use theword ‘quiescence’ to describe this capabil-ity, reserving ‘dormancy’ for specific physi-cal or physiological mechanisms that delayseed germination, even when hydrationand the typical temperature requirementsare satisfied (e.g. Baskin and Baskin, 1998).As we discuss below, there is a generalinverse relationship between dormancyand desiccation sensitivity (an exception isAesculus hippocastanum).

8.2.2.1. Araucariaceae

This family includes 41 species acrossthree genera, Agathis, Araucaria andWollemia (Farjon, 1998). Wollemia is repre-sented by one species, the Wollemi pine(Wollemia nobilis) discovered in the last 10years (see Offord et al., 1999). The familyprimarily has a southern hemisphere distri-

bution (South America, Australia, NewZealand, New Caledonia, New Guinea andother South Pacific Islands) (Setoguchi etal., 1998). In a comprehensive survey ofseed storage responses in this family,Tompsett and Kemp (1996) showed thatfour of 14 species investigated had recalci-trant seeds. All the recalcitrant speciesbelong to the genus Araucaria, which con-stitutes about two-thirds of the species inthe family. The genus can be split intoSections, with Eutacta being the largest. Allinvestigated species of this Section (eight intotal) produce seeds that are tolerant of des-iccation to low levels, from 15 to 2%. Incontrast, species investigated in the SectionsAraucaria (Araucaria araucana, Araucariaangustifolia), Intermedia (Araucaria hun-steinii) and Bunya (Araucaria bidwillii) pro-duce recalcitrant seeds. Based on theconsensus tree of the 20 equally parsimo-nious ones for Araucariaceae, the cpDNArbcL sequence information shows a majorbranching of Agathis from Araucaria after asplit from Wollemia (Setoguchi et al., 1998).However, there is no agreement as towhether Wollemia is closer to eitherAraucaria or Agathis (Gilmore and Hill,1997; Setoguchi et al., 1998). Interestingly,Wollemia, Agathis and the non-EutactaAraucaria have two cotyledons, whilst theEutacta Araucaria have four. All araucar-ian fossil species from the Mesozoic (c.245–65 mya) have dicotyledonous embryos,suggesting that the common ancester of theSection Eutacta had two cotyledons. AsAgathis, the Eutacta Araucaria andWollemia have desiccation-tolerant seeds,we can postulate that the increase from twoto four cotyledons was not associated withthe loss of desiccation tolerance.

Desiccation tolerant seeds (� 15% moisture content) of the Section Eutactavary in size or mass from about 300 mg forAraucaria cunninghamii to 1870 mg forAraucaria heterophylla (Tompsett andKemp, 1996). Similarly, desiccation-tolerantseeds of Agathis sp. are around 200 mg andthose of W. nobilis measure about 1.1 �0.9 cm (Offord et al., 1999). In contrast, the desiccation-sensitive seeds of theSections Araucaria, Intermedia and Bunya


are generally bigger, varying from 580 mg forA. hunsteinii to c. 10,000 mg in A. bidwillii(Dooley, 1990). There are abundant palaeo-botanical data for this family, which haveallowed an assessment to be made betweenthe evolution of seed size and current trendsin the level of seed desiccation tolerance.

The fossil record for Section Bunyaseeds indicates a relatively small seed sizefor Araucaria mirabilis (c. 1 � 0.4 cm),Araucaria brownii (0.8 � 0.3 cm) andAraucaria sphaerocarpa (1.6 � 0.7 cm)(see Setoguchi et al., 1998, and referencestherein). This is in stark contrast to theseeds of the extant species A. bidwillii at c.5 � 3 cm, and it has been suggested, on thebasis of other morphological features, thatthis species should be treated separatelyfrom Mesozoic (c. 245–65 mya) araucariansassigned to the Section Bunya.

The current molecular data suggesting amonophyletic origin for the SectionsAraucaria, Intermedia and Bunya agreewith the fossil record, which suggests theirevolution into Sections was possibly com-plete before South America separated fromAntarctica during the Eocene at the latest.Thus, it is possible that desiccation-sensi-tive seeds in these Sections of the familymay have been present at least 40–60 mya.Molecular data also suggest that SectionEutacta (containing extant species with desiccation-tolerant seeds) is older than theother Sections. Although there is the needto review Mesozoic fossils of Eutacta, ourworking hypothesis is that desiccation tolerance may have been associated with an ancestral small-seeded state inAraucariaceae.

8.2.3. Angiosperms

From comparative studies of DNA amounts(Leitch et al., 1998), it seems likely thatangiosperms at least originated as rapid-cycling species of ephemeral habitats(Midgley and Bond, 1991), where seed des-iccation tolerance would have been a dis-tinct advantage. It is also likely that theearliest angiosperms were early succes-sional herbs or shrubs with relatively small

seeds (Collinson, 1999; Eriksson et al.,2000). At least for angiosperms, the behav-iour of modern species would predict thatsuch ancient seeds were orthodox, i.e. tol-erant of desiccation.

In the angiosperms, all but two of themonophyletic orders recognized by the APGare covered in the Compendium dataset (seeFig. 8.1), and all of those contain somespecies with orthodox seeds. Seed desicca-tion sensitivity, or recalcitrance, is spreadacross all major clades, but, with the excep-tion of Ericales, is quite rare in the asterids.Examples of ‘hot spots’ for this state areMalvales, Arecales and Laurales in theeudicots, monocots and basal angiosperms,respectively, and others are indicated inFig. 8.1. Of the most basal ‘ANITA’ group(Amborellaceae, Nymphaeales, Illicales,Trimeniaceae and Austrobalyaceae; seeBarkman et al., 2000; Qiu et al., 2000), rep-resentation is restricted to Nymphaea spp.(see below), and the other families andorders should be targeted for study. Movingto the other basal orders, less than half thespecies listed from the Magnoliales haverecalcitrant seeds and, of the few Piperales(see also Vázquez-Yanes and Orozco-Segovia, 1982) examined, none has them.Seeds of Ceratophyllum demersum(Ceratophyllales), while difficult to germi-nate, are almost certainly desiccation-tolerant (Hong et al., 1998b; Hay et al.,2000; F. Hay, Ardingly, 2001, personal com-munication). Thus, the extant members ofthe basal groups do not show anything likeexclusively, or even predominantly, recalci-trant behaviour.

At the family level, Table 8.2 shows thepercentages of the three types of seed stor-age behaviour occurring in a selection (44)of the largest angiosperm families. Theseare ordered by % species coverage in theCompendium, to give some idea of the cur-rent sampling levels. In all, about half of allangiosperm families are represented, andof these only c. 25% have members withdesiccation-sensitive seeds. Mean coverageof genera in families is around 18%, rang-ing from 88% in the Fagaceae to less than2% in the Melastomataceae. Species cover-age within family goes from a low of




CeratophyllalesLauralesMagnolialesPiperales(Acorales)AlismatalesAsparagalesDioscoreales(Pandanales)LilialesArecalesPoalesCommelinalesZingiberalesRanunculalesProtealesCaryophyllalesSantalalesSaxifragalesGeranialesMalpighialesOxalidalesFabalesRosalesCucurbitalesFagalesMyrtalesBrassicalesMalvalesSapindalesCornalesEricalesGarryalesGentianalesLamialesSolanalesAquifolialesApialesAsteralesDipsacales

Euasterid II

Euasterid I

Eurosid II

Eurosid I

Commelinoids

Ast

erid

sR

osid

s

Cor

e eu

dico

ts

Eud

icot

s

Ang

iosp

erm

s Mon

ocot

s

0/1

28/42

12/42

0/7

4/31

3/199

0/18

0/22

25/97

4/584

0/10

1/15

0/158

2/81

2/404

2/7

0/80

0/17

31/200

4/10

15/1133

25/338

4/69

69/161

22/314

2/569

97/296

75/285

0/30

32/202

0/1

6/137

5/430

0/181

0/8

4/107

0/489

0/62

Fig. 8.1. Seed storage behaviour at ordinal level. No data for the two orders in parentheses. All other orderscontain some species with desiccation-tolerant seeds. Numbers of desiccation-sensitive species are shown asfractions of total no. examined – orders having at least one sensitive sp. are shown in bold. Tree derived fromseveral cladistic analyses by the Angiosperm Phylogeny Group – see www.rrz.uni-hamburg.de/biologie/b_online/apg/APG.html

0.04%, again in the Melastomataceae, to ahigh of 17% in the Brassicaceae, and amean value between 2 and 3%. The overallpercentage occurrence of species with des-iccation-sensitive seeds is around 10% per

family. Families with a high incidence (> 10% of species examined) of recalcitrantspecies are comparatively rare (ten, or lessthan a quarter of those listed) and widelyscattered taxonomically (examples in Table



Table 8.2. Representation of seed storage ‘groups’ in 44 plant families in relation to the number ofgenera and species for which there are data. Bold signifies families with the highest percentage(10.9–80.2%) of recalcitrant species out of the total species investigated.

Representation Species’ seed storage behaviour

Genera Species Orthodox Intermediate RecalcitrantFamily (%) (%) (%) (%) (%)

Brassicaceae (incl. Capparaceae) 37.5 16.9 99.6 0.2 0.2Fagaceae 87.5 8.2 17.4 1.2 80.2Amaranthaceae(incl. Chenopodiaceae) 20.7 7.1 100.0 0.0 0.0Caryophyllaceae 27.6 7.1 100.0 0.0 0.0Rosaceae 46.3 7.0 96.5 0.0 1.5Fabaceae 34.1 6.3 98.6 0.2 1.2Sapindaceae (incl. Aceraceae,

Hippocastanaceae) 16.8 5.8 63.1 1.2 31.0Cucurbitaceae 18.2 5.4 95.1 0.0 4.9Poaceae 22.6 5.3 98.6 0.6 0.8Solanaceae 23.2 4.9 100.0 0.0 0.0Proteaceae 23.4 4.7 90.7 0.0 2.7Malvaceae (incl. Sterculiaceae,

Bombaceae, Tiliaceae) 24.5 4.2 91.1 1.2 5.9Polygonaceae 26.5 4.0 93.2 0.0 2.3Arecaceae 30.5 3.6 27.8 12.4 25.8Ranunculaceae 29.0 3.6 100.0 0.0 0.0Moraceae 28.9 3.6 51.2 0.0 48.8Rutaceae 16.7 3.6 50.0 28.1 10.9Myrtaceae 25.6 3.1 83.9 0.0 14.7Clusiaceae 20.0 2.8 57.9 0.0 42.1Gentianaceae 12.0 2.5 100.0 0.0 0.0Sapotaceae 18.9 2.4 3.8 23.1 65.4Scrophulariaceae 12.8 2.3 100.0 0.0 0.0Apiaceae 11.2 2.2 98.7 0.0 0.0Ericaceae 15.9 2.1 100.0 0.0 0.0Lamiaceae 17.5 2.0 100.0 0.0 0.0Convolvulaceae 10.9 1.8 100.0 0.0 0.0Boraginaceae 15.4 1.8 100.0 0.0 0.0Asteraceae 12.6 1.8 99.6 0.0 0.0Iridaceae 11.0 1.4 100.0 0.0 0.0Lauraceae 28.8 1.2 5.7 0.0 77.1Celastraceae 9.1 1.2 80.0 13.3 6.7Bromeliaceae 22.0 1.0 100.0 0.0 0.0Cyperaceae 10.6 0.9 100.0 0.0 0.0Apocynaceae (incl. Asclepiadaceae) 6.3 0.9 88.9 0.0 4.4Urticaceae 8.3 0.7 100.0 0.0 0.0Annonaceae 7.8 0.6 62.5 0.0 12.5Euphorbiaceae 8.2 0.5 85.4 0.0 6.3Cactaceae 3.1 0.4 100.0 0.0 0.0Rubiaceae 4.6 0.4 83.9 5.4 7.1Orchidaceae 5.5 0.3 63.9 25.3 0.0Araceae (incl. Lemnaceae) 10.2 0.3 83.3 0.0 8.3Zingiberaceae 6.3 0.3 66.7 33.3 0.0Piperaceae 12.5 0.2 83.3 16.7 0.0Melastomataceae 1.1 0.0 100.0 0.0 0.0



8.2 indicated by bold type). This ‘top ten’list is quite familiar to students of seed des-iccation sensitivity – Fagaceae, Lauraceae,Sapotaceae, Moraceae, Clusiaceae, Sapin-daceae (including Aceraceae), Arecaceae(= Palmae), Myrtaceae, Annonaceae andRutaceae; and all of these also have at leastsome, and usually many, orthodox species.Other ‘hotspots’ for recalcitrant seeds insmaller families, not shown in the table,include Anacardiaceae, Dipterocarpaceae,Meliaceae and Rhizophoraceae. All theextant Nymphaeaceae (a basal family – seeabove) are aquatic herbs, and intuitivelymight be expected to have desiccation-sen-sitive seeds (see remarks on ecologybelow), yet Nymphaea gigantea is reportedto have orthodox seeds (Ewart, 1908, citedin the Compendium).

Below the family level, there are alsosome recognizable patterns in the distribu-tion of seed storage types. Intergeneric variation in seed desiccation tolerance ispresent in c. 25% of families for whichthere are data (i.e. c. 12% of all families).By overlaying the data from theCompendium on the APG cladogram, wehave been able to identify a number of fam-ilies of particular interest with respect tothe level or proportion of species with des-iccation-sensitive seeds. These include themonocotyledon family Arecaceae (c. 4% ofspecies investigated and c. 26% recalci-trant) and the dicotyledon family Fagaceae(of the 8% of species investigated, 80% arerecalcitrant).

8.2.3.1. Fagaceae

The family Fagaceae belongs to the orderFagales, which has 43% recalcitrant species(i.e. 69 out of 161 species investigated) (Fig.8.1). The vast majority of these recalcitrantspecies (53) belong to this family. TheFagaceae are distributed across temperateparts of the northern hemisphere and SouthAmerica, and as far east as Malesia,Australia and New Zealand, and adjacentareas. In most parts of the range, the familyis a very prominent part of broadleavedforests (Govaerts and Frodin, 1998).

The Fagaceae consist of eight or ninegenera (depending on authority) and closeto 1000 species. What is particularly inter-esting about this family is that desiccationtolerance is mainly delimited by genera(note that this is the case in a number ofother families). Species in the genera Fagusand Nothofagus are desiccation-tolerant,whilst those in Quercus and Castanea aredesiccation-sensitive – although one speciesof Quercus (Quercus emoryi) appears to berelatively desiccation-tolerant (Hong et al.,1998b).

The last complete descriptive treatmentof the family dates from about 150 yearsago and various infrafamilial schemes havebeen proposed, with notable character par-allelisms and disagreement on the originand evolution of the cupule. Thus, it is notpossible to be categorical about the evolu-tion of the family. None the less, there isfossil evidence from North America(Buchannan locality) to suggest that thetwo subfamilies – Castaneoideae andFagoideae – may have split no later thanthe Palaeocene (c. 58 mya), and that theFagaceae probably originated in the lateCretaceous (c. 65 mya) (Nixon and Crepet,1989). Fagus (desiccation-tolerant) appearsto be basal for the subfamily Fagoideae,with Quercus arising later (Crepet andNixon, 1989). Thus, there is the possibilitythat the ancestral seed state in this subfam-ily may well have been desiccation-tolerant. Moreover, the palaeoclimate atthis locality is generally considered to havebeen warm temperate to tropical with sea-sonal drought. This type of environment isalso consistent with the modern distribu-tion of many species of Castaneoideae,including Castanopsis (variable desicca-tion tolerance!) in Asia. The mean seedweight for the > 50 species with recalci-trant seeds (principally in the generaQuercus and Castanea) is close to 4 g. Incontrast, seed weight for the handful oforthodox species (in Fagus, Castanopsisand Nothofagus) is around 0.3 g. Thus,orthodox Fagaceae seeds tend to be smallerthan the recalcitrants, as was found to bethe case in Araucariaceae.


8.3. Seed Desiccation Tolerance andEcology sensu lato

The ability of seeds of the vast majority ofextant spermatophyte species (so far exam-ined) to tolerate desiccation is presumablya trait of major adaptive importance to theirsurvival and dispersal role. Assuming thatit is an ancient trait (see above), what eco-logical trade-offs have induced certainspecies to relinquish it from time to timeduring evolution? Seed desiccation sensi-tivity is not a problem for those species thathave it, only for people wishing to store theseeds. From a practical point of view, theassociation between seed storage behaviourand plant ecology and structural character-istics has long been of interest (e.g. Robertsand King, 1980) in relation to the search forpredictors of seed storage behaviour. Thisissue is bound up in the complex of inter-acting factors defining the ‘regenerationniche’ (Grubb, 1977) of particular species.As such, simple associations with singlefactors will not have broad applicability,and this has been recognized, for exampleby Hong and Ellis (1997, 1998), in propos-ing the use of multiple criteria. With partic-ular reference to Meliaceae they suggestedfour – seed size, shape and moisture con-tent at maturity, together with ‘plant ecol-ogy’. The latter is not a single criterion, butpresumably means ‘habitat’ in this context.Desiccation-sensitive seeds, so-called recal-citrants, are widely held to be generallylarge and ‘fleshy’, more likely to occur inforest-tree species and more frequently inthe moist tropics, or in aquatic species, andpossibly in certain taxonomic groups. Thefirst part of this review has confirmed thatseed desiccation sensitivity is more fre-quent in certain plant families, but thatthose families are not exclusively recalci-trant, nor is there any systematic pattern tothat distribution. The occurrence of recalci-trance is much more likely to result fromconvergence in relation to ecological condi-tions. This section of the chapter examinesthe validity of such broad statements bylooking at the available evidence in the fol-lowing, sometimes obviously overlapping,categories.

8.3.1. Are recalcitrant seeds bigger thanorthodox seeds?

Seed size varies over ten orders of magni-tude (Harper et al., 1970), with a number ofpotential ecological and evolutionarycauses, as well as phylogenetic constraints.The case has been made above, for twofamilies, that seeds of the recalcitrantspecies are generally larger than those ofthe related orthodox species. But is this thecase across all species so far investigated?The answer is ‘yes’, with intermediateseeds somewhere in between. Figure 8.2shows the seed mass distributions for 1080species for which ‘seed’ weights are givenin the Compendium (1000-seed weightscited). The mean seed weight for recalci-trant seeds (3958 mg) is greater than thatfor intermediates (900 mg), which is greaterthan for orthodox seeds (329 mg). However,as in all large-scale comparisons of seedmass, the degree of overlap is considerable(see Leishmann et al., 2000), and except atthe extremes it would be a relatively poorpredictor of seed storage behaviour. ‘Seed’mass is easy to measure. However, it is abroad term, including true seeds as well asa variety of indehiscent fruits, confoundedby variation in the proportion of coveringstructures removed in postharvest clean-ing, leading to the use by some authors ofterms such as ‘dispersule’ and ‘germinule’(e.g. Grime et al., 1988). It is strongly asso-ciated with a number of seedling character-istics, habitat type and dispersal mode, andthe strength of the relation between growthform depends on location, but woodyplants and climbers generally have heavierseeds than graminoids and forbs (seeLeishmann et al., 2000). Seed size is alsolarger in tropical floras, independent ofgrowth form and dispersal mode (Lord etal., 1997). Thus, everything that might bepositively associated with seed desiccationsensitivity is associated with seed weight,which in turn is broadly associated withseed desiccation sensitivity.

The very earliest seeds appeared in thelate Devonian–Mississippian era (c. 400mya) on seed ferns and similar plants and,while there may be doubts about the types



of environment in which they evolved,they were relatively quite small (volumemostly less than 10 mm3) (Tiffney, 1986),and thus, by reference to extant species,more likely rather than less likely to bedesiccation-tolerant.

8.3.2. Are desiccation-sensitive seedsmorphologically or anatomically distinct

from tolerant ones?

Are recalcitrant seeds generally ‘fleshy’?This argument centres to some extent on


Seed mass (mg) (log scale)

0.01 0.1 1 10 100 1000 10,000 100,000

Orthodoxn = 839

30

20

10

0

30

20

10

0

30

20

10

0

Intermediaten = 46

Recalcitrantn = 205

% F

requ

ency

Fig. 8.2. Seed size distributions for storage types in the Compendium. The number of species per storagecategory is shown. NB: seed mass values shown are central between the ticks.


size and degree of hydration at dispersal,as well as the proliferation of certain tis-sues. Many orthodox seeds are borne infleshy fruits, presumably animal-dispersed,and are at high moisture content at matu-rity/fruit shedding (e.g. tomato), though theseeds themselves are not especially largeand many of them are known to be ortho-dox, e.g. apples (Dickie and Bowyer, 1985).Among gymnosperms there is also at leastone example, Taxus brevifolia (Walters-Vertucci et al., 1996), where seeds sur-rounded by a fleshy false fruit (aril) arenevertheless orthodox. Hong and Ellis(1998) regarded moisture content at thetime of dispersal as a good marker of seedstorage type, along with other factors, butof itself it is unlikely to be particularlyinformative, and Dussert et al. (2000)found that it was not correlated with thelevel of seed desiccation tolerance in nineCoffea spp.

Corner (1951, 1976) introduced the term‘overgrown seeds’ initially to describe seedsof a number of leguminous species that arerelatively large, non-endospermic and havea poorly developed testa. The list includesgenera such as Castanospermum andPentaclethra, known to have recalcitrantseeds, along with others such as Bauhinia,Millettia and Pithecellobium, from whichdesiccation-sensitive seeds have so far notbeen recorded (Hong et al., 1998b). VonTeichman and van Wyk (1991, 1994)attempted to relate recalcitrant seed behav-iour to large size resulting from over-developed chalazal tissue, along with severalother seed structural characters presumed tobe primitive (e.g. bitegmic, crassinucellateovules, presence of substantial endosperm).However, Corner (1992) doubted that pachy-chalazy itself is primitive.

There is no evidence that the gross mor-phological disposition of reserve materials,i.e. either endospermic or cotyledonous, isassociated with seed storage behaviour. InArecaceae (Palmae), Cocos nucifera isendospermic and recalcitrant, Washingtoniasp. are endospermic and orthodox (Dickie etal., 1992). In Fabaceae (Leguminosae)Pisum spp. are non-endospermic and ortho-dox, whilst Inga spp. are non-endospermic

and have recalcitrant embryos (see Pritchardet al., 1995).

The morphology of the dispersal unitcoat (testa and/or pericarp, depending onspecies) may offer some resistance to dehy-dration in some recalcitrant seeds, e.g.Dipterocarpus tuberculatus (Tompsett,1992) and Acer pseudoplatanus (Dickie etal., 1991), compared with their more ortho-dox relatives, such as Dipterocarpus alatusand Acer platanoides, respectively. Such aproperty may have provoked the sugges-tion that recalcitrant seeds are homoiohy-drous (Berjak et al., 1989), but recalcitrantseeds do equilibrate to ambient relativehumidities, even if equilibration times aresometimes longer than expected. Short termavoidance of desiccation is likely to be aviable alternative to tolerance, as a survivalmechanism under certain conditions.

8.3.3. Are desiccation-sensitive seedsassociated with particular habitats?

There are general associations between habi-tat or macroclimate and seed storageresponses, at least for some species.Tompsett (1994) noted that the more ortho-dox-seeded members of the Aracauriaceaeand Dipterocarpaceae were found in season-ally dry tropical woodland. Dickie et al.(1992) drew a similar conclusion from a lim-ited survey of seed storage behaviour in theArecaceae, and Dussert et al. (2000) haveexamined the relation between seed desicca-tion sensitivity and climate in the nativeenvironments of nine Coffea species. Thedesiccation-tolerant species of Meliaceae areoften located in savannah regions of Africa(Tompsett, 1994; Hong and Ellis, 1998).However, recalcitrant-seeded species areknown in the tropical drylands, e.g.Vitellaria paradoxa (Pritchard and Daws,1997). It could be that such species are his-torical relics of an earlier more hydric envi-ronment and/or that the reproductivestrategy of the species is finely tuned to thedynamics of the local microenvironment,including the availability of seed dispersers.Vázquez-Yanes and Orozco-Segovia (1984)and Vázquez-Yanes et al. (2000) have com-



piled much of the available evidence show-ing that perhaps a majority of species, espe-cially the dominant trees, of humid moistforests have seeds that germinate rapidly toproduce a carpet of dormant or very slowlygrowing seedlings. Many of these may berecalcitrant, but a number do persist as soilseed banks (e.g. Cao et al., 2000), especiallypioneer and gap species (Vázquez-Yanes etal., 2000). The current Compendium datasetalmost certainly under-represents tropicalmoist forest species and more data areneeded. That such vegetation will have qual-itatively more recalcitrant species than mostother vegetation types is perhaps beyonddispute, but by how much more is anotherquestion. There are a number of differenttypes of tropical moist forests and many gra-dations into drier vegetation types, wherethe proportions of different functional typesof trees (e.g. pioneer and gap versus canopyand emergent) may vary considerably.

Aquatic vegetation is the other main typeassumed to contain a high proportion ofspecies with desiccation-sensitive seeds.This is largely on the grounds that there isplenty of water around, seeds would notneed to survive drying out and there may beselection pressure for very rapid germinationand establishment (Farnsworth, 2000). Infact, this is frequently not the case, and theexample of Caltha palustris has been citedabove. Hay et al. (2000) have shown that, ofall the aquatic and marsh species investi-gated, over 90% are orthodox, with the per-centage recalcitrant at 7%, with theremainder having limited desiccation toler-ance, e.g. Najas flexilis (Hay and Muir, 2000).These proportions are no different from thosein seed plants as a whole (see earlier).Perhaps this is less surprising when it isremembered that the seeds of many aquaticspecies are dispersed over long distances onthe feet of birds, for instance, and others inmarshes may have to survive fluctuatingmoisture levels and seasonal desiccation.

8.4. Future Directions

The existence of congeneric species withdistinctly different levels of desiccation

tolerance, e.g. in Fagaceae, Arecaceae andRubicaceae (e.g. Coffea sp.), opens up theprospect of creating a molecular cladogramfor desiccation (in)tolerance in closelyrelated species using existing methodolo-gies. Some of the families identified hereappear suitable for further investigation inthis context, and in particular the basalgroups or species in those families.

The existence of variation in seed desic-cation response among species within agenus has been remarked upon above (seealso Hong and Ellis, 1995). Of particularinterest recently has been information onthe inheritance of desiccation tolerance ininterspecific crosses of Coffea (Dussert et al.,1999). Furthermore, there is growing evi-dence of the potential for variation withinspecies. This follows the isolation of a grow-ing number of viviparous and seed desicca-tion-sensitive mutants in Arabidopsis (e.g.Ooms et al., 1993) and several crop species(maize, rice, peppers), of which the wild-types bear orthodox, desiccation-tolerantseeds. However, there are no reports of seeddesiccation-tolerant mutants in species withrecalcitrant seeds. The work on desiccation-sensitive mutants (e.g. Ooms et al., 1993)suggests that relatively few genes seem to beassociated with sensitivity. Moreover, it canbe assumed that desiccation tolerance in allorthodox seeds is limited to a ‘developmen-tal window’ from maturation to germination(see Hong and Ellis, 1992b), more or lesscoincident with embryo ‘dormancy’ (used inthe broad sense and including quiescence(e.g. Mapes et al., 1989; Farnsworth, 2000)).In addition, it is known that many specieshave variable desiccation tolerance betweentissues of the sporophyte (seed and vegeta-tive tissues) and gametophyte (pollen), indi-cating that transcriptional, translational orpost-translational control of existing genesis most probably involved in the expressionof desiccation tolerance. It is probable thatseeds of the few angiosperm resurrectionplants capable of surviving vegetative desic-cation in the adult phase are desiccation-tolerant. Published data showing whether ornot the desiccation tolerance of the sporo-phyte continues unbroken from matureembryo to adult plant have been lacking.



However, J.M. Farrant (Cape Town, 2001,personal communication) has found thatseedlings of Craterostigma wilmsii,Xerophyta viscosa and Eragrostis nindensisgo through a desiccation-sensitive phasepost-germination. Interestingly, very prelim-inary results suggest that in E. nindensis atleast one gene expressed in the desiccation-tolerant seed and adult phases is notexpressed during the desiccation-sensitiveseedling stage (J.M. Farrant, Cape Town,2001, personal communication). Moreover,it appears that species vary in the level ofdesiccation sensitivity displayed during theseedling window, with relative tolerance inC. wilmsii possibly related to high levels ofup-regulation of protection systems in thatspecies compared with others (Farrant et al.,1999).

Clearly, the identification of a set of spe-cific (homologous) genes or products asso-ciated with the loss or acquisition ofdesiccation tolerance, e.g. through genemicro-array technology, will permit com-parative physiology and molecular screen-ing of diverse species to give us greaterinsight into the evolution of regulatorypathways for stress tolerance. Some mole-cules of potential value, e.g. late embryoge-nesis abundant proteins, are already underextensive investigation in plant and micro-bial systems (see Chapters 1, 5 and 10).Such comparative physiological studiesneed to be to a common standard to permitincorporation into a taxon-based database.Such information will come from manysources, including the Millennium SeedBank project (Royal Botanic Gardens Kew,UK), which may contribute to an increasein seed storage information for speciesfrom an existing level of around 70 familiesto around > 200 families by the year 2010.

Greater understanding of the ecology ofspecies with desiccation-sensitive seeds will

help with understanding as well as diagnos-ing and predicting the condition. Apart fromthe highlighting by Hong and Ellis (1997) ofthe possible role of moisture content atmaturity, little has yet been done to compilethe large but widely scattered amount ofinformation on seed and fruit dispersal (see,for example, Jordano, 2000; Stiles, 2000;Willson and Traveset, 2000) in the context ofseed desiccation tolerance. This will be asignificant component of the Royal BotanicGardens Kew’s (Millennium Seed Bank)Seed Information Database (Tweddle et al.,2002). Another profitable area to pursuewould be the ecological trade-offs involvedin seed size, provisioning and protection andseedling establishment in relation to desicca-tion sensitivity (see, for example, Garwood,1996; Mazer, 1998; Leishmann et al., 2000).Likewise, there is evidence of increasinginterest in the importance of ripening phe-nology in relation to environment at seeddispersal (e.g. Dussert et al., 2000; Rodríguezet al., 2000).

8.5. Conclusion

Seed desiccation tolerance is a complex,labile trait, which is at least as likely to beancestral as is sensitivity in the evolutionof seed plants. In view of its frequent andwide occurrence in both gymnosperms andangiosperms, especially among extantmembers of basal groups, parsimony dic-tates that it is the likely ancestral state inseed plants. The analysis here gives con-siderable support to a similar suggestion byFarnsworth (2000). Applying Dollo’s law(see Judd et al., 1999, p. 21), parallel originseems unlikely, and it is quite conceivablethat this trait has been lost repeatedly, pos-sibly through single gene loss, in responseto one or more ecological trade-offs.


8.6 References

APG (Angiosperm Phylogeny Group) (1998) An ordinal classification for the flowering plants.Annals of the Missouri Botanical Garden 85, 531–553.

Barkman, T.J., Chenery, G., McNeal, J.R., Lyons-Weiler, J., Ellisens, W.J., Moore, G., Wolf, A.D. anddePamphilis, C.W. (2000) Independent and combined analyses of sequences from all threegenomic compartments converge on the root of flowering plant phylogeny. Proceedings of theNational Academy of Sciences USA 97(24), 13166–13171.


Baskin, C.C. and Baskin, J.M. (1998) Seeds: Ecology, Biogeography and Evolution of Dormancy andGermination. Academic Press, New York, 666 pp.

Berjak, P. and Pammenter, N.W. (1994) Recalcitrance is not an all-or-nothing situation. Seed ScienceResearch 4, 263–264.

Berjak, P., Farrant, J.M. and Pammenter, N.W. (1989) The basis of recalcitrant seed behaviour. In:Taylorson, R.B. (ed.) Recent Advances in the Germination and Development of Seeds. PlenumPress, New York, pp. 89–108.

Bonner, F.T. (1990) Storage of seeds: potential and limitations for germplasm conservation. ForestEcology and Management 35, 35–43.

Bowe, L.M., Coat, G. and dePamphilis, C.W. (2000) Phylogeny of seed plants based on all threegenomic compartments: extant gymnosperms are monophyletic and Gnetales’ closest relativesare conifers. Proceedings of the National Academy of Sciences USA 97(8), 4092–4097.

Cao, M., Tang, Y., Sheng, C.Y. and Zhang, J.H. (2000) Viable seeds buried in the tropical forest soils ofXishuangbanna, SW China. Seed Science Research 10, 255–264.

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Part IV

Mechanisms of Damage and Tolerance



9 Desiccation Stress and Damage

Christina Walters,1 Jill M. Farrant,2 Norman W. Pammenter3 andPatricia Berjak3

1USDA-ARS National Seed Storage Laboratory, 1111 South Mason Street, Fort Collins,CO 80521, USA; 2Department of Molecular and Cellular Biology, University of CapeTown, 7700, South Africa; 3School of Life and Environmental Sciences, University of

Natal, Durban 4041, South Africa

9.1. Introduction 2639.2. Water Stress 264

9.2.1 Drought vs. desiccation 2649.2.2. Exacerbating stresses 2659.2.3. Degrees of stress 265

9.3. Desiccation Damage 2699.3.1. Mechanical strains and structural damage 269

9.3.1.1. Cellular and subcellular scales 2699.3.1.2. Molecular scale 273

9.3.2. Metabolically derived damage 2789.4. Perspectives on the Kinetics of Desiccation Damage 2809.5. Conclusion 2819.6. Acknowledgements 2829.7. References 282


9.1. Introduction

Terrestrial plants became established in theSilurian Period (459–409 million yearsago), a few hundred million years after thefirst appearance of multicellular organismson earth (Late Precambrian Period:900–545 million years ago) (Strickberger,2000). The time required for the necessaryadaptions to arise attests to the harshnessof a water-limited environment. The twomajor challenges were maintaining cell and

organism structures when water was notavailable to provide physical support andacquiring nutrients when the lack of waterlimited the movement of both organismsand the necessary resources. The loss ofmobility also required plants to developmechanisms to tolerate a spectrum of otherstresses associated with life on land, partic-ularly temperature extremes and high lev-els of radiation. The requirements for waterare so fundamental (and obvious) that mostresearch has focused on the strategies used


to address the challenges of life in non-aquatic environments (i.e. protective mech-anisms) rather than the physical evidenceof failure – collapse and starvation.

Studies of cellular responses to waterstress mostly focus on what cells need totolerate or resist water loss. Direct evidenceconcerning the damaging process is sparse,with the mechanisms of damage oftenmade by inference from the presence ofputative protectants. Often it is unclearwhether a change in morphology, ultra-structure or metabolism is a simple conse-quence of drying, a protective strategy or asign of damage. For example, cessation ofmetabolism is considered a component ofall three possibilities (Vertucci andLeopold, 1984; Leprince et al., 1999, 2000;Salmen Espindola et al., 1994, respec-tively). Damage by desiccation is oftenmeasured by an irreversible change or afailure of the organism to revive once wateris plentiful again. These rather crudeassays do not detect damage that is repara-ble, though the suite of repair enzymes pro-duced de novo upon rehydration attests tothe turnover of cellular constituents (Oliveret al., 1998). A better understanding of thenature of desiccation stress and the result-ing strains is required if we are to under-stand fully the nature of protection andrepair and, ultimately, exploit millions ofyears of evolutionary adaptation to pro-duce plants more capable of withstandingthe basic challenges of life in a water-limited environment.

9.2. Water Stress

9.2.1. Drought vs. desiccation

Most terrestrial organisms can grow (bymitotic divisions and cell expansion) atwater potentials greater than about �1 MPa(Levitt, 1980; Vertucci and Farrant, 1995,and references therein). Organisms thatsuccessfully deal with lower water poten-tials can either cope with limited wateravailability while maintaining high inter-nal water concentration, or cope withwater loss. The former class constitutes

what are generally considered to be‘drought-tolerant’ organisms, which usu-ally resist water loss by having imperme-able outer coverings and reducing surfacearea-to-volume ratios. Drought-stressedorganisms may grow relatively slowly, per-haps because of the reduced turgor pres-sure for cell expansion, but also because ofthe tremendous metabolic costs of main-taining structures that block water loss tothe environment (e.g. Pimienta-Barrios andNobel, 1998), supporting root structuresthat seek water, and accumulating compati-ble solutes that keep osmotic potentialslow (Jones and Gorham, 1983). The degreeof drought tolerance can be based on howmuch the organism resists water loss (i.e.the minimum water potential sustained),the duration that the organism sustains lowwater potentials, or the productivity(growth) of the organism during the stress.When drought-tolerance mechanisms fail,the organism either loses water essentialfor structure or compromises metabolismto an unsupportable level. Either of theseconsequences is considered a subset of thestrains associated with desiccation damage.The distinction between drought and des-iccation tolerance lies in the protectionmechanisms – mechanisms conferring tol-erance of drought avoid water removalwhile mechanisms conferring tolerance ofdesiccation enable the organism to survivein spite of the water loss.

In the above context, drought toleranceis really desiccation avoidance. Becausethe mechanisms required to scavenge andsequester water may differ from those thatenable the organism to exist without it, tol-erance of drought does not necessarilyimply tolerance of desiccation. None theless, both desiccation- and drought-tolerantorganisms accommodate life at low waterpotentials (� �1 MPa). Mild drops inwater potential (from �1 to about �3 MPa)coincide with a series of metabolic changesthat make cells more tolerant of the waterstress (Ingram and Bartels, 1996; Bray,1997; Oliver et al., 1998; see Chapter 11).The products of these metabolic changes(antioxidants, low-molecular-weight carbo-hydrates, late embryogenesis abundant

264 C. Walters et al.


(LEA)-like proteins, heat-shock proteins)are putative protectants for both droughtand desiccation, even though the mecha-nism of protection is quite different for thetwo types of stress. Future research shouldbe directed towards resolving this apparentcontradiction.

9.2.2. Exacerbating stresses

Water-stressed plants (�w � �1 MPa) arepredisposed to damage by other stresses.Free-radical production appears to be acommon effect of numerous stressesincluding drought and desiccation, ageing,freezing, pollution, temperature extremesand radiation (Elstner et al., 1988;McKersie et al., 1988; Puntarulo et al.,1991; Hendry, 1993; Leprince et al., 1993;Foyer et al., 1994; Wise, 1995; Bowler andFluhr, 2000), and so it is likely that thesestresses may be cooperative or synergistic.Stressed plants are particularly susceptibleto photo-oxidative damage (Elstner et al.,1988; Foyer et al., 1994; Wise, 1995). Lightenergy, which was efficiently harvested,transduced and assimilated in non-water-stressed cells, may be absorbed by the pho-tosynthetic apparatus and dissipated asreactive oxygen molecules that damage cel-lular constituents (Bewley and Krochko,1982; Vertucci et al., 1985; Kaiser, 1987;Elstner et al., 1988; McKersie et al., 1988;Smirnoff, 1993; Foyer et al., 1994; Tuba etal., 1996, 1998; Sherwin and Farrant, 1998;Csintalan et al., 1999; Farrant, 2000;Vander Willigen et al., 2001). Desiccation-tolerant plants initiate many processes thatare considered to be protective against pho-tochemical damage. These processesinclude dismantling of the photosyntheticapparatus (Gaff and Hallam, 1974;Hetherington et al., 1982a,b; Öquist andStrand, 1986; Gaff, 1989; Demmig-Adamsand Adams, 1992; Vertucci and Farrant,1995; Tuba et al., 1996, 1998; Sherwin andFarrant, 1998; Farrant et al., 1999; Farrant,2000), chlorophyll shading by leaf foldingor rolling (Dalla Vecchia et al., 1998;Sherwin and Farrant, 1998; Farrant et al.,1999; Farrant, 2000), accumulation of pro-

tective pigments such as anthocyanins(Smirnoff, 1993; Foyer et al., 1994;Sherwin and Farrant, 1998; Farrant, 2000;Vander Willigen et al., 2001), increases inxanthophyll pools and conversion to thede-epoxide forms (Smirnoff, 1993; Foyer etal., 1994; Kranner and Grill, 1997) and pro-duction of free-radical-scavenging enzymes(Foyer et al., 1994; Wise, 1995; Pammenterand Berjak, 1999).

Low temperatures tend to intensifywater stress. The classic case describestemperatures at which water freezes extra-cellularly, thereby creating a water poten-tial gradient and forcing intracellular waterto migrate out of the cell (Meryman, 1974;Steponkus, 1979). Lowering the tempera-ture requires an increase in the water con-tent of cells to maintain a constant �w(�w � �10 MPa). This requirement for morewater at lower temperatures is related tothe exothermic nature of water condensa-tion on macromolecular surfaces at lowwater potentials (Walters, 1998).Consistently, critical water contents thatlead to desiccation damage are greater atlower temperatures (Kovach and Bradford,1992; Vertucci et al., 1995; Eira et al.,1999a). Indeed, the moisture content givingrise to changes in membrane phase behav-iour, the most often cited consequence ofdesiccation stress in model systems,increases with decreasing temperature(Crowe et al., 1989; Crowe and Crowe,1992; Hoekstra and Golovina, 1999;Hoekstra et al., 1999; Bryant et al., 2001).

9.2.3. Degrees of stress

There are many ways to measure water lossin cells. Water content (absolute or relative)(e.g. Berjak et al., 1992; Sun et al., 1994;Farrant, 2000), water potential and relatedfunctions (Roberts and Ellis, 1989; Vertucciand Roos, 1990; Tompsett and Pritchard,1993; Vertucci and Farrant, 1995; Vertucciet al., 1995; Farrant and Walters, 1998), cellvolume (Meryman, 1974; Steponkus, 1979;Murai and Yoshida, 1998a,b), intracellularviscosity (Vertucci and Roos, 1990; Koster,1991; Williams et al., 1993; Leopold et al.,

Desiccation Stress and Damage 265


1994; Buitink et al., 1998b; Leprince andHoekstra, 1998; Bryant et al., 2001) inter-molecular proximity (Lis et al., 1982;Steponkus et al., 1995; Wolfe and Bryant,1999; Bryant et al., 2001) and structuralwater (Ladbrooke and Chapman, 1969;Vertucci and Leopold, 1984, 1987; Croweet al., 1990; Pammenter et al., 1991) allchange with desiccation (Fig. 9.1; seeChapter 2). Each of these parameters hasbeen used to define the level of waterstress, but it is unclear which parameter(s)causes the stress and which is merely acorrelate of the stress. The distinction isimportant as it reveals the nature of thestrain and the damage. A better under-standing of the nature of desiccation stressand strain will also reveal whether damageaccrues continuously, whether it occurswhen the stress or strain reaches a thresh-old, and whether numerous differentstrains result from the removal of waterfrom the cell. In other words, is damage bydesiccation a single event, a continuousevent or a series of insults to the cell ororganism?

Desiccation tolerance/sensitivity has tra-ditionally been regarded as a qualitativefeature: cells either do or do not survivedrying. The definition of ‘dry’ varies amonglaboratories or experiments (i.e. 90% waterloss; water contents � 10% (0.10 g H2O g�1

dry weight or fresh weight); water contentsin equilibrium with 75% or maybe even15% relative humidity (RH); water con-tents achieved after a material has beenfreeze-dried or held in a laminar flow hoodfor some period of time), and most studiesuse just one drying level. The binaryapproach suggested that damage was a sin-gle event that either happened or did nothappen. Seeds were assigned to one of twocategories, orthodox or recalcitrant, to dis-tinguish between those that survived or didnot survive drying (Roberts, 1973; seeChapter 5). Classification of seeds of cer-tain species as recalcitrant has been dis-puted as laboratories around the worldhave demonstrated variable success in dry-ing them. For example, some groups reportsurvival of Zizania palustris at water con-tents as low as 0.07 g H2O g�1 dw (Kovach

and Bradford, 1992) while other laborato-ries show detrimental effects of drying atmuch higher water contents (Probert andLongley, 1989; Vertucci et al., 1995).Similar discrepancies are reported for cof-fee (Coffea arabica), lemon (Citrus limon),neem (Azadirachta indica) and tea(Camellia sinensis) (e.g. Ezumah, 1986;Ellis et al., 1990; Chaudhury et al., 1991;Berjak et al., 1993; Hong and Ellis, 1995;Dussert et al., 1999; Eira et al., 1999a;Sacandé et al., 2000). Even when the recal-citrant category is undisputed, there aredifferences among species in how muchwater can be removed and how long awater-stressed seed can survive (Berjak etal., 1989, 1990; Farrant et al., 1989, 1997;Vertucci and Farrant, 1995; Pammenter andBerjak, 1999). To accommodate the vari-ability in desiccation tolerances observedamong species, the categories of seedbehaviour were further divided to distin-guish highly recalcitrant, recalcitrant,intermediate, sub-orthodox and orthodox;and Pammenter and Berjak (1999) havesuggested that, in reality, a continuumexists among seed species, based on desic-cation response.

Not only are there differences amongspecies in response to desiccation, butthere are also differences for seeds of thesame species and among tissues as a func-tion of developmental stage. Studies of theacquisition and loss of desiccation toler-ance during embryogenesis and germina-tion have demonstrated that tolerance todesiccation progressively increases withseed maturation (Berjak et al., 1990, 1992,1993; Finch-Savage, 1992b; Farrant et al.,1993; Sun and Leopold, 1993; Sun et al.,1994; Tompsett and Pritchard, 1993;Vertucci et al., 1995; Farrant and Walters,1998; reviewed by Vertucci and Farrant,1995; Pammenter and Berjak, 1999; seeChapter 5) and decreases with germination(Sargent et al., 1981; Senaratna andMcKersie, 1983; Leprince et al., 1990;Reisdorph and Koster, 1999). Correlativeevidence suggests that there are similarstages of tolerance in vegetative tissues ofdesiccation-tolerant angiosperms (Farrant,2000; Vander Willigen et al., 2001; J.M.




Str

ess

Str

ain

Vis

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ty

(N s

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imm

atur

em

atur

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631.5

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mat

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Volu

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Dam

age

Bin

din

gsi

tes

Sta

te

Osmotic excursionsMembranesappressed

Membrane shearing anddeletions

Demixing

Lipid phasetransitionsand fusion

Metabolic imbalance

Stress proteinsproduced

Proteindeformations?

Mechanicalstructures fail

Unprotected oxidations

chargedhydrophilichydro-phobic

capillarynone

solution syrup rubber leather glassporousglass?

IIIIIIIVV

Fig.

9.1

.Sc

ales

of w

ater

str

ess

(wat

er p

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hum

idity

(RH

)), w

ater

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(wat

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sim

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Wol

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des

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998)

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for

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the

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the

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Rup

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Farrant, unpublished data). Vegetative tis-sues from resurrection and cold-tolerantangiosperms require time to adapt to water-stress situations (Steponkus et al., 1995;Oliver et al., 1998; Farrant et al., 1999; seeChapter 7), presumably to induce protec-tive mechanisms. It is surmised that in vege-tative tissues of angiosperms there is also adevelopmental programme in response tostress that leads to progressively greater des-iccation tolerance. The variability in criticalwater contents among different species, dur-ing maturation or germination of embryosand during adjustment of vegetative tissues,leads to the general conclusion that toler-ance/sensitivity is a quantitative feature.

The quantitative nature of desiccationtolerance/sensitivity suggests two broadpossibilities for the mechanism(s) of desic-cation damage in organisms. Desiccationdamage may occur by a single mechanismwith species and tissue types differing inhow much damage they can accrue or howmuch stress they can endure. Alternatively,damage by different mechanisms mayoccur at multiple levels of stress andspecies and tissue types differ in whichstresses, or combinations of stresses, theycan withstand. The former possibility sug-gests that a desiccation-tolerant organismrequires only a simple cadre of protectants,while the latter situation suggests that acomplex suite of protectants, each with adifferent function, is required for an organ-ism to be truly tolerant of dehydration.

Evidence is accumulating to suggest thatdesiccation damages cells and organisms bymany mechanisms and that different typesof damage occur at different levels of stress.When expressed in terms of water poten-tial, developing embryos acquire tolerancestepwise (Vertucci and Farrant, 1995;Farrant and Walters, 1998). During histodif-ferentiation, embryos are damaged by waterpotentials less than about �1.2 to �2 MPa(Vertucci and Farrant, 1995, and referencestherein). During dry matter accumulation,embryos tolerate water potentials as low asabout �4 to �5 MPa ( Farrant et al., 1992,1993; Farrant and Walters, 1998). Followingvascular separation, the water potential atwhich damage is first measured (i.e. critical

�w) in most embryos (Avicennia marina,and perhaps other highly recalcitrant seeds,excepted (Farrant et al., 1992, 1993)) wasfound to decline to about �10 to �15 MPa(Pritchard, 1991; Finch-Savage, 1992a;Tompsett and Pritchard, 1993; Vertucci andFarrant, 1995; Farrant and Walters, 1998).Embryos that are recalcitrant cannot bedried below this level. During the finalstages of maturation, species defined ashaving intermediate postharvest physiology(e.g. coffee, citrus and papaya) acquire theability to tolerate between about �60 and�80 MPa (Dussert et al., 1999; Eira et al.,1999a; Sacandé et al., 2000) (neem is con-sidered to be in the intermediate categoryas it has diminishing longevity at lowerwater potentials (see Ellis et al., 1990)).Truly orthodox species survive the immedi-ate effects of complete water loss, but suc-cumb more rapidly if they are dried belowabout �190 to �250 MPa (Vertucci andLeopold, 1987; Vertucci and Roos, 1990;Walters, 1998). Vegetative tissues of resur-rection plants acquire the same degree ofextreme tolerance (Bewley, 1979; Gaff,1989; Oliver et al., 1998). Cells that havethe genetic capacity to induce tolerancemechanisms progress towards toleranceand, with sufficient time, complete thedevelopmental programme leading to fulltolerance of desiccation (e.g. Finch-Savage,1992b).

The differing levels of desiccation sensi-tivity among developmental stages and seedcategories appear to correspond to levels ofphysiological activity documented in desic-cation-tolerant organisms with studies ofmetabolic activity and properties of theaqueous medium (Vertucci and Farrant,1995). Five hydration levels designate thecells’ ability to support growth (Level V, 0to �1.5 MPa), to photosynthesize and effectstress-related metabolism (Level IV, �1.8 to�4 MPa), to respire (Level III, �5 to about�12 MPa), to carry out catabolic reactions(Level II, about �15 to �190 MPa), and tobe almost in stasis (Level I, � �220 MPa)(Fig. 9.1) (Clegg, 1986; Roberts and Ellis,1989; reviewed by Vertucci and Farrant,1995; Farrant, 2000; Vander Willigen et al.,2001). These hydration levels correspond to



other parameters that measure the extent ofdesiccation (Fig 9.1) (Chapters 2 and 4). InHydration Level V, turgor pressure is posi-tive and water behaves as it would in adilute solution. At lower hydration levels,cells shrink (Meryman, 1974; Steponkus,1979; Steponkus et al., 1995), the propertiesof water change (Rupley et al., 1983;Vertucci, 1990) and the aqueous matrixbecomes more viscous having properties ofsyrups (Level IV), rubbers (Level III) andleathers and glasses (Level II) (Vertucci andRoos, 1990; Slade and Levine, 1991;Williams et al., 1993; Leopold et al., 1994;Buitink et al., 1998b; Leprince andHoekstra, 1998). Viscosity is minimized atthe transition from Level II to I (Vertucciand Roos, 1990; Buitink et al., 1998b), amoisture level that also corresponds to adiscrete change in the heat capacity ofwater (Rupley et al., 1983; Vertucci, 1990;Buitink et al., 1996; M.T.S. Eira, unpub-lished data) and poorly understood charac-teristics of water sorption (Vertucci andLeopold, 1987; Vertucci and Roos, 1990;Vertucci et al., 1994; Buitink et al., 1998a,b;Walters, 1998; Eira et al., 1999b). With theexception of Hydration Level I (Vertucciand Leopold, 1987; Eira et al., 1999b;M.T.S. Eira, L.S. Caldas and C. Waltersunpublished data), the relationshipsbetween physical properties of water andwater potential appear similar amongdiverse cells (Fig. 9.1), perhaps with onlysubtle differences to distinguish desiccation-tolerant from less tolerant materials (Koster,1991; Berjak et al., 1993; Farrant andWalters, 1998; Leprince et al., 1999). If des-iccation stress occurs when cells traversecritical water potentials or hydration levels,then the stresses experienced by desiccat-ing cells are similar among organisms, butthe responses to those stresses (i.e. damageversus protection) differ with desiccationtolerance (Pammenter et al., 1991).

9.3. Desiccation Damage

As water is removed from cells, the physi-cal and physiological properties of the cellschange. These changes, often characterized

by a reduced cell size or lack of integratedmetabolism, do not in themselves implydamage. They may be purely consequencesof water removal and may be completelyreversible once water is added back to thesystem. Therefore, damage from desicca-tion is not indicated by differencesbetween the hydrated and dry state, butrather by the resumption of normal activityupon rehydration.

The number of different stresses thatcan be associated with removal of waterfrom cells can be attributed to the multipleroles that water plays in supporting life.Water plays a structural role: at the cellularscale, water, fills spaces and provides tur-gor, while, at the molecular scale, waterprovides hydrophilic and hydrophobicassociations and controls intermoleculardistances that determine the conformationof proteins, polar lipids and the partition-ing of molecules within organelles. Withwater present, reactive surfaces of metalsor molecules are not as exposed, and thislimits reactivity among molecules. Wateralso plays a role in controlling metabolism,as it is a reactant and product of manyreactions. As a dilutant, water affects thechemical potential of other molecules,potentially shifting the likelihood of reac-tions. Water also provides the fluid matrixthat allows diffusion of substances to reac-tive sites. Changes in water concentrationaffect viscosity of the matrix and the over-all mobility of dissolved or suspended mol-ecules. The drier the medium becomes, themore viscous it becomes, until it is essen-tially a solid matrix, trapping molecules(Slade and Levine, 1991; Williams et al.,1993; Leopold et al., 1994; Buitink et al.,1998b; Wolfe and Bryant, 1999). As onewould expect from all the roles of water,there will be a number of strains that thetissues undergo when water is removed.

9.3.1. Mechanical strains and structuraldamage

9.3.1.1. Cellular and subcellular scales

The first sign of desiccation/drought stressis the loss of turgor pressure. This occurs at



water potentials of about �1 to �2 MPa,coinciding with the water potential rangedesignated as ‘permanent wilting point’ fornon-transpiring vegetative tissue (Levitt,1980). At lower water potentials, cells losewater and shrink (Meryman, 1974,Steponkus, 1979; Levitt, 1980; Steponkusand Lynch, 1989; Steponkus et al., 1995).Osmotic adjustments, which lessen thewater potential difference between cellsand the environment and augment theamount of dry matter in cells, can preventwater loss and cell contraction at waterpotentials between �1 and �2.5 MPa(Levitt, 1980; Jones and Gorham, 1983).Osmotic adjustments are fairly ineffectiveat reducing strains when cells are exposedto lower water potentials (Wolfe andBryant, 1999). In slow-freezing experi-ments, believed to mimic dehydrationstress, protoplasts can undergo reversiblecontraction–expansion cycles, or ‘osmoticexcursions’, when slowly cooled andwarmed from > 0°C (�w ≈ �0.5 MPa) totemperatures of �2 to �5°C (�2.5 � �w� �6 MPa) (Meryman, 1974; Steponkus,1979; Steponkus and Lynch, 1989). A60–80% reduction in cell volume occurswhen the water potential of cells decreasesfrom about �0.5 MPa to about �4.5 to �6 MPa (�4 to �5°C) (Meryman, 1974;Steponkus, 1979; Steponkus and Lynch,1989). Similar contraction was calculatedfor immature embryo cells in which 88%of the cell volume was occupied by water(Fig. 9.1). However, cells filled with drymatter reserves (mature embryos in Fig.9.1) do not contract as much as highly vac-uolated cells (immature embryos in Fig.9.1). For a similar reduction in waterpotential to �5 MPa, the cells of fullymature bean axes contract only by about18%, and complete desiccation only causesa 24% reduction in volume in these cells(Fig. 9.1). When cells that have not beenacclimatized to the water stress shrink by50–80%, they burst when returned to theoriginal water potential. This observationled to the concept of ‘minimum criticalvolume’ (Meryman, 1974), which describesthe limits to which a cell can contract in areversible osmotic excursion. As seen for

mature embryos (Fig. 9.1), this strain of cellcontraction can be avoided by accumulat-ing dry matter.

Differences in the degree to which cellwalls contract compared with protoplasmmay cause mechanical stress and damageto the plasmalemma or plant cells duringdehydration. The tight attachment of theplasmalemma to the cell wall is believed tocreate tension to the membrane in shrink-ing cells (e.g. Murai and Yoshida, 1998b),which is most profound at the cellwall–plasmalemmma attachments near theplasmodesmata (Iljin, 1957; Bewley andKrochko, 1982). Plasmolysis, where theplasma membrane separates from the cellwall, appears to mitigate damage to wholecells during severe water stress (Murai andYoshida, 1998b), and there is some evi-dence to suggest that cells in desiccation-tolerant seeds are slightly plasmolysed(Perner, 1965). Observations of plasmolysismay be an artefact of the aqueous fixativesused to study dry organisms (Öpik, 1985;Platt et al., 1997; Wesley-Smith, 2001). Instudies using anhydrous chemical fixation(Öpik, 1985) or freeze substitution (Wesley-Smith, 2001, Wesley-Smith et al., 2001),the plasma membrane remained closelyappressed to the cell walls, and both thecell wall and the plasmalemma becamehighly convoluted during desiccation oftolerant cells. Öpik (1985) demonstratedthat the plasmalemma separated from thecell wall during rehydration as a result ofdifferential swelling or weakening of thecell wall–plasmalemma association causedby detergents such as dimethylsulphoxide.The mechanical properties of the cell wall,including its elasticity, ability to fold andassociations with plasmodesmata, influ-ence the degree of plasma membrane dis-ruption consequent upon contraction orexpansion (Webb and Arnott, 1982; Öpik,1985; Murai and Yoshida, 1998b; Vicré etal., 1999).

Cell membranes must fold or vesiculateto accommodate the volume changes dur-ing cell contractions. Conservation of mem-brane surface area during contraction iscritical for successful rehydration. If thesurface area of the plasmalemma is



reduced too much, the cell bursts uponrehydration, suggesting that there is a criti-cal minimum surface area, rather than acritical minimum volume, to which cellscan survive (Steponkus, 1979; Steponkusand Lynch, 1989; Steponkus et al., 1995).Protoplasts from cells that are not acclima-tized to the cold contract through invagina-tions of the plasma membrane, whicheventually form endocytotic vesicles thatcannot be reincorporated into the plas-malemma upon warming (Steponkus andLynch, 1989; Steponkus et al., 1995). Theplasma membrane of protoplasts from cellsmore tolerant of water stress (i.e. acclima-tized by low temperatures) contractsthrough exocytotic extrusions whichremain continuous with the plasma mem-brane and help to conserve the membranesurface area (Steponkus and Lynch, 1989;Steponkus et al., 1995). High phospho-lipid:sterol ratios and high amounts ofdiunsaturated fatty acids in the plas-malemma appear to facilitate exocytoticfolding in shrinking protoplasts and greaterelasticity of the expanding membranes(Steponkus and Lynch, 1989; Steponkus etal., 1995). Protoplasts with these propertiestend to survive to lower water potentials(Steponkus et al., 1995).

The mechanism by which membranesurface area is conserved in intact cells islargely unknown. There are some studiesof the effect of dehydration on cell volumeand membrane configuration in cells fromplant embryos, but these are often con-founded by problems associated with usingaqueous fixatives (Platt et al., 1997;Wesley-Smith et al., 2001). In addition, thestudies often use mature embryos (recalci-trant or orthodox) where > 50% of the cellvolume is occupied by dry matter (e.g.Farrant et al., 1997). These cells will notexperience the same degree of shrinkage ashighly vacuolated cells (Fig. 9.1), and sothe need for conserving membrane surfacearea is not as critical. Circumventing theproblem of cell shrinkage may explain whymost orthodox and recalcitrant embryos(except for A. marina and other recalcitrantseeds with highly vacuolated cells (Farrantet al., 1992, 1993)) are fairly tolerant of

water stress, surviving to water potentialsof �12 MPa or less, compared with thebenchmark of �5 MPa described above forprotoplasts from non-acclimatized cells.None the less, drying results in somedegree of cell contraction, which is mostlycompleted when the water potential of thecell is reduced to �12 MPa (Fig. 9.1). Incells that survive water potentials of about�12 MPa but not lower, both endo- andexocytotic vesicles have been observed (P.Berjak and N.W. Pammenter, unpublisheddate). These observations are not reportedin extremely dried embryos, perhapsbecause of technical problems of fixation.In severely dried cells of fully desiccation-tolerant seeds, the plasmalemma staysintact and closely attached to the cell wallas this folds, suggesting that the membranesurface area remains relatively constantduring drying even though the cell volumeis diminished (Öpik, 1985). Some mem-brane constituents may be removed duringcell contraction as evidenced by whorls ofmembrane close to the plasmalemma inseed cells (Webster and Leopold, 1977;Öpik, 1985; Wesley-Smith et al., 2001) andcircular membrane structures and plas-toglobuli within chloroplasts in sections ofleaf tissue from desiccation-tolerantangiosperms (Farrant et al., 1999; Farrant2000; Mundree et al., 2000). These mem-brane bodies have been proposed to pro-vide additional membrane reserves uponrehydration (Webster and Leopold, 1977;Farrant et al., 1999; Mundree et al., 2000),although mechanisms by which theywould be reinserted are not clear and theirvery presence may be artefacts of aqueousfixation. Alternatively, these membraneabnormalities may arise from otherorganelles, such as endoplasmic reticula,and may participate in autophagy or vac-uole formation (Wesley-Smith et al., 2001).The shapes of nuclei, mitochondria andplastids in dried cells of desiccation-toler-ant seeds are irregular and convoluted, sug-gesting that the surface area of themembranes of these organelles are alsoconserved simply by folding (Öpik, 1985).

The membranes of cell vacuoles arelikely to experience tensions similar to



those described for protoplast membranesduring osmotic excursions, and so areprone to rupture, with lethal consequences,following exposure to water potentials of�2.5 to �5 MPa (Murai and Yoshida,1998a). Highly vacuolated cells of imma-ture seeds (Berjak et al., 1984, 1994;Farrant et al., 1989, 1997) and desiccation-sensitive vegetative tissue (Farrant andSherwin, 1997; Farrant, 2000) are particu-larly sensitive to tonoplast dissolution.Replacing the water in vacuoles with solidmaterial reduces the degree to which vac-uoles must contract, thereby lessening thetension on tonoplast membranes duringdrying. Dry matter reserves naturally accu-mulate during embryogenesis in orthodoxand some recalcitrant seeds, and mayexplain the progressive tolerance to lowwater contents in developing embryos(Vertucci and Farrant, 1995; Farrant et al.,1997; Farrant and Walters, 1998). There isalso accumulation of dry matter in vac-uoles of vegetative tissues in many of thedesiccation-tolerant angiosperm speciesduring acclimatization to water stress(Farrant, 2000).

Water loss results in a general contrac-tion of cell volume. The plasmalemmae ofplant cells can be damaged if they aresheared from cell walls, which contract lessthan protoplasm, or if contraction resultsin an irreversible loss of membrane surfacearea. In addition to protection by fillingcells with dry matter (described above), theconsequences of volume changes can alsobe lessened by initial high surface area-to-volume ratios of cells and vacuoles (Iljin,1957; Bewley, 1979) and may explain whycells from non-vascular plants, which usu-ally have small vacuoles and lack plasmo-desmata, do not appear to suffer physicaldamage upon contraction (reviewed byBewley and Krochko, 1982). Damagingeffects of cell contraction are usually mani-fested during rehydration, suggesting thatthe stress and damage are not direct effectsof desiccation, but rather indications ofrehydration stress and mechanical failure.

A dismantling of mitochondria andchloroplasts is associated with severewater stress. Mitochondria observed in

mature orthodox seeds lack defined cristae(Bergtrom et al., 1982; Thomson and Platt-Aloia, 1984; Farrant et al., 1997) and mito-chondrial proteins are easily extractablefrom dried pollen (Hoekstra and vanRoekel, 1983). Conversely, mitochondriafrom immature embryos and recalcitrantseeds are more defined, and the greater dif-ferentiation has been linked to greater sen-sitivity to desiccation (Farrant et al., 1997).Chloroplast structure also degrades duringwater stress. Dried leaves of the desicca-tion-tolerant grasses Borya nitida andXerophyta humilis become yellow, concur-rent with the loss of grana stacks in thechloroplasts (Gaff and Hallam, 1974;Farrant, 2000). Using fluorescence-induc-tion kinetics to study partial processes ofphotosynthesis, researchers found adecrease in the efficiency of photosystem IIat water potentials between �3 and �4MPa (Wiltens et al., 1978; Hetherington, etal., 1982b; Vertucci et al., 1985; Sherwinand Farrant, 1998; Tuba et al., 1998;Csintalan et al., 1999) or during acclimati-zation to winter (Öquist and Strand, 1986).This decline could be a consequence ofphotochemical damage, but is more likelyto be a reflection of protective dismantlingof photosystem II (Demmig-Adams andAdams, 1992; Farrant, 2000). Indeed, thedismantling of the photosynthetic appara-tus during drying of B. nitida and X.humilis is required for survival: plantsdried too rapidly stay green and do notrecover (Gaff and Hallam, 1974; Farrant etal., 1999).

Slight water stress (�1 � �w � �3 MPa)enhances the protein synthesis that isbelieved to be important for conferring tol-erance (Ried and Walker-Simmons, 1993;Vertucci and Farrant, 1995; Ingram andBartels, 1996; Oliver et al., 1998; Mundreeet al., 2000; Whittaker et al., 2001). Furtherdrying reduces the rate of protein synthesisin both tolerant and sensitive cells (Bewleyand Krochko, 1982; Salmen Espindola etal., 1994; Ingram and Bartels, 1996; Oliveret al., 1998; Mundree et al., 2000;Whittaker et al., 2001), perhaps because ofa dismantling of endoplasmic reticulum,dictyosomes and polysomes (Webster and



Leopold, 1977; Thomson and Platt-Aloia,1984; Farrant et al., 1997; Wesley-Smith etal., 2001).

Indirect evidence from recalcitrantseeds suggests that, during dehydration,the cytoskeleton is disrupted at fairly highwater potentials (�3.8 MPa for Trichiliadregeana and �3.5 MPa for Quercus robur)leading to an abnormal distribution oforganelles within cells (Berjak et al., 1999;Mycock et al., 2000). Although it is tacitlyassumed that cytoskeletal disassemblymust occur during dehydration in desicca-tion-tolerant seeds (and vegetative tissues),it is its failure to reconstitute that charac-terizes this aspect of dehydration-relatedinjury in recalcitrant material (Mycock etal., 2000).

There is clearly a general trend towardscontraction or disassembly of cellularmachinery during water stress to about �5MPa. In most desiccation experiments,plant materials are stressed further and cellsurvival is assayed by whether or notorganelles reassemble upon rehydration. Indesiccation-sensitive cells that do not rup-ture, the protein-synthesizing machinerydoes not recover, nor do mitochondria andchloroplasts resume normal function;organelles become irregularly shaped anddisorganized (reviewed by Bewley andKrochko, 1982; Farrant et al., 1989; Berjaket al., 1990; Mycock et al., 2000). The con-traction and dismantling of organellesdescribed above are clearly signs of waterstress, but it is unclear whether thesechanges are symptoms of damage occurringat �5 MPa, or means of protection whenwater stress intensifies. It is also unclearwhether the failure to reconstituteorganelles indicates a primary site of dam-age or a general debilitation when cells die.These cause and effect arguments have ledresearchers to study the primary effects ofdehydration on the structure of macromol-ecules.

9.3.1.2. Molecular scale

Removing water from cells pushes cellularconstituents together, causing them tointeract in ways that might not otherwise

occur. A consequence of these molecularaggregations is an increased ordering ofmolecular structures, and it may seemironic that primary lesions during dryingare directly attributed to order rather thanto loss of it. Drying-induced compaction ofmolecules requires greater packing effi-ciency, resulting in localized enrichmentsof similar-type molecules in a processknown as demixing (Lis et al., 1982; Bryantand Wolfe, 1989; Rand and Parsegian,1989; Bryant et al., 1992). Molecules remixupon rehydration, but the reactions thatoccurred in the desiccated state may haveirreversible consequences.

Intermolecular associations of polarlipids are intrinsically linked to the watercontent of the medium. Under aqueousconditions, polar lipids spontaneouslyalign to form micelles or bilayer structuresdepending on the polar head group of thelipid. Acyl chains within bilayers aremore-or-less mobile, giving considerablefluidity to the structure and allowing pro-teins and other constituents to be inserted.Drying brings membrane bilayers into closeproximity and causes membrane con-stituents to segregate laterally into differentdomains enriched with particular lipidclasses or proteins (Lis et al., 1982; Bryantand Wolfe, 1989; Rand and Parsegian,1989; Bryant et al., 1992; Crowe andCrowe, 1992; Steponkus et al., 1995;Hoekstra and Golovina, 1999) (Fig. 9.2).The closer packing between membranesand among membrane constituents resultsin greater rigidity of the fatty acid domainwithin the bilayer. There are two mecha-nisms, based on either intra- or interlamel-lar events, used to explain why fatty aciddomains become more rigid. If water mole-cules are removed from between adjacentpolar head groups, the associated fatty acidscompress because of the increased strengthof van der Waals attractions (Crowe et al.,1990; Crowe and Crowe, 1992; Hoekstraand Golovina, 1999). Alternatively, as dif-ferent bilayers come into close apposition,strong repulsive hydration forces keepthem separate, but create isotropic tensionsthat lead to lateral compression within theacyl domain (Lis et al., 1982; Wolfe, 1987;



Rand and Parsegian, 1989; Bryant andWolfe, 1992; Wolfe and Bryant, 1999).Increased rigidity eventually leads to phasetransitions within the membrane from afluid to a gel state (Ladbrooke andChapman, 1969; Cullis and de Kruijff,1979). While these phase transitions arecompletely reversible, they are believed tointerfere with the semi-permeable proper-ties of membranes. Permanent damagecomes from the exclusion of proteins fromparts of the bilayer (Rand and Parsegian,1989; Bryant and Wolfe, 1992; Crowe andCrowe, 1992; Hoekstra and Golovina, 1999)(Fig. 9.2). Transient damage occurs uponrehydration: the rush of water on to aninelastic membrane may cause it to rupture(Murphy and Noland, 1982; Steponkus etal., 1995; Hoekstra et al., 1999) or imper-fect packing among different domains maycause leakage of cellular constituents(Crowe and Crowe, 1992; Hoekstra et al.,1999).

The close approach of membrane systemsand the lateral demixing of membrane com-ponents can lead to an even greater threat tomembranes than lamellar fluid-to-gel transi-tions. Membranes can fuse together, causingthe complete loss of compartmentationwithin the cell (Crowe et al., 1986; Croweand Crowe, 1992; Steponkus et al., 1995)(Fig. 9.2). Fusion is known to occur amongliposomes and native membrane fractions,although the mechanism that causes polarlipids to cross over to a different bilayer isunclear. In principle, the hydration charac-teristics of individual lipids and lipids in amixture, the intrinsic curvature of differenthead groups, the water content and the tem-perature allow the formation of invertedmicelles within closely appressed bilayers(Cullis and de Kruijff, 1979; Crowe et al.,1986; Steponkus et al., 1995) (Fig. 9.2). Indomains enriched with non-bilayer-forminglipids such as phosphatidylethanolamine-diglycerides or monogalactosyl-diglycerides,the polar head groups coalesce into rings andthe acyl chains extend radially outwards inwhat is known as a hexagonal phase (Cullisand de Kruijff, 1979; Siegel et al., 1994;Steponkus et al., 1995). Fusion via hexago-nal-phase changes is rare in native mem-

branes, but has been demonstrated in cellsfrom non-acclimatized leaves that werelethally cooled to �5°C (oat, �w ≈ �6 MPa)or �10°C (rye, �w ≈ �12 MPa) (Steponkus etal., 1995) and more frequently in animalcells (Cullis and de Kruijff, 1979; Crowe andCrowe, 1992). Evidence of cell fusion, butnot via hexagonal-phase changes, is commonin desiccation-damaged cells, protoplastsand liposomes (e.g. Crowe et al., 1986;Steponkus et al., 1995). In oat and rye leavesacclimatized to cold (but clearly not fullydesiccation-tolerant), fusion of plasmalemmaand endomembrane systems is suggested attemperatures between �10 and �40°C (�12� �w � �48 MPa), depending on the level ofcold tolerance achieved (Steponkus et al.,1995). Upon rehydration, improperly fusedmembranes produce vesicles that excludecell constituents or are combinations of dif-ferent membrane systems (e.g. the plas-malemma fuses with chloroplast outermembrane or with endoplasmic reticulum)(Fig. 9.2). Because the osmotic balanceinside and outside the cells has been com-pletely disrupted, vesicles produced frommembrane fusions are identified by theirinability to expand during rehydration(Steponkus et al., 1995).

Most of our understanding of how polarlipids behave in water-stressed situationscomes from model studies of liposomeswith known composition. In these systems,phase transitions are usually studied, eventhough they may only be harbingers of realdamage. Phase transitions of preparedmembrane systems occur at a range ofwater contents and temperatures depend-ing on the saturation of the acyl chains andthe presence of non-phospholipids (e.g.Ladbrooke and Chapman, 1969; Cullis andde Kruijff, 1979; Crowe et al., 1989;Steponkus et al., 1995). A water potentialof about �12 MPa is often cited as critical.It has been suggested that structural waterneeded for the proper spacing of polarhead groups is removed at �w � �12 MPa(Ladbrooke and Chapman, 1969; Crowe etal., 1990). Also, at �w ≈ �12 MPa, large,potentially deforming hydration forcesresult from the close approach of mole-cules (Wolfe, 1987).




Hydratedmembrane

systems

Dehydratedmembranes

Dehydratedmembranes

Rehydratedmembranes

–H2O

Demixing

Cytoplasm

Plasma membrane

Endomembrane

Exclusion ofintrinsic proteins

Plasmamembrane

Membraneappression

Endomembrane

Endomembrane

Non-bilayer phase formationand membrane fusion

Plasma membrane

Cytoplasm

EndomembraneLeakage of cellcontents

Plasma membrane

+H2O

Fig. 9.2. Schematic drawing of the effect of dehydration on cellular membranes. Different membranesystems may become closely appressed, leading to demixing of lipids and proteins and the loss of proteinsfrom parts of the bilayer. Closely appressed membranes may then form non-bilayer structures that lead tofusion between different membrane systems. Upon rehydration, cellular contents leak out and fusedmembrane particles do not swell (i.e. they are ‘osmotically unresponsive’). (Adapted from Steponkus et al.(1993), with permission.)


There is little information for comparingmembrane phase behaviour among ortho-dox and recalcitrant embryos, maturingembryos as they become more tolerant ofdesiccation, or leaves from desiccation-tol-erant angiosperms as they adjust to lowwater potentials. Changes in bilayer spac-ings or lamellar fluid-to-gel transitionshave been detected in both desiccation-tolerant and sensitive plant cells duringdehydration, with little difference inbehaviour detected with degree of toler-ance (McKersie and Stinson, 1980;Seewaldt et al., 1981; Priestley and deKruijff, 1982; Singh et al., 1984; Kerhoas etal., 1987; Crowe et al., 1989; Hoekstra etal., 1991, 1992; Sun et al., 1994; Hoekstraand Golovina, 1999). In tolerant soybeancotyledons, a gel-like transition occurredwhen seeds were dried to less than 0.2 gH2O g�1 dry mass (Seewaldt et al., 1981), awater content that corresponds to a waterpotential of about �12 MPa (e.g. Vertucciand Roos, 1990) (Fig. 9.1). Water potentialsbetween �10 and �15 MPa also mark thesurvival limit for recalcitrant seeds(described above). A membrane-mediatedmechanism is often invoked to explaindamage in desiccation-sensitive embryosand pollen because the membrane integrityof these cells appears to be compromisedupon rehydration (McKersie and Stinson,1980; Senaratna and McKersie, 1983;Vertucci and Leopold, 1987; Berjak et al.,1992, 1993; Poulsen and Eriksen, 1992;Sun and Leopold, 1993; Sun et al., 1994;Wolkers et al., 1998a).

The different views of dehydrationstress (i.e. removal of structural water ver-sus enhancement of hydration forces) havepromoted different ideas for the mecha-nisms of protection. According to the‘Water Replacement Hypothesis’, if struc-tural water is removed, small hydrophilicmolecules such as sugars must be insertedbetween polar lipid head groups to main-tain proper intermolecular spacings andmembrane integrity (Clegg, 1986; Crowe etal., 1990; Crowe and Crowe, 1992). Analternative, but not mutually exclusive,model suggests that high concentrations ofcompatible solutes can help resist water

loss between molecular surfaces, relievingthe size of hydration forces (Wolfe andBryant, 1999; Koster et al., 2000; Bryant etal., 2001). As dehydration proceeds, theconcentration within the interfacesincreases, with a concomitant increase inviscosity (Fig. 9.2). The high viscosity ofthese interfacial solutions providesmechanical resistance to the further com-pression of macromolecules (Wolfe andBryant, 1999; Koster et al., 2000; Bryant etal., 2001). In both protective models, thegoal is to keep molecules separated so thatharmful interactions are prevented. Sugarsaccomplish this capably in model mem-brane systems (Crowe et al., 1986, 1989,1990; Crowe and Crowe, 1992; Wolfe andBryant, 1999; Koster et al., 2000; Bryant etal., 2001). However, the presence of ade-quate quantities of sugars in cells and thevitrification of cellular constituents do notappear to prevent polar lipid phasechanges in desiccation-tolerant cells(Seewaldt et al., 1981; Priestley and deKruijff, 1982; Crowe et al., 1989; Hoekstraet al., 1989, 1992, 1999; Leopold et al.,1994) or damage in desiccation-sensitivecells (Berjak et al., 1992, 1993; Sun andLeopold, 1993; Still et al., 1994; Sun et al.,1994; Vertucci and Farrant, 1995; Vertucciet al., 1995; Farrant and Walters, 1998;Wolkers et al., 1998a; Hoekstra andGolovina, 1999; see Chapter 10). Changingthe composition of membranes (reviewedby Steponkus et al., 1995) and reducingtheir surface area by dismantlingendomembrane systems (described above)may be the important tools for maintainingcompartmentation in drying cells.

Structural changes of proteins withhydration have received wide attention inthe literature. Early work using a variety ofproteins showed that protein structure wasconserved during drying to extremely lowlevels (Schneider and Schneider, 1972;Kuntz and Kauzmann, 1974; Ruegg andHani, 1975; Ruegg et al., 1975; Fujita andNoda, 1978; Careri et al., 1980; Takahashiet al., 1980; Jaenicke, 1981; Rupley et al.,1983). In parallel studies, it was demon-strated that some proteins even maintainedfunctional activity (albeit at low levels)



when dry (Acker, 1969; Potthast, 1978;Labuza, 1980; Rupley et al., 1983).Secondary structure of cytoplasmic pro-teins (extracted from desiccation-tolerantpollen) was conserved upon drying in theabsence of protectant sugars, demonstrat-ing innate stability perhaps because of thehigh degree of �-helical structures (Wolkersand Hoekstra, 1995). The reversibility ofsorption–desorption isotherms of numer-ous proteins supported the idea that con-formational changes of proteins duringhydration were slight and reversible, mak-ing proteins an ideal model for studyinghydration properties of biological materials(Bull, 1944; D’Arcy and Watt, 1970). Slight,reversible changes in protein structure,particularly secondary structure, have beenattributed to volumetric changes from theloss of water rather than to changes in thenative structure of proteins. These changesoccur at fairly low moisture levels(between 0.2 and 0.1 g H2O g�1 dry mass or�70 to �200 MPa) (Ruegg and Hani, 1975;Griebenow and Klibanov, 1995). Drying, infact, stabilizes protein structures, makingthem particularly resistant to ageing(Franks et al., 1991; Costantino et al., 1998)and heat denaturation (e.g. Echigo et al.,1966; Ruegg et al., 1975; Fujita and Noda,1978; Takahashi et al., 1980; Jaenicke,1981; Leopold and Vertucci, 1986; Wolkersand Hoekstra, 1997). The extreme stabilityof protein structure with low hydrationmay be attributed to stronger intramolecu-lar associations compared with the situa-tion of polar lipids. Such interactionswould reduce the need for hydrogen bond-ing with water to maintain structuralintegrity (obviating the need for waterreplacement by sugars as suggested byCrowe and co-workers (e.g. Crowe andCrowe, 1992)) and/or provide mechanicalstrength that resists deformation whenmolecules are compressed (obviating theneed for mechanical barriers to compres-sion as suggested by Wolfe and co-workers(e.g. Wolfe and Bryant, 1999)).

The conformations of some proteins andpolypeptides are irreversibly damaged bydrying or freeze-drying in the absence ofprotectants (Hanafusa, 1969; Carpenter et

al., 1987, 1990; Franks et al., 1991;Prestrelski et al., 1993). Enzymes such aslactate dehydrogenase and polypeptidessuch as poly-L-lysine are particularly labile(Prestrelski et al., 1993), and damage isexacerbated if molecules are freeze-driedrather than air-dried (Franks et al., 1991).Rate of drying also has a large effect on theconservation of protein structure, withgreater preservation achieved by rapid dry-ing conditions (Wolkers et al., 1998a,b).Often, desiccation-labile proteins are usedto study the effects of protectants(Carpenter et al., 1987, 1990; Prestrelski etal., 1993). Clearly, these studies are essen-tial to the pharmaceutical industry, butsimilar mechanisms of protection must notbe presumed to apply in vivo in dehydrat-ing plants. A tremendous amount of workhas demonstrated that proteins are ratherrobust; thus, a need for protection must bedemonstrated before a protective mecha-nism is implied. Studies must show thatdesiccation-labile enzymes exist in vivo,that they are not produced de novo duringrehydration and that they are irreversiblydamaged in desiccation-sensitive cells.

The structure and activity of proteins arecompromised if they are stored underextremely dry conditions of approximately0.1 g H2O g�1 dry matter or about �200MPa or less (Kuntz and Kauzmann, 1974;Luscher-Mattli and Ruegg, 1982; Sanches etal., 1986; Labrude et al., 1987). Substantialdeterioration of the lattice of protein crystalswas attributed to the refolding of polypep-tide chains to increase packing efficiency(Kuntz and Kauzmann, 1974; Luscher-Mattliand Ruegg, 1982). Other studies have shownthat severe drying exposes haem groups onproteins, promoting free radical production(Sanches et al., 1986; Labrude et al., 1987).At such low water contents, protonexchanges among charged amino acidscould be measured, suggesting that thesesites were exposed (Careri et al., 1980;Rupley et al., 1983). Deterioration at similarwater potentials and in similar time framesis observed in stored seeds and pollen (e.g.Vertucci and Leopold, 1987; Vertucci andRoos, 1990; Buitink et al., 1996). Althoughthese organisms survive the initial stress of



complete water removal, they age progres-sively more rapidly when stored at�w � �220 MPa (< 20% RH). Perhaps mech-anisms suggested to cause damage in pro-teins at low water contents (e.g. exposure ofreactive sites on the proteins, increasedrelaxation of molecular structures as theyfill voids left by water, or relaxation of theglassy matrix that embeds the proteins) areresponsible for the deterioration of storedseeds and pollen. Protein structure is stablein seeds stored at about 30% RH (Golovinaet al. 1997), but stability of protein struc-tures in seeds stored at lower humiditieshas not been documented. Increased ageingrates of seeds and pollen stored below a crit-ical water content have also been attributedto reduced viscosity of the aqueous mediumin cells that are almost completely dry(Buitink et al., 1998b).

Upon dehydration, the same destabiliz-ing forces that perturb lipid and same pro-tein structures may also affect nucleic acidstructure (Rau et al., 1984). DNA is a partic-ularly stable molecule (Wayne et al., 1999)which maintains its structure in the absenceof water and reversibly unfolds at high tem-peratures (Bonner and Klibanov, 2000). Theintermolecular distances of dehydratingDNA strands are comparable to those ofcondensed DNA in hydrated nuclei (Rau etal., 1984), suggesting that DNA structuresare resistant to perturbations resulting fromdense packing. When DNA is replicated andso is decondensed during germination, thecells concomitantly become susceptible todesiccation injury (Deltour and Jacqmard,1974; Crèvecoeur et al., 1988) and rapidlydividing cells during embryogenesis alsoappear to be sensitive to desiccation (Myerset al., 1992). Desiccation did not affect thestructure of condensed or decondensedchromatin in desiccation-tolerant or sensi-tive maize embryos, respectively (Leprinceet al., 1995a). However, in those studies, thechelation of Ca2+ (and other divalentcations) by the ethylenediamine tetra-aceticacid (EDTA) present in the medium used forchromatin spreading, may have relaxed pre-viously condensed chromatin, possiblyaccounting for the reportedly similar resultsfrom desiccation-tolerant and sensitive

material (Pammenter and Berjak, 1999) (seealso Chapter 12).

When unprotected cells are dried,organelles and macromolecules experiencemechanical or structural damage. This typeof desiccation damage is termed sensustricto because the primary stress is waterremoval (Pammenter and Berjak, 1999;Walters et al., 2001). Membrane structuresappear more prone to desiccation damagesensu stricto than do proteins or DNA, per-haps because of the intense hydrogenbonding within proteins and nucleic acidstructures. Protection from damage oftenlies in the ability of the structure or thesurrounding medium to offer mechanicalresistance to the stress or accommodate thestress through enhanced elasticity.

9.3.2. Metabolically derived damage

Loss of turgor precipitates a number ofchanges in metabolic pathways of plantcells. Assimilation of CO2 (if the tissue isphotosynthetic) and growth are impaired.Often protein synthesis is temporarilystimulated during mild water stress(reviewed by Farrant et al., 1989; Ingramand Bartels, 1996; Oliver et al., 1998), witha switch in metabolism believed to lead tothe production of putative protectionmechanisms (reviewed by Vertucci andFarrant, 1995; Ingram and Bartels, 1996;Oliver et al., 1998; Chapters 1, 5 and 11).Observations of increased polysomes andrough endoplasmic reticulum in slightlywater-stressed recalcitrant embryos suggestthat certain (possibly similar) metabolicpathways may also be induced in seedsthat do not acquire full tolerance of desic-cation (Berjak et al., 1984; Farrant et al.,1989; Pammenter et al., 1998). Thesechanges in metabolism do not indicate thatcells have already experienced damage;when briefly stressed, most organismsresume normal metabolism once the waterstress is relieved. However, prolonged mildstress (which could be considered akin todrought) is deleterious to both vegetativeand embryonic tissues. Many recalcitrantseeds lose viability if maintained for long



periods at constant high water contents(e.g. Chin and Roberts, 1980; Berjak et al.,1989; Pammenter et al., 1994; Walters etal., 2001), and similar damage is observedin orthodox seeds (Walters et al., 2001).The loss of viability has been associatedwith the continuation of metabolism(including cell division) (Farrant et al.,1989), which will ultimately lead to agreater demand for water to maintain highwater potentials (Berjak et al., 1989;Pammenter et al., 1994).

Metabolism slows at water potentialsless than about �2 MPa, but not all reac-tions are affected by dehydration in thesame way. Protein synthesis slows down atrelatively high water potentials (reviewedby Bewley and Krochko, 1982; Clegg, 1986;Salmen Espindola et al., 1994; Ingram andBartels, 1996; Mundree et al., 2000;Whittaker et al., 2001), while respirationcontinues to much lower levels (Vertucciand Leopold, 1984; Vertucci and Roos,1990; Salmen Espindola et al., 1994;Leprince and Hoekstra, 1998; Leprince etal., 1999; Farrant, 2000; Walters et al.,2001). Various reactions within photosyn-thetic (Wiltens et al., 1978; Hetherington etal., 1982b; Vertucci et al., 1985; Vertucciand Leopold, 1986; Farrant, 2000) and res-piratory (Vertucci and Leopold, 1986;Leprince and Hoekstra, 1998; Leprince etal., 2000) pathways respond differently tolow water contents. The differing responsesto water stress among and within metabolicpathways can lead to imbalances in metab-olism. Metabolic imbalances may be con-founded by the respiration of fungi thatoccurs at water potentials as low as �20MPa in orthodox and recalcitrant seed tis-sues (Mycock and Berjak, 1990; Goodman,1994; Calistru et al., 2000). Damage bymetabolic stress is most pronounced incells at water potentials between �2 and�5 MPa with a diminishing effect as cellsare dried to �12 MPa (Leprince et al., 2000;Walters et al., 2001). Both desiccation-sen-sitive and -tolerant organisms are damagedwhen stored at intermediate water poten-tials, though the time-dependency of thedamage varies considerably among speciesand tissues (Walters et al., 2001).

A by-product of continued respirationand light harvesting when other metabolicprocesses are shut off is the accumulationof high-energy intermediates that leak outof mitochondria and plastids and formreactive oxygen species (ROS) and free rad-icals (Puntarulo et al., 1991; Dean et al.,1993; Hendry, 1993; Leprince et al., 1993,1994, 1995b; Smirnoff, 1993; Foyer et al.,1994; Halliwell and Gutteridge, 1999).Reactive oxygen species and free radicalsreact with proteins, lipids and nucleicacids, causing permanent damage toenzymes (Wolff et al., 1986; Dean et al.,1993; Halliwell and Gutteridge, 1999),membranes (Senaratna and McKersie,1983, 1986; Chan, 1987; McKersie et al.,1988, 1989; Finch-Savage et al., 1996;Halliwell and Gutteridge, 1999; Leprince etal., 2000) and chromosomes (Dizdaroglu,1994). Peroxidation of lipids decreases thefluidity within membranes (McKersie etal., 1988, 1989), interfering with theirselective permeability upon rehydration (asdescribed above). Upon dehydration, highlevels of free radicals have been detected indesiccation-sensitive embryos (Senaratnaand McKersie, 1983, 1986; McKersie et al.,1988; Hendry et al., 1992; Leprince et al.,1993, 1994, 1995b, 1999, 2000; reviewedby Vertucci and Farrant, 1995; Pammenterand Berjak, 1999). The origin and sequenceof events following the appearance of thesetoxic compounds are still unclear. Theymay be produced by the water-stressed cell(Leprince et al., 1994, 1995b, 1999, 2000;Leprince and Hoekstra, 1998) or as a resultof the associated fungi (Goodman, 1994;Finch-Savage, 1999), and they may precede(or precipitate) damage (Finch-Savage etal., 1996; Leprince et al., 2000) or ariseafter the cell has already died (Finch-Savage, 1999).

There are several ways that cells canprotect themselves from metabolic imbal-ance and ROS-mediated damage. At highermoisture levels, free-radical-scavengingenzymes efficiently detoxify ROS (Bewley,1979; Dhindsa, 1987; Hendry, 1993;Smirnoff, 1993; Foyer et al., 1994; Krannerand Grill, 1997; Sherwin and Farrant, 1998;Pammenter and Berjak, 1999; Farrant,



2000). These enzymes appear ineffective atlow water contents, and tocopherol andascorbic acid may be more effective(reviewed by McKersie et al., 1988;Pammenter and Berjak, 1999).Amphipathic molecules such as tocopherolcan partition between aqueous and lipiddomains according to the water content ofthe cell and polarity of the molecule(Golovina et al., 1998). A controlled shut-down of metabolism upon drying may alsomitigate the consequences of unbalancedmetabolism (as reviewed by Leprince et al.,1993; Vertucci and Farrant, 1995; Handand Hardewig, 1996; Pammenter andBerjak, 1999). Cells with more organellesand greater definition of organelle structureappear to be more sensitive to desiccation(Bewley, 1979; Hetherington, 1982a; Gaff,1989; Berjak et al., 1990; Farrant et al.,1997; Farrant and Walters, 1998; Farrant,2000), either because there are more mem-brane structures to protect (describedabove) or because the higher metabolismleads to greater ROS production.Conditions that reduce metabolism such aslow temperature (Leprince et al., 1995b) orhighly complex substrates (Leprince et al.,1990) also tend to reduce sensitivity to des-iccation. Desiccation-sensitive cells respireat comparatively greater rates than tolerantcells at the same water content (Leprince etal., 1999; Walters et al., 2001), which mayreflect properties of the mitochondriathemselves or of the cellular matrix. It hasbeen suggested that changes in viscositywith dehydration are not as marked in des-iccation-sensitive cells, and so metabolismis not as restricted (Leprince and Hoekstra,1998). It has also been suggested that thepacking of macromolecules during dehy-dration of desiccation-sensitive cells is notas dense (Wolkers et al., 1998a,c), and thismight facilitate the diffusion of oxygenthrough the cell matrix.

9.4. Perspectives on the Kinetics ofDesiccation Damage

This chapter has described how desicca-tion damage, incurred from structural

changes of cellular constituents, may leadto metabolically derived damage and viceversa. Most model studies suggest thatchanges in molecular conformations withdehydration are reversible. Dehydrationslows chemical reactions and so organismsthat are dried sufficiently rapidly shouldexperience few, if any, changes in thechemistry of their cells. Thus, it seemslikely that the primary lesions resultingfrom water stress, whether they are physi-cal or chemical, are minor. It also appearsthat the primary lesions are not exclusiveto desiccation-sensitive cells. Gel-phaselipid transitions occur in both tolerant andsensitive pollens (e.g. Hoekstra andGolovina, 1999; Hoekstra et al., 1999);metabolic imbalances occur in both desic-cation-tolerant pea and desiccation-sensitive tea (Walters et al., 2001); changesin the secondary structures of proteins arecomparable in both mature and immaturemaize embryos (Wolkers et al., 1998a).Irreparable desiccation damage must thenresult from a cascade of reactions, initiatedby primary but subtle lesions, which per-turb organization within the cell and ulti-mately lead to cell death. The extent ofdesiccation damage can then be viewed as afunction of the rapidity at which the cas-cade of deleterious reactions occurs. Fromthis perspective, desiccation damage is atime-dependent process – an ageing phe-nomenon.

Dehydration has contrasting effects onthe kinetics of both physical and chemicaldeteriorative reactions. Removing waterfrom cells increases the concentrations ofreactants but slows the molecular motionsnecessary for reactions to occur. The degreeto which cells are damaged by desiccation,and by extension the critical moisture levelto which they survive drying, is deter-mined by the treatment duration, the con-centration of the reactants and the physicalbarriers (e.g. viscosity and compartmenta-tion) to thermodynamically favourablereactions. Given the same experimentaltime, cells that tolerate more stress haveeither fewer reacting substances or greaterbarriers to harmful reactions. For example,according to the Water Replacement



Hypothesis, the concentration of reactingphospholipids is reduced by inserting sug-ars between head groups (Clegg, 1986;Hoekstra et al., 1989, 1991; Crowe andCrowe, 1992; Hoekstra and Golovina,1999). Alternatively, vitrification betweenbilayer interfaces provides a mechanicalresistance to the compression of bilayers(Wolfe and Bryant, 1999; Koster et al.,2000; Bryant et al., 2001). According tothese two hypotheses, more tolerant organ-isms have either proportionally more sug-ars to insert (Hoekstra et al., 1989) or morestrength to repel (Wolfe and Bryant, 1999).Continuing this line of thinking may helpto further distinguish between the twohypotheses. According to the WaterReplacement Hypothesis, once a finitenumber of sites is filled, the membrane orcell should be able to tolerate completedrying and maintain structure through timesince reactive sites are protected. On theother hand, vitrification is likely to provideimperfect protection, and macromolecularstructures will eventually be compromisedgiven sufficient time or stress. This latterpossibility is consistent with observations ofnaturally occurring desiccation-tolerant systems: desiccation-tolerant seeds (Vertucciand Roos, 1990), pollen (Buitink et al.,1998b) and other phylogenetically diverselife forms such as Artemia cysts, leaves fromresurrection angiosferms, and yeast (C.Walters, unpublished data) are progressivelydamaged when dried to water potentialsless than about �200 MPa.

The effect of time on desiccation-relateddamage has been explored at higher mois-ture levels (�12 � �w � �3 MPa) wheremetabolism is believed to play a role(Pammenter et al., 1998; Leprince et al.,1999, 2000; Walters et al., 2001) and atextremely low moisture levels (�w � �220MPa) where mechanical properties arecompromised and/or reacting substancesare exposed (Vertucci and Roos, 1990;Buitink et al., 1996, 1998a,b; Walters,1998). In these systems, the time scale formeasuring damage is days or years, respec-tively. However, some damaging reactionsmay appear to be almost instantaneous. Forexample, membrane-phase transitions lead-

ing to fusion occur in milliseconds (Siegelet al., 1994). Drying within 2–3 minreduces protein denaturation of purifiedprotein (Wolkers et al., 1998a,b), and dry-ing within a few hours allows recalcitrantembryos to survive greater amounts ofwater loss (Pammenter et al., 1991, 1998;Walters et al., 2001). Despite valiantattempts, we have not been able to dryrecalcitrant embryos in less than 10 minand have yet to observe a recalcitrantembryo survive �15 MPa. This failure toextend the lower limits of survivable watercontents in recalcitrant seeds suggests thatthe damaging reaction that occurs between�10 and �15 MPa is extremely fast in des-iccation-sensitive cells and relatively slow(and perhaps non-existent) in tolerantcells. The low rate of damage at this waterpotential in tolerant cells cannot beexplained by glass formation per se (e.g.Leopold et al., 1994), as glasses form inseeds and pollen at lower water potentials(�w � �70 MPa) (see Chapter 10). However,the viscosity of leathery and rubbery states(Fig. 9.1) may effectively impede molecularmovements leading to membrane damage athigher moisture levels. The greater viscos-ity measured in more desiccation-tolerantcells (Leprince et al., 1999) may contributeto greater structural stability.

9.5. Conclusion

Clearly desiccation sensitivity is not an ‘allor nothing’ or qualitative feature as it wasonce treated. Macromolecules, cells andorganisms succumb at a range of stress lev-els over a range of times. The primarylesion may be slight and reversible by ourstandards of measurement and may occurin both tolerant and sensitive cells. Thislesion, which arises from the contraction ofthe aqueous volume and the consolidationof cellular constituents, may lead to theirreversible loss of plasmalemma surfacearea, disruption of normal metabolism,fusion of unrelated membrane systems andprotein aggregation. As deleterious reac-tions cascade, it becomes difficult to deter-mine the earliest sources of damage.



Protectants either mitigate unbalancedmetabolism or prevent the contraction andconsolidation of cellular constituents. Indoing so, a level of quiescence andmechanical rigour is imposed. Theseappear to be the most distinguishingcharacteristics of desiccation-sensitive and-tolerant angiosperms.


The authors gratefully acknowledge DrsPeter L. Steponkus (Cornell University),Folkert Hoekstra (Wageningen University)and, especially, Karen L. Koster (TheUniversity of South Dakota) for helpful andthought-provoking discussions.


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10 Biochemistry and Biophysics of Tolerance Systems

Julia Buitink,1 Folkert A. Hoekstra2 and Olivier Leprince11UMR Physiologie Moléculaire des Semences, Institut National d’Horticulture,

16 Bd Lavoisier, F49045 Angers, France; 2Laboratory of Plant Physiology, Departmentof Plant Sciences, University of Wageningen, Arboretumlaan 4, 6703 BD Wageningen,

The Netherlands

10.1. Introduction 29310.2. Repression of Metabolism 29410.3. Antioxidant Defence 29510.4. Partitioning of Amphiphilic Compounds 29610.5. Macromolecule Stabilization 298

10.5.1. Preferential hydration 29810.5.2. DNA integrity and chromatin condensation 29910.5.3. Water replacement hypothesis 29910.5.4. Vitrification 302

10.5.4.1. Role of vitrification in desiccation toleranceand longevity 303

10.5.4.2. Composition of glasses in desiccation-tolerantorganisms 305

10.6. Roles of Specific Compounds in Stability 30610.6.1. Sucrose/oligosaccharides 30610.6.2. Late embryogenesis abundant (LEA) proteins 30710.6.3. Heat-shock proteins (HSPs) 309

10.7. Conclusion and Outlook 31010.8. References 311


10.1. Introduction

Most living cells only survive dehydrationto a limited extent. Water has a profoundinfluence on the association of amphiphilicphospholipids in bilayers and the folding ofproteins (Tanford, 1978). These molecular

arrangements are lost when the water inwhich they are formed is removed. Forinstance, when a biological membrane isdehydrated, irreversible changes occur in itsstructural and functional integrity (reviewedby Crowe et al., 1997a). Similarly, manylabile proteins lose their functional and


probably structural integrity when they aredesiccated (reviewed by Carpenter et al.,1992). Also, removal of water from desicca-tion-sensitive organisms leads to metabolicimbalances, resulting in the production offree radicals and oxidative damage. WhereasChapter 9 discussed the damage linked todesiccation, this chapter deals with the biochemical and biophysical changes thatare part of the mechanisms by which desiccation-tolerant organisms (anhydro-biotes) cope with dehydration. The acquisi-tion of desiccation tolerance is correlatedwith the accumulation of considerablequantities of non-reducing di- andoligosaccharides, compatible solutes andspecific proteins such as the late embryoge-nesis abundant (LEA) and heat-shock pro-teins (HSPs) (see Chapters 1, 5 and 11).

Two strategies that confer desiccationtolerance have been identified. The firststrategy involves an initial avoidance of theaccumulation of desiccation-induced dam-age accompanied by the presence of pro-tecting factors such as sugars or LEAproteins. The protecting factors can be pre-sent before or synthesized during drying(Vertucci and Farrant, 1995). Desiccationtolerance probably depends on theseresponses acting in synergy during drying.For example, it has been shown that pro-tective disaccharides do not exert their pro-tective effects on dried membranes thatcontain more than 15% free fatty acids, aproduct that accumulates during desicca-tion-induced oxidative stress (Senaratnaand McKersie, 1986; Crowe et al., 1989b).

The second strategy is based on activa-tion of efficient repair mechanisms uponrehydration. This second strategy is dealtwith in Chapter 12. This chapter will give acritical assessment as to how desiccation-tolerant organisms have adapted theirstrategies to cope with the stresses thatoccur during dehydration. The differentstrategies will be discussed in sequence ofhydration level at which they are thoughtto be active, from the hydrated to the drystate, starting with the avoidance of oxida-tive damage by means of regulation ofmetabolism (Section 10.2) and the presenceof antioxidant systems (Section 10.3), fol-

lowed by partitioning of amphiphilic com-pounds (Section 10.4). The mechanismsthrough which the macromolecules are sta-bilized at different hydration levels are dis-cussed in Section 10.5, together with anoverview of the protective substances thathave been identified so far in relation todesiccation tolerance (Section 10.6).

10.2. Repression of Metabolism

The most reported degradative reactionslinked with desiccation sensitivity in seedsare the extensive peroxidation and de-esteri-fication of phospholipids, leading to the lossof membrane integrity (Senaratna et al., 1987;Hendry et al., 1992; Leprince et al., 1994).The cause of peroxidative damage is thoughtto originate from an increased formation ofreactive O2 species (ROS) as a result of theimpairment of the electron transport chainsduring drying (see Chapter 9).

Characteristically, desiccation-toleranttissues suffer less from oxidative damagethan do sensitive tissues (Leprince et al.,1993, 1994; Vertucci and Farrant, 1995;Pammenter and Berjak, 1999). The produc-tion rate of ROS is dependent on a numberof factors (Skulachev, 1996). It increaseswith an increased lifetime of the electroncarriers in the reduced state, with thedepletion of ADP and with an increase inrespiration rate. It has been surmised thatdesiccation-tolerant organisms reduce oradapt their metabolic activities to diminishthe chance of generating ROS (Leprince etal., 1994; Vertucci and Farrant, 1995;Pammenter and Berjak, 1999). Evidencesupporting this hypothesis is increasing,but has been indirect so far. Rogerson andMatthews (1977) observed for garden peaseeds that during maturation the ability towithstand desiccation was preceded by afall in respiration rate. Furthermore, desic-cation-tolerant axes of pea and cucumberwere found to exhibit a much reduced CO2production before dehydration comparedwith germinated, desiccation-sensitiveaxes, and this difference was maintainedduring drying (Leprince et al., 2000; seeTable 10.1). Respiration rates in the desicca-

294 J. Buitink et al.


tion-sensitive cotyledons of Castanea sativaMill. increased at the onset of drying anddecreased only with the loss of membraneintegrity. In contrast, dehydration of themore desiccation-tolerant axes resulted in arapid decline in O2 uptake rates at the onsetof drying (Leprince et al., 1999). In general,mature recalcitrant seeds have relativelyhigh respiration rates (Salmen Espindola etal., 1994; Leprince et al., 1999; Pammenterand Berjak, 1999). Thus, the high respira-tion rates in desiccation-sensitive seedsmay promote free-radical-induced injuryduring drying. The relationship betweenthe extent of metabolic activity and desicca-tion sensitivity is strengthened by theobservation that treatments that limit ratesof metabolism also reduce the incidence ofdesiccation injury (Leprince et al., 1995b).

If metabolism must be down-regulatedfor desiccation tolerance to be acquired, thenature of this regulation is unknown.Leprince et al. (1994, 1995b, 2000) sug-gested that a coordinated down-regulationof energy metabolism in seeds early duringdrying may play an important role in avoid-ing oxidative stress conditions and/or accu-mulation of by-products of metabolism totoxic levels. During drying, the O2 availabil-ity decreases with the increase in cellularviscosity. It is likely that to avoid metabolicimbalances there will be a fine tuningbetween repression of metabolic activityand O2 availability (Leprince et al., 2000).Down-regulation of metabolism appears tobe an ancient and widespread regulatorymechanism that allows aerobes to with-stand severe environmental stresses, such

as anoxia, freezing and dehydration (Handand Hardewig, 1996; Hardie et al., 1998).However, thus far there is only limited evi-dence for metabolic depression in develop-ing seeds (Kollöffel and Matthews, 1983)and somatic embryos (Tetteroo et al., 1995).Some indications that coordinated down-regulation might be essential to confer des-iccation tolerance in seeds comes fromLeprince et al. (2000). Dehydration of desic-cation-sensitive axes resulted in an increasein the emission rates of acetaldehyde andethanol, which peaked well before theonset of membrane damage. Tolerant axesdid not exhibit acetaldehyde or ethanolproduction. The question remains as towhether controlled down-regulation isexerted before drying (i.e. during seed mat-uration) or also during drying.

10.3. Antioxidant Defence

Since susceptibility to peroxidation mayincrease with drying (see Vertucci andFarrant, 1995, for a review; Chapter 9), onemay reason that free-radical-scavenging sys-tems are an important component amongthe mechanisms of desiccation tolerance.ROS are natural by-products of the metabo-lism, which are particularly present inchloroplasts and mitochondria. Thus,plants are well endowed with antioxidantmolecules and scavenging systems (Larson,1988; Hendry, 1993). Enzymatic free-radical-processing systems include super-oxide dismutase (SOD), which catalyses thedismutation of superoxide (O2

�) into H2O2

Biochemistry and Biophysics of Tolerance Systems 295

Table 10.1. CO2 production rates during drying of desiccation-tolerant cucumberaxes (24 h imbibed at 20°C; DT), desiccation-sensitive axes (72 h imbibed at20°C, radicle 3 mm protruded; DI) and germinated cucumber axes that wereincubated in polyethylene glycol (PEG) for 7 days to reinduce desiccationtolerance (72 h imbibed at 20°C with a radicle 3 mm protruded, then 7 days inPEG (�1.5 MPa) at 10°C; DT PEG). (Adapted from Leprince et al., 2000.)

CO2 production rate (�l h�1 g�1 dry weight)

g H2O g�1 dry weight DT DI DT PEG

3.0 1.7 4.5 1.12.0 1.3 3.2 0.51.0 0.7 1.5 0.4


and O2, and those that are involved in thedetoxification of H2O2 (i.e. catalase, glu-tathione reductase and peroxidases).Several studies have demonstrated the linkbetween tolerance of oxidative stressinduced by water deficit and rise in antioxi-dant concentrations in photosyntheticplants (Winston, 1990; Price and Hendry,1991). In vegetative tissues, removal of thecytotoxic products resulting from oxidativeevents is considered to be of prime impor-tance for survival of drought stress becausegenes encoding enzymatic antioxidantsbecome up-regulated during drying (Ingramand Bartels, 1996). In the resurrection plantCraterostigma plantagineum, an inhibitorof lipoxygenase (the activity of whichresults in lipid hydroperoxide formation)accumulates in the leaves during desicca-tion (Bianchi et al., 1992). In Craterostigmawilmsii and Xerophyta viscosa, the activityof ascorbate peroxidase increases duringdehydration. During rehydration, the activ-ity of SOD and glutathione reductaseincreases (Sherwin and Farrant, 1996).Furthermore, C. wilmsii was also found toaccumulate large amounts of anthocyanins,which have antioxidant capabilities.

The protective role of antioxidants indesiccation tolerance of seeds is far fromresolved (reviewed by McKersie, 1991;Hendry, 1993; Leprince et al., 1993).Vertucci and Farrant (1995) have suggestedthat it is particularly in the water contentrange corresponding to water potentials of�3 to �11 MPa that unregulated metabolicevents result in the first wave of free-radi-cal generation, although the upper limitcould be higher. Thus, it is assumed thatantioxidant systems should be maximallyeffective during the initial stages of thematuration drying of developing orthodoxseeds (Arrigoni et al., 1992).

Despite several studies that assessed invitro (i.e. under hydrated conditions) theactivity of free-radical-processing systemsextracted from drying tissues, it remains tobe ascertained whether these systems arealso active at low water contents in vivo.Considering that the metabolism is virtu-ally nil in cells containing less than 0.3 gH2O g�1 dry weight (dw), and that free-

radical-processing systems in seeds rely onenzymatic activities, it is likely that they aremainly efficient early during drying andmay not function in reduced hydration con-ditions. At the lower water contents, onemay expect molecular antioxidants (e.g. glu-tathione, ascorbate, tocopherol) to play apreponderant role in alleviating oxidativestress. This is supported by a study on ger-minating seeds of soybean, which showedthat their desiccation tolerance was associ-ated with the presence of an unknown lipid-soluble antioxidant in extracted membranes(Senaratna and McKersie, 1986). However,in germinating maize, no correlation wasfound between the presence of lipid-solubleand hydrophilic antioxidants and desicca-tion tolerance (Leprince et al., 1990b). Acomparative study on ascorbate and dehy-dro-ascorbate showed that the concentra-tions of these antioxidants were higher inrecalcitrant seeds than in orthodox seeds(Tommasi et al., 1999). Future studies aimedat establishing the importance of free-radi-cal-processing systems in relation to desic-cation tolerance should take into accountboth the hydration level during drying andthe fact that free-radical production will belocalized within the mitochondria and/ormicrosomal membranes. Thus, effortsshould concentrate on antioxidant systemswithin these organelles. Furthermore, coin-cidental antioxidants such as quinones,polyols, carbohydrates, amphipathic mole-cules (flavonoids, phenolics) and proteins(e.g. peroxiredoxin) also deserve particularattention.

10.4. Partitioning of AmphiphilicCompounds

Cells may contain various cytoplasmicmetabolites that have amphiphilic proper-ties. Hoekstra et al. (1997) proposed thatdesiccation may increase the transfer ofthese amphiphiles from the polar cyto-plasm into the lipid phase, i.e. the mem-branes and lipid bodies. On the one hand,such partitioning into membranes couldseriously perturb membrane structure, witheffects on permeability properties as the




result. On the other hand, partitioning intomembranes might be extremely effective atautomatically inserting amphiphilicantioxidants into membranes upon dehy-dration, which could promote desiccationtolerance and extend storage longevity. Thepresumed transfer of amphiphiles has beenexperimentally tested in pollen and seedswith electron paramagnetic resonance(EPR) spectroscopy, using amphiphilicspin probes inserted into the cytoplasm(Golovina et al., 1998; Buitink et al.,2000d; Hoekstra and Golovina, 2000). Theadvantage of this method is that the EPRspectra can be used to derive where thespin probe resides – in the cytoplasm or inthe lipid phase. It has been demonstratedthat amphiphilic probes partition into thelipid phase with drying, in proportion tothe reduction of the cytoplasmic volume,and vice versa with rehydration (Golovinaet al., 1998; Hoekstra and Golovina, 2000).

A similar partitioning behaviour withdrying is plausible for endogenous cyto-plasmic amphiphiles. Extracted endoge-nous amphiphiles reversibly partitionedinto liposomal membranes with drying,which made the liposomes transientlyleaky for entrapped polar compounds(Golovina et al., 1998). Thus, partitioninginto membranes is possible and has beenused to explain the transient leakage ofcytoplasmic solutes from rehydrating anhy-drobiotes. Plasma membrane permeabilityof pollen, for example, was indeed elevatedas long as the amphiphiles resided in thelipid phase. On sufficient rehydration,when the amphiphiles had mainly returnedto the aqueous cytoplasm, permeabilityreturned to the low level observed beforedehydration, and leakage rates became low.This transiently increased permeabilityupon imbibition lasted approximately 10 sin the case of pollen (Hoekstra et al., 1999).

There are more indications that in dryanhydrobiotes endogenous amphiphilesreside in membranes. In situ Fourier trans-form infrared (FTIR) spectroscopy data hasindicated that the average rigidity of mem-branes in the gel phase is less when theorganism is dry than when it is hydrated.When comparing dried isolated membranes

or liposomes with hydrated ones, the aver-age rigidity was similar, even in the pres-ence of a protective sugar. This has beeninterpreted to mean that dry membranesinside anhydrobiotes are fluidized, presum-ably by amphiphilic guest molecules(Hoekstra and Golovina, 1999). In isolatedmembranes, such amphiphiles become lostduring the isolation procedure. Recent databased on the number of spin-probe mole-cules in either phase during dehydration(Golovina and Hoekstra, 2001) indicate thatthere is already a considerable transfer ofamphiphiles from the cytoplasm to mem-branes at the beginning of drying. Thiswould mean that fluidization occurs earlyduring dehydration. Indeed, an increase inmolecular mobility at the membrane surfacewas observed early during dehydration,probably brought about by endogenousamphiphiles. While this mobility decreasesin desiccation-tolerant organisms on furtherdrying, it remains high in desiccation-sensi-tive organisms until almost all water hasdisappeared (Golovina and Hoekstra, 2001).

Endogenous biologically relevantamphiphiles that might undergo partition-ing similar to the spin probes are phenolicacids and flavonoids. These molecules areabundantly present in dry seeds, pollen andresurrection plants (Larson, 1988; Oliver etal., 1998; Shirley, 1998) and may play sev-eral roles in relation to desiccation toler-ance. A wide array of amphiphiles presentin plants are known to be potent antioxi-dants (Larson, 1988; Saija et al., 1995; Rice-Evans et al., 1997). Partitioning of suchamphiphilic antioxidants from the cyto-plasm into membranes during drying mightprevent desiccation-induced oxidativedamage. A dehydration test with the antiox-idant flavonoid rutin on liposomes indi-cated that rutin depresses the averagephase-transition temperature of the lipo-somes (Hoekstra and Golovina, 2000). Fromthis it has been inferred that amphiphilesmight have a role in controlling the mem-brane-phase transition temperature (Tm),along with the non-reducing di- andoligosaccharides (see Section 10.5.3).

Although partitioning seems beneficial,as indicated above, it appears that the trans-


fer must be controlled in order to conferdesiccation tolerance (Buitink et al., 2000d).This is because amphiphiles can perturbmembrane functions (Herbette et al., 1983;Takahashi et al., 1998), which might be par-ticularly detrimental at high water contents.For example, if mitochondrial respiration isaffected, there is the likelihood of anenhanced production of free radicals.

10.5. Macromolecule Stabilization

Macromolecule stabilization, such as theretention of phospholipid bilayers orproper folding of proteins, depends on thepresence of water. Upon dehydration, thesemacromolecules need to be protected fromthe deleterious effects that are normallyassociated with the removal of water. Thestabilization of the molecules in the cells isrealized through one or more of the mecha-nisms that are discussed below, in theorder of the mechanisms that are func-tional at high hydration levels to those thatare operational at low water contents.

10.5.1. Preferential hydration

Many plants and microorganisms accumu-late organic osmolytes in response to envi-ronmental stresses that cause cellulardehydration, such as drought, freezing andosmotic shock. This accumulation corre-lates with improved stress tolerance.Evidence of a causal relationship betweenelevated levels of these so-called compati-ble solutes and stress tolerance has comefrom the results of enrichment experiments(e.g. Saranga et al., 1992) and from thebehaviours of transgenic organisms engi-neered to accumulate these compounds(e.g. Takagi et al., 1997; Strom, 1998).Among these compatible solutes are pro-line, serine, glutamate, glycine-betaine, car-nitine, mannitol, sorbitol, fructans, polyols,trehalose, sucrose and oligosaccharides.The absolute osmolyte concentrations,however, are unlikely to mediate osmoticadjustment (Hare et al., 1998).

It has been shown in model experi-ments that these substances stabilize pro-tein structure and activity against thermaldenaturation (Arakawa and Timasheff,1985). Further, they protect isolated pro-teins (Carpenter and Crowe, 1988;Carpenter et al., 1990) and (functional)vesicles (Rudolph and Crowe, 1985;Anchordoguy et al., 1987, 1988) duringfreeze–thawing by minimizing denatura-tion and the vesicles by preventing fusion.Coming from chemically dissimilarclasses, these substances have in commonthat they are preferentially excluded fromcontact with the surface of proteins inaqueous solution, which makes it thermo-dynamically unfavourable for proteins tounfold (Arakawa and Timasheff, 1985;Carpenter and Crowe, 1988; Carpenter etal., 1992). In other words, these substanceskeep the macromolecules preferentiallyhydrated. In contrast, compounds such asurea and guanidine–HCl, which preferen-tially bind with proteins, destabilize pro-teins in solution. This means that whenthe bulk water is removed (below 0.3H2O g�1 dw), this mechanism would fail towork because there is no water left forpreferential hydration (Crowe et al., 1990).Indeed, most of the compatible solutes,except a few sugars, are unable to protectproteins and membranes against air-dryingor freeze-drying. In the case of sugars, it isenvisaged that, after the bulk water is lost,hydrogen bonding and glass formation arethe mechanisms by which proteins andmembranes are structurally and function-ally preserved (see Sections 10.5.3 and10.5.4).

One has to realize that dehydratinganhydrobiotes pass through stages ofdrought during which the mechanism ofpreferential exclusion may be effective. Itis, therefore, not surprising that anhydro-biotes are endowed with large amounts ofcompatible solutes, e.g. proline andsucrose in pollen (Zhang et al., 1982;Hoekstra et al., 1992), di- and oligosaccha-rides in seeds (Amuti and Pollard, 1977),and proline, glycine-betaine and trehalosein microorganisms and yeast.



10.5.2. DNA integrity and chromatincondensation

The maintenance of genetic information iscentral to survival upon dehydration andrehydration (see Chapter 12). As a dynamicand hydrated molecule in vivo, DNA canassume different conformational structures,depending on the water activity, the basesequence and the presence of specific bind-ing proteins (Osborne and Boubriak, 1994).Determination of the integrity of extractedDNA in embryos of seeds and in wind-dis-persed pollen during the transition fromthe desiccation-tolerant to the sensitivestage showed that only DNA from desicca-tion-tolerant cells retained integrity whencells were subjected to drying regimes. Itwas further proposed that the attainment ofstable secondary structures that are resistantto degradation in vivo at low water poten-tials is a likely accessory to desiccation tol-erance (Osborne and Boubriak, 1994).

Another genetic factor that changes as afunction of hydration is the structure ofchromatin. In the desiccation-tolerantphase of developing and germinating ortho-dox seeds, the chromatin is in a condensedstate. This state appears to be retained dur-ing early rehydration, while seeds are stilldesiccation-tolerant (Leprince et al., 1995a;Pammenter and Berjak, 1999). During ger-mination, the rehydration stage correspond-ing to the resumption of DNA replication isassociated both with chromatin deconden-sation and loss of desiccation tolerance(Deltour, 1985). Orderly, reversible chro-matin compaction and rehydration-inducedredispersion of condensed chromatin thusappear to be associated with desiccationtolerance. Evidence for this comes fromelectron microscope observations on seeds(Crèvecoeur et al., 1976; Deltour, 1985) andresurrection plants (Hallam and Luff, 1980),and from a biophysical characterization ofisolated chromatin from germinating maizeembryos (Leprince et al., 1995a). Electronmicroscopy studies show that irreversiblechromatin compaction accompanies injuri-ous dehydration of desiccation-sensitivematerial. Extracted chromatin from dried,desiccation-sensitive tissues was unable to

undergo reversible changes in condensationstates, indicating that certain protectivesubstances are necessary to confer stabilityto the chromatin during drying in desicca-tion-tolerant tissues (Leprince et al., 1995a).

While the stability of DNA and chro-matin in the dehydrated state must be aprerequisite for desiccation tolerance inseeds and resurrection plants, muchremains to be ascertained about factorscontributing to the genetic stability and theresponses to dehydration in both desicca-tion-tolerant and sensitive material.

10.5.3. Water replacement hypothesis

Studies on model liposome systems com-posed of pure phospholipids (PLs) showthat drying induces a phase transition inthe membranes from the fluid liquid-crys-talline phase to the solid gel phase (Croweet al., 1997a; see Chapter 9). The rise of themembrane-phase transition temperature(Tm) with drying commences with the dis-sipation of the last 10–12 water moleculesper PL molecule, i.e. below 0.2–0.3 gH2O g�1 dw. The removal of water mole-cules from the head groups leads to a reduc-tion in the lateral spacing between the PLmolecules and, consequently, to the forma-tion of a gel phase. Thus, the Tm of modelmembranes composed of phosphatidyl-choline increases by as much as 70°C duringdehydration. A liquid-crystal-to-gel-phasetransition of membrane PLs during dehy-dration has been detected in intact pollen,using FTIR spectroscopy (Crowe et al.,1989a). Similar to the model liposome sys-tems, the average Tm values of membranesin pollen increased with dehydration.Comparison of the Tm value in dried cattailpollen (32°C) with that of dry membranesisolated from this pollen (58°C) indicatedthat the Tm in intact pollen was depressedby 26°C (Hoekstra et al., 1991). Apparently,in the dry, intact organism there is a mech-anism that depresses the dehydration-induced increase of Tm, which is lost whenthe membranes are isolated. Upon addingsucrose to the isolated membranes beforedrying, the Tm of the dried membranes was



again depressed from 58 to 31°C. Themechanism by which this depression isconsidered to work is discussed below.

Experiments on model systems havegiven some insight into the mechanism bywhich sugars depress the Tm of dry mem-branes. It has been established that duringdesiccation soluble sugars interact with thepolar head groups and replace the watermolecules. Phospholipid molecules thuslargely retain the original spacing betweenone another, and the desiccation-inducedincrease in Tm is circumvented (Fig. 10.1).Interaction with sugars can even lead todepression of Tm to values considerablybelow the Tm of the hydrated specimens(Crowe et al., 1996). As the size of the solu-ble carbohydrate molecule increases, thechance of interaction between the phos-phate and the carbohydrate decreases, andthe depression of Tm is less. Thus, it hasbeen demonstrated that hydrogen-bondingbetween the head group PO and sugar OHgroups is pivotal for the depression of Tmin dry membranes (Crowe et al., 1996).

In the presence of trehalose (a commondisaccharide often found in anhydrobioticmicroscopic animals and yeasts (Crowe etal., 1984)) polar compounds trappedinside the liposomes do not leak duringdrying and rehydration (Crowe et al.,1986). Also, other disaccharides (sucrose)and oligosaccharides such as stachyoseand raffinose are effective at retainingcompounds trapped inside the liposomes.It is thought that the leakage does notoccur because a phase change is prevented(Fig. 10.1). In the light of these in vitrostudies, the presence of sucrose andoligosaccharides in desiccation-tolerantorganisms is thought to be similarlyinvolved in the depression of Tm in situand the prevention of leakage.

From air-drying experiments with lipo-somes in the presence of disaccharides, ithas become clear that a mass ratio of 5 : 1,sugar : PL, satisfies the head group hydrogen-bonding capacity (Hoekstra and Golovina,1999). Such mass ratios often occur inanhydrobiotic pollen and seeds, but excep-


Water

Sugars

Hydratedliquid crystal

Desiccation

– sugars

+ sugars

Rehydration

No leakage,no fusion

Hydrated,liquid crystal

Rehydration

Extensiveleakage + fusion

Dry gel

Fig. 10.1. Schematic representation of the phase behaviour of phospholipids in a bilayer as influenced bydesiccation in the presence or absence of a disaccharide. The disaccharide maintains the original spacingbetween the phospholipid molecules and, thus, prevents the transition from the liquid crystalline phase tothe gel phase.


tions with lower mass ratios have beenfound, which are nevertheless desiccation-tolerant (Hoekstra et al., 1997). The effec-tive availability of sugars to PLs in situmight be less than expected, because sug-ars also interact with other constituents ofthe cells and with one another. Thisrestricted availability of the sugars forinteraction with the head groups would bein line with the observation that depres-sion in situ of Tm in dry organisms is oftennot to the low value measured in thehydrated specimens (Hoekstra andGolovina, 1999). It has been suggested that,besides sugars, other molecules might aidin the depression of Tm in desiccation-tol-erant organisms, for example by the trans-fer of fluidizing compounds from thecytoplasm into membranes (see Section10.4).

The question remains as to why post-poning or preventing the rise in Tm withdehydration might be beneficial. The factthat dry pollen and seeds can be safelystored below �20°C (i.e. under conditionsthat make the gel-phase formation particu-larly likely) indicates that the presence ofthe gel phase per se is not detrimental. Aphase change leads to transiently increasedpermeability, and a loss of solutes may beexpected under these conditions. Theextent of this loss will depend on the vis-cosity of the surrounding cytoplasmic envi-ronment. Below 0.8 g H2O g�1 dw, theviscosity increases exponentially andbecomes extremely high when the cyto-plasm enters a glassy state (Leprince andHoekstra, 1998) (see Section 10.5.4). Noloss of solutes is to be expected when thephase change of the membranes occurswhile the cytoplasm is close to reaching aglassy state. However, leakage may resultwhen the phase change occurs when thecytoplasm is in the liquid state. Data oncattail pollen have indicated that, duringdehydration at 20°C, the gel phase and theglassy state are formed simultaneously(Hoekstra and Golovina, 1999). Any upliftof the phase diagram (i.e. the curve repre-senting the relationship between Tm andwater content) by ineffective depression ofthe Tm would result in gel-phase formation

during drying before the formation of aglass, thereby increasing the chance ofsolute loss. Thus, depression of Tm mayprevent a loss of membrane permeabilityduring drying. Furthermore, depression ofthe Tm in dry anhydrobiotes is also impor-tant to reduce the risk of leakage duringrehydration (also called imbibitional leak-age), apart from evading leakage duringdrying (see Chapter 12).

Another role for sugars in relation todesiccation tolerance is their stabilizingeffect on dried proteins (see Chapter 9).Sugars are special in that they allow theremoval of the closely associated waterfrom proteins without this leading to con-formational changes and loss of enzymaticfunction. Above 0.3–0.5 g H2O g�1 dw,other (compatible) solutes can also beeffective (see also Section 10.5.1).Employing phosphofructokinase (PFK), anextremely labile protein when dried orfreeze-dried, Carpenter and co-workershave shown that the disaccharides sucrose,maltose and trehalose are very effective sta-bilizing agents, particularly in the presenceof certain divalent cations (Carpenter et al.,1987a,b, 1990). FTIR spectroscopy studiesindicate that sugars act as a water substi-tute by satisfying the hydrogen-bondingrequirement of polar groups on the surfaceof the dried protein (Carpenter and Crowe,1989; Prestrelski et al., 1993; Wolkers etal., 1998b). This mechanism is analogousto the water replacement hypothesis men-tioned above for membranes, although fixa-tion of the protein’s secondary structurealso occurs via glass formation (see Section10.5.4) (Franks et al., 1991). Thus, proteinunfolding and aggregation during dehydra-tion are prevented. In dry, orthodox seeds,proteins retain their native secondarystructure for decades, long after the seedshave died (Golovina et al., 1997). Recently,Wolkers et al. (1998b) contributed to thequestion of whether glass formation orhydrogen bonding of sugar with protein isinvolved in the stabilization during slowair-drying. The sugars having the besthydrogen-bonding properties give the beststructural protection, but have the lowestglass transition temperature (Tg), which



supports the hydrogen-bonding mechanismof protection.

10.5.4. Vitrification

Upon drying of desiccation-tolerant tis-sues, as the concentration of solutesincreases there is an increase in the viscos-ity of the cytoplasm and a decrease in mol-ecular mobility of molecules. For example,in embryonic tissues below 0.8 g H2O g�1

dw, the cytoplasm becomes increasinglyviscous (Leprince and Hoekstra, 1998;Buitink et al., 2000d). Drying below 0.3 gH2O g�1 dw leads to a decrease in the mol-ecular mobility in the cytoplasm of overfive orders of magnitude (Buitink et al.,1999). Finally, at around 0.1 g H2O g�1 dw,the cytoplasm vitrifies and exists in a so-called glassy state. A glass is defined as anamorphous metastable state that resemblesa solid, brittle material, but retains the dis-order and physical properties of the liquid

state (Franks et al., 1991). A glass is ahighly viscous solid liquid. Its high viscos-ity has been shown to slow down severelymolecular diffusion and decrease the prob-ability of chemical reactions (see Slade andLevine, 1991; Roos, 1995, for reviews).Glass formation has been detected in seeds(Williams and Leopold, 1989; Bruni andLeopold, 1992; Leopold et al., 1994;Leprince and Walters-Vertucci, 1995),pollen (Buitink et al., 1996) and the resur-rection plant C. plantagineum (J. Buitink,unpublished results). Apparently, glass for-mation is a characteristic typical of all des-iccation-tolerant tissues.

The presence of a glassy state is depen-dent on three factors: water content, temper-ature and chemical composition. A decreasein the water content of the tissue results inan increased glass transition temperature(Tg), as demonstrated by a state diagram,which depicts the relationship between thewater content and the Tg (Fig. 10.2). Themagnitude of Tg is also dependent on the


60

30

0

–30

–60

Gla

ss tr

ansi

tion

tem

pera

ture

(�C

)

Fast dryingSlow drying

0.00 0.08 0.16 0.24 0.32

Water content (g H2O g–1 dry weight)

Fig. 10.2. State diagram depicting the relationship between water content and glass transition temperatureof developing bean axes (O. Leprince and C. Walters, unpublished data). Seeds were harvested near massmaturity. After slow drying (i.e. 3 days), isolated axes were found to elongate and grow when cultured invitro on agar. They were considered to be desiccation-tolerant. In contrast, isolated axes failed to elongateafter fast drying (5 h) and were considered to be desiccation-sensitive. The different water contents wereobtained by drying isolated axes for different times. Glass transition temperatures were measured at the mid-point using a differential scanning calorimeter (DSC) during heating at a scanning rate of 10°C min�1

according to Leprince and Walters-Vertucci (1995). Data points represent three to five axes in the DSC pan.


composition of the amorphous state. Forone-component model systems, Tg is knownto vary with Mr in a characteristic and theo-retically predicted fashion (Slade andLevine, 1991). For instance, a sugar of a highMr (like stachyose) exhibits a higher Tg overthe entire range of water contents than asmall Mr sugar such as glucose.

10.5.4.1. Role of vitrification in desiccationtolerance and longevity

Intracellular glasses were, originally, sug-gested to play a role in desiccation tolerance.Considering the glass-forming capability ofsugars, several studies have appearedattempting to link changes in sugar composi-tion during the acquisition of desiccationtolerance with the existence of glasses upondrying (Koster, 1991; Williams and Leopold,1995). For instance, sugars similar to thosefound in desiccation-tolerant seeds (sucrose,raffinose) are capable of forming glasses atambient temperatures, whereas sugar mix-tures similar to those found in axes that donot tolerate desiccation were found only toform glasses at sub-zero temperatures(Koster, 1991). Williams and Leopold(1995) showed that, after 50 h of imbibition,the Tg of desiccation-sensitive pea embry-onic axes was remarkably lower than thatof desiccation-tolerant axes imbibed for 14h, accompanying the loss of oligosaccha-rides and replacement by monosaccha-rides. However, in other studies, glasseshave also been found in desiccation-sensi-tive tissues (Sun et al., 1994; Buitink et al.,1996). For instance, the state diagrams ofdeveloping embryonic axes of bean afterfast and slow drying (which renders themdesiccation-sensitive and -tolerant, respec-tively) were found to be identical (Fig. 10.2).This questions the exclusive role of glassesin desiccation tolerance. It is important torealize that the water content at which glassformation occurs during drying at roomtemperature in seeds (~ 10% moisture; Fig.10.2) is much lower than the critical watercontent most desiccation-sensitive speciesexhibit. Apparently, dehydration-induceddamage in these seeds occurs at water con-tents far above those at which protection of

the glassy state can be effective. Althoughglass formation is not a mechanism thatinitially confers tolerance to desiccationduring drying, its formation is indispens-able for surviving the dry state, as dis-cussed below.

An important consequence of the forma-tion of the glassy state in tissues is theabsence of crystallization (Leopold et al.,1994; Sun and Leopold, 1997). It has beensuggested that loss of viability could bedue to time-dependent crystallization lead-ing to loss of membrane structure and cel-lular integrity (Caffrey et al., 1988; Sun andLeopold, 1993). However, so far there is noexperimental evidence that demonstratescrystallization events in desiccation-sensitive tissues (Sun and Leopold, 1993).It is surmised that the complex cytoplas-mic composition is most probably respon-sible for preventing crystallization(Walters, 1998; Buitink et al., 2000e). Themajor function of intracellular glasses indry seeds may be their contribution to thestability of macromolecular and structuralcomponents during storage.

One of the most studied functions ofglasses is maintenance of the structural andfunctional integrity of macromolecules (seeSlade and Levine, 1991, 1993; Roos, 1995,for reviews). Glasses are known to slowdown detrimental reactions, such as the rateof browning reactions (Karmas et al., 1992),to increase the stability of enzymes (Changet al., 1996) and to prevent conformationalchanges in proteins (Prestrelski et al., 1993).

It has also been shown in model systemsthat glasses are capable of preventing thefusion of membranes. Leakage from lipo-somes can be the result of a phase change(Section 10.5.3), but also because of fusionbetween the liposomes. In the former case,the size of the liposomes remains the same,but, in the latter case, the size increasesconsiderably. Protection of liposomes hasalso been shown to depend on how effec-tively sugars can form glasses (Crowe et al.,1998). In this respect, monosaccharides arepoor protectants, despite their generallyexcellent capability to interact with thepolar head groups (Section 10.5.3).Monosaccharides differ from the di- and



oligosaccharides in having low Tgs (belowroom temperature). This means that duringdrying the liposomes remain in a liquidenvironment, which results in fusion-induced release of the entrapped contents.The importance of both the ability to formglasses at ambient temperature and directinteraction with the polar head groups toprotect membranes (see Section 10.5.3) hasbeen demonstrated by drying liposomes inthe presence of two different compounds,hydroxy-ethyl starch and glucose (Crowe etal., 1997b). Hydroxy-ethyl starch has ahigh Tg, thus forming a glass at ambienttemperature, but it does not depress Tmbecause it is too large to fit between thehead groups to efficiently interact. Glucose,by contrast, depresses Tm in dry lipids butwill not easily form a glass at ambient tem-perature. Thus, hydroxy-ethyl starch pre-vents the leakage associated with fusion(below 1.5 g H2O g�1 dw), but cannot pre-vent the leakage associated with the phasetransition (below 0.25 g H2O g�1 dw).Together, however, hydroxy-ethyl starchand glucose protect the dry liposomes, butneither compound is effective alone. Bothproperties – timely glass formation duringdrying and interaction with the polar headgroups – appear to be required for preser-vation. Disaccharides combine these twoessential properties within one compound.

The effect of glasses on the stability ofmacromolecular and structural componentsduring storage has led to the concept thatglasses play an essential role in thelongevity of seeds and pollen. For example,the storage stability of Arabidopsis thalianaseeds has been found to correlate with themolecular density of the cellular matrix(Wolkers et al., 1998a). Sun and Leopold(1994) have found that seed deteriorationappears to be accelerated when seeds arenot in the glassy state, as estimated throughthe viability equation of Ellis and Roberts(1980). Probably the most compelling evi-dence to suggest that ageing rates areaffected by the viscosity of the intracellularglass has come from the linear relationshipbetween ageing rate and cytoplasmic mole-cular mobility found for many different tis-sues over a wide range of temperatures and

water contents (Buitink et al., 2000c). Allthese arguments suggest that ageing ratesand, consequently, life span of germplasmare influenced by the molecular stability ofthe cytoplasm, signifying the pivotal func-tion of intracellular glasses in conferringsuch stability during storage.

Recently, the consequences of being inor near the glassy state have gained furtherphysiological significance. A study on themolecular mobility in desiccation-toleranttissues indicated that the special composi-tion of biological glasses might have a rolein survival (Buitink et al., 2000e). Based onsaturation transfer-electron paramagneticresonance (ST-EPR) spectroscopy measure-ments, Buitink et al. (2000e) observed a sec-ond kinetic change in mobility at a definitetemperature above Tg, referred to as the crit-ical temperature (Tc). The occurrence of Tchas been coupled to the collapse tempera-ture of sugar glasses (Tg + 15°C; Fig. 10.3), awell-documented phenomenon, which isattributed to a reduction in viscosity suchthat a flow on a practical time scale isobserved. Although the viscosity decreasesaround Tg, it is not until the temperature Tcis reached that the viscosity abruptly drops(see � Fig. 10.3). This is contrary to thebelief that the viscosity decreases abruptlyat Tg, as is often assumed. The Tc in desic-cation-tolerant organisms occurs at temper-atures as high as 55°C above Tg (Fig. 10.3).A high Tc implies high stability as a resultof high viscosity (> 108 Pa s) far above Tg.This high Tc in biological tissues hasimportant implications for the survival ofgermplasm in its natural environment.Under ambient conditions, for example20°C and 50% relative humidity (RH), seedtissues are around their Tg. This means thatany environmental fluctuation that resultsin an increase in RH or temperature willbring the tissue above its Tg. However, theunique properties of the intracellular glassprotect the tissue from dramatic changescaused by environmental fluctuations. If theintracellular glass were composed ofsucrose alone, a small increase in RH ortemperature would bring the sucrose glassabove its Tc (Fig. 10.3), resulting in crystal-lization and loss of macromolecule function




and integrity (see Roos, 1995, for review).Therefore, the characteristically high Tc ofintracellular glasses serves as an ecologicaland physiological advantage.

10.5.4.2. Composition of glasses indesiccation-tolerant organisms

When the concept of glasses was intro-duced in seed science, sugars were thoughtto play an important role in the composi-tion of the glass. This assumption wasbased on the fact that sugars are present inlarge amounts in desiccation-tolerant tissues and are known to be excellent glass-formers. The correlation between oligosac-charides and longevity (Horbowicz andObendorf, 1994; Bernal-Lugo and Leopold,1995; Steadman et al., 1996; Sun andLeopold, 1997) and the knowledge thatoligosaccharides increase Tg in model sys-tems (Slade and Levine, 1991; Roos, 1995)added to the notion that sugars are impor-tant for in vivo glass formation.

However, several reports do not supportthe contention that sugars play a dominant

role in intracellular glass formation. Sunand Leopold (1993) found that the magni-tude of the glassy signal and Tg, both mea-sured by the thermally stimulateddepolarization current (TSDC) method,decreased during accelerated ageing of soy-bean seeds. Yet no differences wereobserved in sucrose, raffinose and stachyosecontents during the same period of time.Despite different sugar compositions in soy-bean axes compared with oak cotyledons,their state diagrams are similar (Sun et al.,1994). Also, the state diagrams of immatureand mature soybean axes are similar,despite the accumulation of oligosaccha-rides during maturation. Another indicationthat sugars alone are not sufficient toexplain the formation of the vitreous state inseeds came from Sun and Leopold (1997),who showed that the state diagram of maizeembryos is different from that of a represen-tative carbohydrate mix. An extensivecalorimetric study on the glass transition inbean axes revealed the complexity of intra-cellular glasses. Correspondence of differ-ential scanning calorimetry (DSC) data

Fast

Slow

Mol

ecul

ar m

obili

ty

Sucrose

Poly-L-lysine

Bean embryonic axis

–20 0 20 40 60 80 100 120

T – Tg (�C)

Fig. 10.3. The effect of melting the glassy state on the molecular mobility of a guest molecule (spin probe)incorporated in dry glasses composed of sucrose or poly-L-lysine, and in the intracellular glass of driedembryonic axes of bean. Molecular mobility was measured by electron paramagnetic resonancespectroscopy (Buitink et al., 2000e). The � indicates the critical temperature, Tc, where the dynamics of thesystem changed from solid-like to liquid-like (signified by an abrupt drop in viscosity).


from beans with a model that predicts theeffects of glass components on Tg has sug-gested that intracellular glasses could becomposed of a highly complex oligomericsugar matrix, such as, for instance, malto-dextrin (Leprince and Walters-Vertucci,1995). Buitink et al. (2000a,b) observedthat a change in sugar composition uponpriming did not change Tg or the molecularmobility in the intracellular glass. All thesedata suggest that, besides sugars, othermolecules play a crucial role in intracellu-lar glass formation.

In this respect, the role of proteins inintracellular glass formation has receivedrecent attention. Wolkers et al. (1998a,b)have found that the molecular density (i.e.hydrogen-bonding strength) of dry seeds ofA. thaliana was quite different from that ofa sugar glass, but much more comparable tothat of a protein–sugar glass. Investigationson the glass properties in biological sys-tems using EPR spectroscopy also point toa role for proteins in intracellular glass for-mation (Buitink et al., 2000e). The temper-ature dependence of molecular mobility inintracellular glasses is much more compa-rable to that in protein glasses than that insugar glasses (see Fig. 10.3). Although moreresearch is needed to elucidate what typesof protein may play a role in the glass for-mation, LEA proteins are possible candi-dates (Sun and Leopold, 1997). Wolkers etal. (1999) suggested that LEA proteins thatare embedded in the glassy cellular matrixconfer stability on slowly dried carrotsomatic embryos. Indeed, LEA proteinschange the hydrogen-bonding properties ofmodel sugar systems comparable to thoseof intracellular glasses, pointing to a possi-ble participation of LEA proteins in intra-cellular glass formation (Wolkers et al.,2000).

10.6. Roles of Specific Compounds inStability

A specific feature of all anhydrobioticorganisms is the accumulation of non-reducing sugars, particularly of the raffi-nose series (Koster and Leopold, 1988;

Leprince et al., 1990a; Blackman et al.,1992) and/or galactosyl cyclitols(Horbowicz and Obendorf, 1994; Obendorf,1997). Also accumulating during the acqui-sition of desiccation tolerance are differentmembers of the LEA protein family (Ingramand Bartels, 1996). The current under-standing is that these molecules seem toact as stabilizing agents through one ormore different mechanisms. Furthermore, arole for HSPs in desiccation tolerance hasrecently been put forward (Wehmeyer andVierling, 2000). The (putative) functions ofthese protective molecules will be dis-cussed in the following sections.

10.6.1. Sucrose/oligosaccharides

The roles of sucrose and trehalose in pref-erential exclusion, water replacement andvitrification have been implicated as dis-cussed above. However, the general abun-dance of oligosaccharides in anhydrobiotichigher plant systems raises the question asto why they too are preferentially accumu-lated (see Chapters 1 and 5).

The accumulation of oligosaccharidesand cyclitols during seed maturation andtheir disappearance during germinationhas led to the hypothesis that these sugarsare important for desiccation tolerance.However, it has been shown that desicca-tion tolerance in seeds can occur in theabsence of oligosaccharides (Hoekstra etal., 1994; Lin and Huang, 1994; Bochicchioet al., 1997; Black et al., 1999; Buitink etal., 2000a). Moreover, the accumulation ofoligosaccharides does not necessarily leadto the establishment of desiccation toler-ance (Still et al., 1994; Black et al., 1999).Although oligosaccharides do not appear tobe pivotal for desiccation tolerance, thereseems to be a significant correlationbetween the oligosaccharide:sucrose ratioand storage longevity of dry seeds(Horbowicz and Obendorf, 1994; Steadmanet al., 1996; Sun and Leopold, 1997). Forexample, Horbowicz and Obendorf (1994)calculated that orthodox seeds of specieswith a sucrose:oligosaccharide ratio of < 1.0 have storability half-viability periods



of > 10 years, whereas those with a ratio > 1.0 have storability half-viability periodsof < 10 years. None the less, there areexceptions where there is no relationbetween the oligosaccharide content andlongevity (Steadman et al., 1996; Buitink etal., 2000a), indicating that molecules otherthan oligosaccharides might have a similarfunction and take over the role of thesesugars in their absence.

How oligosaccharides mediate theincrease in longevity is not yet resolved.Because of their intrinsic high Tg it hasbeen suggested that they play a role in theprotection of cytoplasmic components dur-ing storage by elevating the Tg of the intra-cellular glass, thereby increasing viscosity(see Section 10.5.4). Yet there is no evidencethat oligosaccharides have an effect on theTg or viscosity of intracellular glasses(Buitink et al., 2000a,b). Considering thatoligosaccharides often make up only 4% ofthe dry weight in seeds like Impatiens orbell pepper (Buitink et al., 2000a), it is per-haps unsurprising that no effect on intra-cellular glass properties could bemeasured. An obvious role for sugars inseeds is the protection of macromolecularstructures in the dry state, especially mem-branes. Hydrogen bonding of sugar mole-cules with macromolecules will increasetheir stability (see Crowe et al., 1998, for areview; see Section 10.5.3). However, thisexplanation does not provide a clue to therole of oligosaccharides in particular. Infact, there are indications that oligosaccha-rides are less effective than disaccharidesat hydrogen bonding with the polar headgroups, particularly in the case of saturatedphospholipids (Crowe et al., 1986, 1996).Also, oligosaccharides in seeds have beensuggested to be able to prevent crystalliza-tion. Notwithstanding the argument thatoligosaccharides are indeed capable of pre-venting crystallization in model sugarglasses (Caffrey et al., 1988), this character-istic would most probably not be requiredin vivo because of the complex mixture ofall the different compounds in the cyto-plasm, as mentioned before (see Section10.5.4). Because the maturation stage ofseeds correlates with storage stability, and

oligosaccharides accumulate during matu-ration, it could be possible that oligosac-charides are linked indirectly with storagestability, being merely an indicator of seedmaturity. Future research will have tofocus on roles for oligosaccharides in seedmaturation other than cellular protection.

10.6.2. Late embryogenesis abundant (LEA) proteins

Generally, the presence of LEA proteinscorrelates well with desiccation tolerance(see Chapters 1, 5, 11). They accumulateduring late maturation of developing seeds(Galau et al., 1986; Bartels et al., 1988;Baker et al., 1995; Blackman et al., 1995;Ingram and Bartels, 1996; Kermode, 1997;Oliver and Bewley, 1997; Cuming, 1999),in dehydrating vegetative tissues of thedesiccation-tolerant grass Sporobolus stap-fianus (Kuang et al., 1995), and in the res-urrection plant C. plantagineum(Piatkowski et al., 1990). However, leatranscripts have also been detected inrecalcitrant seeds and in desiccation-sensi-tive tissues submitted to water and/or tem-perature stress (Kermode, 1997). LEAproteins represent a broad class of highlyconserved genes expressed in a wide rangeof plants. Comparisons of the deducedpolypeptide sequences of the various leagenes have led to the establishment of fivesubclasses of LEA proteins, simply desig-nated Group 1, Group 2 (dehydrins), Group3, Group 4 and Group 5 LEA proteins (seeCuming, 1999, for a review, and Chapters1, 5 and 11). Spatial patterns of LEA pro-tein accumulation indicate that they areprimarily localized in the cytosol andnucleus (Asghar et al., 1994; Goday et al.,1994; Blackman et al., 1995; Egerton-Warburton et al., 1997). In the leaves of theresurrection plant C. plantagineum, LEAproteins were found to be both cytosolicand present in the chloroplasts of theleaves (Schneider et al., 1993).Furthermore, the presence of LEA proteinshas been detected at the membranes of pro-tein and lipid bodies of Zea mays kernels(Egerton-Warburton et al., 1997) and at the



plasmalemma of Saccharomyces cerevisiae(Sales et al., 2000). Whereas the moleculargenetics of LEAs will be discussed else-where (Chapter 11), this section will belimited to the hypothetical mechanisms ofhow LEA proteins act in stabilization andprotection during desiccation.

The strongest support for a primary des-iccation-protecting role comes from theobservation that the accumulation of theproteins coincides with the acquisition ofdesiccation tolerance. Bartels et al. (1988)demonstrated that desiccation tolerancecould be induced precociously in imma-ture barley embryos by the application ofabscisic acid (ABA), coinciding with theaccumulation of LEA proteins. Similarly,Blackman et al. (1995) used exogenousABA to elevate the level of heat-solubleLEA-like proteins in axes from immatureseeds of soybean. As the LEA-like proteinsaccumulated in response to ABA, soluteleakage from the dried soybean embryosupon rehydration markedly declined. Bothfactors were apparently dependent on thepresence of ABA. These data are consistentwith the hypothesis that the LEA-like pro-teins contribute to the increase in desicca-tion tolerance in response to ABA.

Since the earliest identification of LEAproteins, hypotheses regarding their func-tions in desiccation tolerance haverevolved around physiological and bio-chemical experiments and predictions onthe structure–function relationship thatcan be deduced from the amino acidsequence. The fact that LEA proteins accu-mulate to a much higher cellular concen-tration than is typically the case forenzymes, together with their predictedstructural flexibility (of Group 2 and 3 LEAproteins), appears to rule out an enzymaticrole. For the Group 1 LEA proteins, it hasbeen calculated that these polypeptideshave a tremendously high potential forhydration, several times greater than thatfor ‘normal’ cellular proteins (McCubbin etal., 1985). This is a function of the remark-ably high number of charged anduncharged polar residues within the struc-ture. Because of these specific properties,LEA proteins potentially bind to intracellu-

lar macromolecules, coating these macro-molecules with a cohesive water layer andpreventing their coagulation during desic-cation (Section 10.5.1; Close, 1996). Uponremoval of their own hydration shell, theseproteins would still be capable of playing arole in stabilizing macromolecular struc-tures, as they could provide a layer of theirown hydroxylated residues to interact withthe surface groups of other proteins, actingas ‘replacement water’ (see Section 10.5.3;Cuming, 1999). It has been shown thatpurified maize dehydrin has potent cryo-protective activity in vitro in a rabbit lac-tate dehydrogenase freeze–thaw assay,especially in combination with solutesincluding compatible solutes such assucrose, proline and glycine-betaine (Close,1996). Rinne et al. (1999) suggested that, incold-acclimatized apices of birch, dehy-drins might create local pools of water inotherwise dehydrated cells, thereby main-taining enzyme function. Under conditionsof low water activity (20% polyethyleneglycol (PEG), corresponding to approxi-mately –0.5 MPa), the activity of �-amylasewas greater in the presence of a partiallypurified dehydrin fraction than in the pres-ence of bovine serum albumin (BSA) as acontrol (Rinne et al., 1999).

For the Group 2 and 3 LEA proteins,hypotheses concerning their biologicalfunction are based on their potential foramphipathic helix formation. It has beensuggested that the amphipathic helices ofthe Group 3 LEA proteins could form bun-dles through hydrophobic interactions,thereby exposing a highly charged surfaceto the exterior to which ions can bind(Dure, 1993). Thus, sequestration of ionscan take place, whose intracellular concen-trations might otherwise become unaccept-ably high within the dehydrating cell.

The nuclear localization of dehydrinsraises the possibility of a dehydrin–chromatin alliance or an association withthe nuclear matrix, since the matrix is thechromatin-organizing structure (Nickersonet al., 1989). In this regard, it is interestingto note that many dehydrins contain a tractof serine residues (the S segment). In maizeRAB17 and tomato TAS14, it has been



demonstrated that the serine residues inthe S segment can be phosphorylated, andit has been proposed that phosphorylationis related to the binding of nuclear localiza-tion signal peptides and, therefore, tonuclear transport (Goday et al., 1994;Godoy et al., 1994). Because the matrix isassociated with regulatory processes, dehy-drins may protect genetically sensitiveareas. As in the nucleus, nucleolar dehy-drins may have a structural role via anassociation with the nucleolar filamentmatrix, or a functional role in protectingtranscriptionally active regions (Godoy etal., 1994).

It has also been speculated that LEAproteins might play a role in glass forma-tion (Sun and Leopold, 1997; Wolkers etal., 1999; Buitink et al., 2000e). Wolkers etal. (1999) suggested that LEA proteins thatare embedded in the glassy matrix mightconfer stability on slowly dried carrotsomatic embryos. Indeed, LEA proteinschanged the hydrogen-bonding propertiesof model sugar systems toward those ofintracellular glasses, pointing to a possibleparticipation of LEA proteins in intracellu-lar glass formation (Wolkers et al., 2000;see Section 10.5.4).

Despite numerous studies on the geneexpression of LEA proteins in plants, atpresent, their biological function remainsto be assessed in vivo. None the less, itappears that, based on their observed local-ization at a number of sites, coupled withtheir accumulation in response to desicca-tion stress, dehydrins have a general role inprotection during drought and/or desicca-tion stress. Recently, Black et al. (1999)found that, in wheat embryos, dehydrinaccumulation is not regulated by factorsthat specifically control the induction oftolerance. Instead, it would appear that thedehydrin-like protein is produced inresponse to grain detachment, even whenthis is not followed by dehydration, as wasalso concluded for soybean (Blackman etal., 1991). Remarkably, recalcitrant seedswere also found to accumulate dehydrinsduring seed development and in responseto dehydration or to ABA treatment(Bradford and Chandler, 1992; Finch-

Savage et al., 1994). It seems that the soleability, or lack thereof, to express LEAs ordehydrin-like proteins cannot be taken asan indication that the seeds of a particularspecies can or cannot withstand dehydra-tion, reflecting the earlier views ofBlackman et al. (1991, 1992) and Leprinceet al. (1993) that desiccation tolerancemust be the outcome of the interplay ofmore than one (and probably many) mech-anisms or processes.

10.6.3. Heat-shock proteins (HSPs)

Another class of proteins that have recentlybeen associated with desiccation toleranceare the HSPs (see also Chapters 1 and 5). Incontrast to those of other eukaryotes, themost prominent HSPs of plants are smallheat-shock proteins (sHSPs). They havemonomeric molecular masses of 15–42kDa, but assemble into oligomers of nine toover 20 subunits, depending on the protein(Waters et al., 1996, and referencestherein). Their implication in desiccationtolerance has been inferred from studies ongene expression in developing seeds and inresurrection plants. In pea, Arabidopsisand sunflower seeds, sHSP expression isalways observed significantly before dis-cernible seed desiccation, and sHSPs areabundant in the dry seeds (Coca et al.,1994; DeRocher and Vierling, 1994;Wehmeyer et al., 1996). During germina-tion, the developmentally regulated sHSPsare relatively abundant for the first fewdays and then decline quickly. However,the precise timing corresponding to theacquisition and loss of desiccation toler-ance was not assessed in the above studies.In vegetative tissues of C. plantagineum,constitutive expression of sHSPs has beendetected (Alamillo et al., 1995).

Further evidence that HSPs may be impli-cated in desiccation tolerance comes fromthe observation that there appears to be acoordinated expression of lea and sHSP tran-scripts during embryo development inresponse to ABA, indicating the existence ofcommon regulatory elements of both LEAproteins, sHSPs and desiccation tolerance



(Almoguera and Jordano, 1992; Wehmeyer etal., 1996). Also, in desiccation-sensitive cal-lus tissue of Craterostigma, no sHSP-relatedpolypeptides could be detected, but sHSPexpression and the concurrent acquisition ofdesiccation tolerance in the callus were initi-ated by exogenous ABA treatment (Alamilloet al., 1995). Recently, a reporter-gene tran-scription assay showed that sHSP expressionexhibits little tissue specificity, but insteadspreads throughout the embryo duringdevelopment until essentially all cells arestained in the mature seeds prior to com-plete desiccation (Wehmeyer and Vierling,2000). The activity of the reporter gene wasstrongly reduced in fus3-3, lec1-2 and wasalmost nil in abi3-6, all desiccation-intolerant mutants of Arabidopsis seeds.This would suggest an overall protectiveeffect of HSPs in the cells during drying. Asfor all protective mechanisms reviewed sofar, sHSPs may be necessary for desiccationtolerance, but they are unlikely to be suffi-cient.

In contrast to other seeds, which typi-cally accumulate low to moderate levels ofsHSPs, recalcitrant chestnut (Castaneasativa) seed cotyledons contain a highlyabundant sHSP (Collada et al., 1997). ThissHSP was shown to exhibit molecularchaperone activity in vitro, as demon-strated by the ability of the sHSP to main-tain soluble cytosolic proteins in theirnative formation during both heat and coldstress (Soto et al., 1999). A model liposomesystem with the encapsulated fluorescentdye calcein was used to investigate the pro-tection of membranes by the LEA-like pro-tein HSP 12 from S. cerevisiae duringdesiccation (Sales et al., 2000). This LEA-like HSP was found to act in an analogousmanner to trehalose and protect liposomalmembrane integrity against desiccation.The interaction between HSP 12 and theliposomal membrane was judged to beelectrostatic, as membrane protection wasonly observed with positively charged lipo-somes and not with either neutral or nega-tively charged liposomes.

So far, there is no direct experimentalevidence that points to a specific role ofsHSPs in desiccation tolerance. Small

HSPs are thought to offer a general protec-tive role in dry anhydrobiotes, based on theobservation that HSPs are molecular chap-erones (i.e. they interact with other pro-teins and, in doing so, minimize theprobability that these other proteins willinteract inappropriately with one another(Waters et al., 1996; Gething, 1997; Federand Hofmann, 1999)).

The function and working mechanismsof HSPs are well investigated in mam-malian systems, although direct evidenceoriginates only from in vitro studies (seeFeder and Hofmann, 1999, for a review). Itis currently known that HSPs recognizeand bind to other proteins when these pro-teins are in non-native conformations,whether because of protein-denaturingstress or because the peptides they com-prise have not yet been fully synthesized,folded, assembled or localized to an appro-priate cellular compartment. Bindingand/or release of these other proteins isoften regulated by association with and/orhydrolysis of nucleotides. Typically, HSPsfunction as oligomers, or as complexes ofseveral different chaperones, co-chaper-ones, and/or nucleotide exchange factors.Interaction with chaperones is variouslyresponsible for: (i) maintaining HSP part-ner proteins in a folding-competent, foldedor unfolded state; (ii) organellar localiza-tion, import and/or export; (iii) minimizingthe aggregation of non-native proteins; and(iv) targeting non-native or aggregated pro-teins for degradation and removal from thecell. Presumably, the last two functions arethe most important in coping with environ-mental stress (Feder and Hofmann, 1999).

10.7. Conclusion and Outlook

From the above evidence amassed so far it isclear that more than one mechanism acts inconferring desiccation tolerance on plantorganisms. Often, the involvement of a spe-cific substance in desiccation tolerance isdifficult to establish, because different sub-stances may substitute for one another.Decreasing water potential appears to neces-sitate successive mechanisms of protection



during drying. In the early stages of dehy-dration, partitioning of amphiphiles intomembranes might cause signalling of thestress, leading to a variety of responses.Among these responses are the productionof antioxidants, compatible solutes, dehy-dration proteins and, probably, sHSPs.Whereas in seeds these substances are pro-duced as a part of the developmental pro-gramme, in vegetative plants that aresensitive to complete dehydration theseresponses often occur upon exposure tomoderate levels of water loss (= drought).Since these substances appear to improvetolerance to drought, it can be implied thatthe mechanisms involved give protectionunder conditions where there is still bulkwater left. Among these mechanisms couldbe depression of metabolism, improved free-radical scavenging and preferential hydra-tion of macromolecules. Oxidative damage,membrane fusion and protein denaturationare thus prevented. It has to be realized thatit is often in these high water contentranges, i.e. when bulk water is present, thatdesiccation-sensitive organisms die.

When most of the bulk water has disap-peared, the interaction between moleculesincreases, which renders mechanisms such

as preferential hydration and enzymaticantioxidant activity ineffective. Then,immobilization in a glassy matrix viahydrogen bonding with water-replacingsubstances gains importance. This preventsexcessive ordering, e.g. crystallization, andprotects the structure of macromolecules.There is an important role for sugars in thishydrogen-bonding process – from interac-tion with proteins to interaction with othersugar molecules in the formation of aglassy matrix. Some of the dehydrationproteins may help improve the stability ofthe glassy matrix. Glasses are considered asparticularly important in slowing molecu-lar mobility and chemical reaction rates.Consequently, they are important indepressing the rate of ageing in the drystate.

The majority of investigations concern-ing anhydrobiosis in plants have beenfocused on phenomena in the dry state.Considering the fact that desiccation-sensi-tive organisms usually die at relativelyhigh water contents of, for example, 2–0.5g H2O g�1 dw, future research should beaddressed more towards mechanisms ofprotection that operate in this particularrange of water contents.


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11 Molecular Genetics of Desiccation andTolerant Systems

Jonathan R. Phillips,1 Melvin J. Oliver2 and Dorothea Bartels31Max-Planck-Institute for Plant Breeding Research, Carl-von-Linné-Weg 10,

D-550829 Köln, Germany; 2USDA-ARS Plant Stress and Germplasm Development Unit, 3810 4th Street, Lubbock, Texas 79415, USA; 3Institute of

Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany

11.1. Definition of Desiccation Tolerance 32011.2. Resurrection Plants: Definition and Distribution 32011.3. Metabolic Changes During the Dehydration–Rehydration Cycle in

Resurrection Plants 32111.4. Molecular Studies with Resurrection Plants 321

11.4.1. LEA proteins 32311.4.2. Carbohydrate metabolism 324

11.5. Regulation of Gene Expression During the Desiccation Process in Resurrection Plants 324

11.6. Desiccation-tolerant Bryophytes 32611.7. Constitutive Cellular Protection 32711.8. Cellular Damage and Recovery Following Rehydration 32811.9. Gene Expression During Recovery 329

11.9.1. Rehydrins 33011.10. Transgenic Approaches towards Improving Plant Dehydration/

Desiccation Tolerance 33011.10.1. Compatible solutes or osmolytes 33111.10.2. Oxygen-scavenging proteins 33311.10.3. LEA proteins 33411.10.4. Regulatory genes 334

11.11. Conclusions and Perspectives 33511.12. Acknowledgements 33611.13. References 336



11.1. Definition of DesiccationTolerance

Desiccation is the drying out of an organ-ism that is exposed to air. Under these con-ditions, most of the protoplasmic water islost and a very low amount of tightlybound water remains in the cell.Desiccation tolerance apparently dependson the ability of the cells to maintain theintegrity of the cell membranes and to pre-vent denaturation of proteins. Tolerance inorgans such as seeds and pollen is wide-spread among higher plants and in fact par-tial desiccation is a prerequisite for thecompletion of the life cycle in most speciesproducing seeds. In contrast, only a fewplants possess mature foliage or vegetativetissue that is desiccation-tolerant. Theseinclude a small group of angiosperms,termed resurrection plants (Gaff, 1971),some ferns and fern allies (Bewley andKrochko, 1982), and several species ofalgae, lichens and bryophytes (Oliver,1996; Oliver and Bewley, 1997; Oliver etal., 2000; see Chapters 1 and 7).

Desiccation has to be distinguished froma mild water deficit, which is a conditionwhere the water status of plants undergoesrelatively small changes (Bray, 1997). Manyplants are able to cope with this challengeeither by reducing water flux through theplant or by increasing their water uptake.Water loss can be avoided by variousmechanisms such as stomatal closure,reduction of leaf growth or production ofspecialized leaf surfaces to avoid transpira-tion, whereas water uptake can only beincreased by the development of specializedroot structures. Many of these mechanismsare common to both desiccation-tolerantand non-tolerant plants; however, little isknown about those mechanisms that, forexample, allow a resurrection plant to sur-vive equilibrium with 0% air humidity.

Desiccation tolerance has been studied atthe molecular level by examining tolerantsystems such as seeds, resurrection plantsand mosses. As a result of these studies, ithas become apparent that tightly regulatedprogrammes of gene expression, both at thespatial and temporal levels, occur in vegeta-

tive tissues during drying. By using differen-tial screening approaches, cDNAs corre-sponding to transcripts expressed only inresponse to water deficit have been isolatedand characterized (Ingram and Bartels,1996; Bockel et al., 1998). However, itshould be emphasized that, due to thepurely descriptive nature of the data, thefunctional role of many gene products isunclear. Using examples from resurrectionplants and the moss Tortula ruralis, thischapter will review what is currentlyknown about the molecular responses thatoccur during the acquisition of desiccationtolerance and how this knowledge may beapplied to improve plant tolerance to limit-ing water conditions.

11.2. Resurrection Plants: Definitionand Distribution

Desiccation tolerance in vegetative tissues ofhigher plants has been most studied in theso-called resurrection plants, which possessthe unique ability to revive from an air-driedstate (Gaff, 1971). Such plants are oftenpoikilohydrous and their water content cor-relates with fluctuations in the relativehumidity (RH) of the local environment.Broadly two types of desiccation-tolerantplants have been reported: those that losechlorophyll during dehydration and thosethat retain chlorophyll (see Chapters 1and 7).

Resurrection plants colonize ecologicalniches with restricted seasonal water avail-ability. Most species are found preferen-tially on rock outcrops at low to moderateelevations below 2000 m in tropical andsubtropical zones and to a lesser extent intemperate climates (Porembski andBarthlott, 2000). The geographic locationswhere resurrection plants have been identi-fied are southern Africa (includingMadagascar), Australia, India and parts ofSouth America (Gaff, 1977, 1987; Gaff andBole, 1986). Although rarely found inEurope, a few species have been observedin the western Balkan mountains (Stefanovet al., 1992).

Desiccation-tolerant plants comprise

320 J.R. Phillips et al.


monocotyledonous and dicotyledonousspecies within the angiosperms. To date,however, no desiccation-tolerant plantshave been reported that belong to the gym-nosperms. The desiccation-tolerantdicotyledonous species appear to be repre-sented mainly in the Gesneriaceae,Myrothamnaceae and Scrophulariaceaeplant families, in contrast to their mono-cotyledonous counterparts, which are morewidely distributed throughout evolution.Many of the described dicotyledonous res-urrection plants belong to theScrophulariaceae, which include tenCraterostigma and 15 Lindernia speciesthat are indigenous to Africa (Fischer,1992) (see Chapter 7).

The acquisition of desiccation tolerancein resurrection plants is complex. This isdue to the multiple stresses that areimposed on plant tissues during severedehydration. Consequently, tolerant plantsmust overcome several problems, includingminimization of mechanical damage asso-ciated with turgor loss, maintenance of thefunctional integrity of macromoleculessuch as proteins and nucleic acids, mini-mization of toxin accumulation and free-radical damage, and initiation of repairmechanisms upon rehydration. The speedof water loss and the events before dehy-dration appear to be critical for the sur-vival, such that, if the speed of dehydrationis too fast, plants do not acquire toleranceto desiccation. This observation suggeststhat the acquisition of desiccation toler-ance is an active process and requires spe-cific biochemical changes and thesynthesis of desiccation-related molecules.The nature of these molecules has recentlybeen described for some species by molec-ular and biochemical studies.

11.3. Metabolic Changes During theDehydration–Rehydration Cycle in

Resurrection Plants

Physiological, morphological, biochemicaland molecular studies have been per-formed with several resurrection plants.This information, largely derived from

physiological and morphological studies,has been recently reviewed in some detail(Oliver and Bewley, 1997; Hartung et al.,1998; Scott, 2000). The emphasis in thischapter will be on summarizing the resultsof biochemical and molecular studies.

11.4. Molecular Studies withResurrection Plants

To date, molecular studies of resurrectionplants are limited to several species: thedicotyledonous species Craterostigmaplantagineum (Bartels et al., 1990; Frank etal., 2000), the monocotyledonous speciesSporobulus stapfianus (Neale et al., 2000)and Xerophyta villosa (Mundree et al.,2000), and the moss T. ruralis (Wood et al.,1999). In C. plantagineum and S. stapfi-anus the majority of changes in geneexpression occur during dehydration, notduring the rehydration phase of the resur-rection process, which in turn leads to avery efficient protection system againstdesiccation. This contrasts with what isobserved for T. ruralis and may be linked todiffering mechanisms of desiccation toler-ance that exist in vascular higher plantsversus bryophytes.

Dehydration induces the expression of alarge number of transcripts in both C. plan-tagineum and S. stapfianus (see Table11.1). Homology analyses reveal a broadspectrum of differentially regulated geneswith diverse putative functional identities,which underlines the fact that desiccationtolerance is the result of a complex interac-tion of different cellular processes. It hasbeen suggested that the dehydration-induced gene products can be associatedwith signal transduction pathways and reg-ulation of stress-specific transcription, withcarbohydrate metabolism or with cellularprotection (Phillips and Bartels, 2000).Comparison between the dehydration-induced genes identified in C. plan-tagineum and S. stapfianus has revealedthat several genes encode homologous pro-teins and thus belong to the same func-tional group. These include gene productswith a putative protective function such as

Molecular Genetics of Desiccation and Tolerant Systems 321



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late embryogenesis abundant (LEA) pro-teins, the thylakoid membrane-associatedearly light-induced protein (ELIP) or �tonoplast intrinsic protein (TIP). Homologyalso extends to cDNAs isolated from T.ruralis (Wood et al., 1999). Expression pat-terns of the isolated genes have beendescribed at the mRNA level. Three basicpatterns of gene expression are observed:class 1 transcripts accumulate to high lev-els from an initial low level during dehy-dration and disappear during rehydration;class 2 transcripts accumulate transientlyduring low-level dehydration; class 3 tran-scripts are down-regulated during dehydra-tion. Most of the class 1 transcripts andsome of the class 2 transcripts also accu-mulate following the application of exoge-nous abscisic acid (ABA), which indicatesa central role for ABA in mediating geneexpression during dehydration.

It has been hypothesized that the tempo-ral and spatial expression patterns wouldprovide information concerning the func-tion of a gene. Data on spatial expressionpatterns and subcellular localization asinvestigated by RNA in situ hybridizationand cell fractionation are only availablefrom C. plantagineum. The analysesrevealed that RNAs and proteins show aspecific tissue and cellular distribution(Schneider et al., 1993). LEA-like proteins,which may have a general protective func-tion, are found in most tissues and celltypes, and accumulate in the cytoplasm orin chloroplasts. Water channel-like pro-teins, which are predicted to be involvedin water flux or transport of small mole-cules, are associated with membranes(Mariaux et al., 1998). Transcripts encodingsucrose synthase are only detected in theexternal phloem of vascular bundles, sug-gesting an association with transportprocesses within the plant (Kleines et al.,1999).

11.4.1. LEA proteins

LEA proteins represent one major group ofproteins that are reported to be expressedin response to dehydration in desiccation-

tolerant and also in desiccation-sensitiveplants (Close, 1997; Bartels, 1999; Cuming,1999; see Chapters 1, 5 and 10). LEA pro-teins comprise a large family of plant pro-teins that accumulate to high levels duringlate stages of embryo development (Galauet al., 1986). Expression studies show thatLEA proteins are generally associated withcellular dehydration in seeds and inresponse to water deficit in vegetative tis-sues. Treating plant tissues with the planthormone ABA can also induce the expres-sion of lea genes. A common feature ofmost LEA proteins is their highhydrophilicity, which permits solubilityafter boiling. Correlative studies and bio-chemical features strongly suggest a protec-tive role in the plant cell duringdehydration.

LEA proteins from different plant specieshave been divided into groups based on pre-dicted biochemical properties and sequencesimilarities (Dure et al., 1989; Ingram andBartels, 1996; Cuming, 1999). The strongconservation of motifs in LEA proteins dur-ing evolution points to domains with func-tional constraints. One such motif, which ischaracteristic for group 1 LEA proteins, is 20amino acids in length and was first found inthe wheat Em protein. Group 2 LEA pro-teins, also referred to as dehydrins, are themost widely studied LEA proteins (Close,1997). Many homologues have been isolatedfrom species ranging from gymnosperms todicotyledonous and monocotyledonousangiosperms. Dehydrins are characterized bya lysine-rich 15-amino-acid motif (termedthe K-segment), which is predicted to forman amphipathic �-helix, a tract of contiguousserine residues and a conserved motif con-taining the consensus sequence DEYGNP,which is found close to the N-terminus of the protein. Group 3 LEA pro-teins share a characteristic repeat motif of 11amino acids, which appears to have under-gone duplication and some substitutionevents. Dure et al. (1989) predicted that the11-amino-acid peptide forms an amphi-pathic �-helix with possibilities for intra-and intermolecular interactions. In relationto the others, LEA proteins belonging to groups 4 and 5 are less frequently



represented in the literature. Group 4 ischaracterized by a conserved N-terminuspredicted to form �-helices and a diverse C-terminal part with a random coil structure.Group 5 LEA proteins contain morehydrophobic residues than groups 1 to 4 andconsequently are not soluble after boiling,leading to the suggestion that they probablyadopt a globular conformation. The lea genesfrom all five groups have been isolated andcharacterized at the molecular level from theresurrection plant C. plantagineum, and aremarkably high level of LEA proteins hasbeen found to accumulate in desiccated veg-etative tissues. This observation leads to thehypothesis that cellular desiccation toler-ance depends on a relatively high concentra-tion of a number of different LEA proteins,which are simultaneously expressed inresponse to water deficit. To test this, a quan-titative comparison of LEA protein accumu-lation in tolerant and sensitive vegetativetissues of two closely related species may beinformative.

Despite extensive studies, biochemicalknowledge of the function of LEA proteinsis scarce. Functional studies have used twoapproaches: in vitro protection assays withpurified proteins, and in vivo studies over-expressing LEA proteins in plants or yeast.Results from both types of experimentssupport a role for LEA proteins in theacquisition of desiccation tolerance.

11.4.2. Carbohydrate metabolism

In addition to de novo synthesis of proteins,major changes in carbohydrate metabolismtake place during the resurrection process.While the nature of dehydration-inducedproteins is broadly similar among differentresurrection species, the abundant carbo-hydrate molecules in the hydrated tissuesappear to be diverse. The accumulation ofsucrose in dehydrated tissues is, however,a common theme (see Table 11.2). Takentogether, resurrection plant species appearto possess different metabolic pathwaysthat result in the synthesis of sucrose. Thissupports a role for sucrose in desiccationtolerance, which may be twofold. One

function may be to protect the cell via glassformation rather than solutes crystallizing(see Chapter 10). Through the presence ofsugars, a supersaturated liquid is producedat desiccation with the mechanical proper-ties of a solid. Secondly, sucrose may main-tain hydrogen bonds within and betweenmacromolecules and maintain the structure.This property has been shown in in vitroexperiments for trehalose, a non-reducingdisaccharide of glucose (�-D-glucopyranosyl(1-1) �-D-glucopyranoside) (Crowe et al.,1992). Trehalose is found in desiccation-tol-erant lower organisms such as yeast orSelaginella, but only in small amounts in theresurrection plants Myrothamnus flabelli-folia and S. stapfianus (Bianchi et al., 1993;Drennan et al., 1993; Albini et al., 1994).When the carbohydrate levels in hydratedand dehydrated tissues in resurrectionplants are compared, it is clear that in somespecies large qualitative and quantitativechanges occur during the dehydration–rehydration cycle but in others only smalldifferences are observed (see Table 11.2).One well-documented change in dryingleaves is the conversion of the highly abun-dant C8-sugar 2-octulose into sucrose,which comprises up to 40% of dry weightin desiccated leaves, as first reported for C.plantagineum (Bianchi et al., 1991a).

11.5. Regulation of Gene ExpressionDuring the Desiccation Process in

Resurrection Plants

Knowledge of regulatory pathways is of par-ticular importance because they determinethe expression of a set of genes in a multi-genic trait. Despite the large body of infor-mation concerning genes that are inducedduring dehydration, knowledge of the regu-latory network(s) is scarce. Most informa-tion concerning the regulation of geneexpression is derived from promoter analy-ses and comes from desiccation-sensitivespecies, in particular Arabidopsis. Mutantshave yet to be fully exploited as a potentialtool for dissecting regulatory pathways.This is mainly due to the facts that desicca-tion tolerance is a polygenic character and




Tab

le 1

1.2.

Con

tent

s of

abu

ndan

t car

bohy

drat

es in

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rate

d an

d de

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ated

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ants

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ar c

onte

nt (

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Spe

cies

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rate

d le

aves

Deh

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ted

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esR

efer

ence

Cra

tero

stig

ma

plan

tagi

neum

(Sco

roph

ular

iace

ae)

Oct

ulos

e62

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se52

0B

ianc

hi e

t al.,

199

1a

Cra

tero

stig

ma

lanc

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tum

(Sco

roph

ular

iace

ae)

Oct

ulos

e11

20S

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se30

4A

. Ric

hter

and

D. B

arte

ls, u

npub

lishe

d da

ta

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ma

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Oct

ulos

e81

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icht

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. Bar

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ulos

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D. B

arte

ls, u

npub

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d da

ta

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npub

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d da

ta

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s fla

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otha

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luco

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8aG

luco

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rol

14a

Bia

nchi

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l., 1

993

Treh

alos

e14

8Tr

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ose

136

Dre

nnan

et a

l., 1

993

Suc

rose

383

Suc

rose

463

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a hy

gros

copi

ca (G

esne

riace

ae)

Suc

rose

14a

Suc

rose

90a

Bia

nchi

et a

l., 1

991b

Ram

onda

myc

oni (

Ges

neria

ceae

)S

ucro

se96

Suc

rose

204

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ler

et a

l., 1

997

Hab

erle

a rh

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s (G

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riace

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Suc

rose

59.7

Suc

rose

165

Mül

ler

et a

l., 1

997

Spo

robu

lus

stap

fianu

s (P

oace

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Suc

rose

236

Suc

rose

772

Alb

ini e

t al.,

199

4Tr

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ose

29Tr

ehal

ose

17.5

Oro

petiu

m th

omae

um (P

oace

ae)

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rose

116

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rose

156

A. R

icht

er a

nd D

. Bar

tels

, unp

ublis

hed

data

Xer

ophy

ta v

illos

a (V

eloc

iace

ae)

Suc

rose

43S

ucro

se83

Gha

sem

pour

et a

l., 1

998

a The

se v

alue

s ar

e ex

pres

sed

as %

sug

ar in

aqu

eous

ext

ract

s.


that many resurrection plants are polyploidand consequently do not lend themselvesto mutational studies. Here, regulation ofgene expression in C. plantagineum will bediscussed with reference to the generalfield, and some recent discoveries concern-ing Arabidopsis will be described later (seeSection 11.10).

The plant hormone ABA is associatedwith dehydration-regulated gene expres-sion. Exposure to exogenous ABA causesthe induction of genes that otherwise areactivated by dehydration. Mutants withaltered sensitivity to ABA or a modifiedABA biosynthesis pathway provide evi-dence for the role of ABA in mediatinggene expression in response to waterdeficit (Leung and Giraudat, 1998). It has,however, also become apparent that ABA-independent regulatory systems functionin gene expression under the same stress(Frank et al., 1998; Shinozaki andYamaguchi-Shinozaki, 2000).

The regulation of gene expression bydehydration and ABA in vegetative tissuesof C. plantagineum involves several sig-nalling pathways. Different types of cis-acting elements are required for stress-responsive, coordinated gene expression.Promoters from different groups of stress-inducible genes have been analysed andcompared. The most extensively studiedpromoters from C. plantagineum regulatethe expression of three ABA-responsivelea-like genes, pcC6-19, pcC27-45 andpcC11-24 (Michel et al., 1993, 1994;Velasco et al., 1998). Despite the fact thatdehydration and ABA regulate all threegenes, no common sequence motifs wereapparent in the promoter sequences.Promoter analyses were performed intransgenic tobacco and Arabidopsis plantsto determine the functional cis elements.All three promoters were found to behighly active in seeds and pollen.However, the pcC6-19 promoter differsfrom pcC27-45 and pcC11-24 since no protein synthesis is required for ABA-mediated transcription. A second differ-ence is that the pcC6-19 promoter isinducible by dehydration or ABA in vege-tative tissues of tobacco or Arabidopsis,

whereas pcC27-45 and pcC11-24 promotersare not active in vegetative tissues. Theectopic expression of the ArabidopsisABI-3 gene product did, however, lead toABA-inducible promoter activity in leavesof Arabidopsis, suggesting that the activityof transcription factors in the leaves of C.plantagineum is absent from the leaves ofArabidopsis or tobacco (Furini et al., 1996).The promoter analysis approach led to theidentification of cis-elements that havesince been used to isolate DNA-bindingproteins.

Molecules with putative transcriptional-activating activities or putative signallingmolecules have been obtained from differ-ential screening experiments. Theseinclude members of the myb transcriptionfactor family (Iturriaga et al., 1996), a heat-shock transcription factor (Bockel et al.,1998), members of the homoeodomainleucine zipper family (Frank et al., 1998),phospholipase D (Frank et al., 2000) and anovel C. plantagineum gene cDT-1 (Furiniet al., 1997). Transcripts encoding thesemolecules are induced by dehydration,suggesting an involvement of the geneproducts in the dehydration process. A dif-ficult challenge is to identify the targetgenes of these putative regulatory mole-cules. The findings from C. plantagineumare extended mainly by studies of theresponse to dehydration in Arabidopsis,which involves transcription factorsbelonging to the Myb, Myc, AP2/EREBPand bZip classes, protein kinases and pro-tein phosphatases. Although the exact roleof most factors in gene regulation isunknown, it is interesting to note thatmembers of different regulator families arepart of the regulatory dehydration network.

11.6. Desiccation-tolerant Bryophytes

Desiccation-tolerant bryophytes are foundworldwide and inhabit a variety of habi-tats, most of which could, during someperiod of the year, be considered asextreme, either on a macro or micro level.In most cases, the extremes that theseplants experience are both in water avail-



ability and temperature (Clausen, 1952; Leeand Stewart, 1971; Norr, 1974; Dilks andProctor, 1976; Alpert and Oechel, 1987; seeChapter 7).

Desiccation-tolerant bryophytes, becauseof their simple architecture, have little orno morphological (or indeed physiological)characteristics or adaptations that can limitwater loss or regulate plant temperature.As a result of this, the internal water con-tent of their photosynthetic tissues rapidlyequilibrates to the water potential of theenvironment once free water is lost fromthe surface of the plant. This in turn meansthat these plants experience drying ratesthat are much faster than those experi-enced by their more complex pteridophyteor angiosperm counterparts. In fact, thedrying rates that desiccation-tolerantbryophytes experience are in the mainlethal to desiccation-tolerant ferns andflowering plants (Bewley and Krochko,1982). The fact that bryophyte tissuesrapidly equilibrate to the water potential ofthe environment means that, in the major-ity of cases where temperatures becomeextreme, hot or cold, these plants are dry. Itis in the dried state that desiccation-toler-ant bryophytes (and most desiccation-toler-ant plants of the less complex clades)tolerate temperature extremes (Norr, 1974;Malek and Bewley, 1978).

The rapid equilibration of protoplasmicwater potential with that of the environ-ment in bryophyte tissues appears todemand a type of desiccation tolerance thatis significantly different from that exhib-ited by the resurrection plants so fardescribed (Oliver and Bewley, 1997).Rather than acquiring desiccation tolerancein response to a dehydration event as seenin Craterostigma, Sporobolus and otherdesiccation-tolerant angiosperms, desicca-tion-tolerant bryophytes appear to expressthis trait constitutively (Bewley and Oliver,1992; Oliver and Bewley, 1997). This formof desiccation tolerance is considered themost primitive of those that have receivedattention so far (Oliver et al., 2000). In thistype of tolerance, the major genetic responseto a desiccation event, at least at the level ofgene expression, occurs after the fact, during

the first hour or two following rehydration.This has led to the suggestion that a majorcomponent of the mechanism of desiccation tolerance in bryophytes is a rehydration-induced cellular repair response (seeBewley and Oliver, 1992; Oliver andBewley, 1997, for reviews). The implicationis that, although cellular protection andhence desiccation tolerance are constitutive,it is not sufficient to prevent some damagefrom occurring (or being manifested) uponrehydration, and thus repair processes areneeded and induced when water returns tothe protoplasm of the cells. Much of the evi-dence for these hypotheses comes from thestudy of a family of desiccation-tolerantmosses, the Tortula complex, and in partic-ular the species T. ruralis (synonymous withSyntrichia ruralis).

11.7. Constitutive Cellular Protection

Observational experiments confirm a pro-tective component to the mechanism ofdesiccation tolerance in bryophytes.Freeze–fracture studies of dried T. ruraliscells (both rapidly and slowly dried)demonstrate that cellular integrity ismaintained during drying (Platt et al.,1994) and plasma membranes, internalmembranes and structures remainundamaged. Physiological experimentsdesigned to elucidate the effect of desic-cation on photosynthesis suggest that theprotection mechanisms that are operatingin Tortula are very effective in protectingthe photosynthetic apparatus and inallowing for the rapid recovery of photo-synthetic activity (Tuba et al., 1996;Proctor et al., 1998; Csintalan et al., 1999;Proctor, 2000). The almost instantaneousphotosystem recovery and the relativelyshort time needed (20 min) to reach apositive carbon balance, as described bythese authors, occur at a time whenchloroplast structure is substantially dis-rupted (see below). How this is achievedis enigmatic but ecologically it makessense for these opportunistic bryophytesselectively to protect their photosyntheticcapability.



Metabolically, desiccation of gameto-phytic tissues of T. ruralis results in a rapiddecline in protein synthesis, as in all desic-cation-tolerant and sensitive mosses testedso far (see Bewley and Oliver, 1992; Oliverand Bewley, 1997, for reviews). In T. ruralis,this loss of protein synthetic capacity ismanifested by a loss of polysomes resultingfrom the run-off of ribosomes from mRNAs,concomitant with their failure to reinitiateprotein synthesis (see Bewley, 1979;Bewley and Oliver, 1992, for reviews). Therapid loss of polysomes during drying andthe apparent sensitivity of the initiationstep of protein synthesis to protoplasmicdrying lead to the conclusion that the pro-tection component of the mechanism oftolerance for these plants does not involvethe synthesis of proteins induced by theonset of a water deficit. This is borne outby the observation that no new mRNAs arerecruited into the protein synthetic com-plex, even if the rate of water loss is slow(Oliver, 1991, 1996). The fact that the mosssurvives rapid desiccation (even when des-iccation is achieved in a few minutes in alyophilizer) also indicates that aninducible protection mechanism is not necessary for survival.

As discussed above, LEA proteins andcarbohydrates are important components ofprotective mechanisms in desiccation-tolerant plants and plant tissues. In desiccation-tolerant mosses, sucrose is theonly free sugar available for cellular protec-tion (Bewley et al., 1978; Smirnoff, 1992).The amount of this sugar in gametophyticcells of T. ruralis is approximately 10% ofdry mass, which is sufficient to offer mem-brane protection during drying, at least invitro (Strauss and Hauser, 1986). Moreover,neither drying nor rehydration in the darkor light results in a change in sucrose con-centration, suggesting that it is importantfor cells to maintain sufficient amounts ofthis sugar (Bewley et al., 1978). The lack ofan increase in soluble sugars during dryingappears to be a common feature of desicca-tion-tolerant mosses (Smirnoff, 1992). Theexistence of dehydrin-type LEA proteins indesiccation-tolerant vegetative tissues ofdesiccation-tolerant bryophytes has only

recently been reported. Protein analysesusing purified antibodies raised against thecommon C-terminus of maize seedlingdehydrins (Close et al., 1993) show that T.ruralis produces two major dehydrins(80–90 kDa and 35 kDa). These are presentin the hydrated state and do not appear toincrease during rapid or slow drying(Bewley et al., 1993). A similar result wasobtained with the desiccation-tolerantmoss Thuidium delicatulum (T.L.Reynolds, M.J. Oliver and J.D. Bewley,unpublished data).

11.8. Cellular Damage and RecoveryFollowing Rehydration

Following rehydration, gametophytic cellsof desiccation-tolerant mosses undergosubstantial and universal disruption of cel-lular integrity including breaches of allmembrane systems (see Oliver and Bewley,1984a, for review). Internal organellesswell and distort and their internal mem-brane systems become dispersed.Nevertheless, the cells do not die, as docells of sensitive species, but return to anormal appearance within 12–24 h. Theamount of cellular disruption that occursduring rehydration clearly depends uponthe rate at which water was lost duringdesiccation. Chloroplasts of T. ruralis driedto air dryness over 4–6 h (a natural rate) areswollen when rehydrated but retain moreof their normal internal structure andexhibit fewer clefts in their membranesthan do the chloroplasts in rehydrated cellsof gametophytes dried within an hour(Tucker et al., 1975). The greater retentionof chloroplast structure allows slow-driedT. ruralis to effect a more rapid recovery ofphotosynthesis, achieving a positive car-bon balance within 20 min following rehy-dration (Bewley, 1979; Tuba et al., 1996).The time required for full photosyntheticrecovery upon rehydration, however, variesconsiderably among species, depending ontheir degree of desiccation tolerance(Proctor et al., 1998). Electrolyte leakageupon rehydration, a measure of membranedamage, is also affected by the speed at



which desiccation occurs. After slow dry-ing, leakage in moss is less than half asgreat as after rapid desiccation and is simi-lar to leakage of hydrated controls, indicat-ing minimal membrane damage (Bewleyand Krochko, 1982). It is these observa-tional studies that first led to the hypothe-sis that the mechanism for tolerance indesiccation-tolerant bryophytes includes arepair-based strategy to recover from thedamage manifested upon rehydration.

11.9. Gene Expression During Recovery

The repair aspect of the mechanism of des-iccation tolerance in these plants, althoughdemonstrated to be a major component oftolerance, is difficult to detail and charac-terize. Most work has focused on the pro-teins whose synthesis is inducedimmediately upon rehydration of desic-cated gametophytic tissue. Early work (seeBewley, 1979, for review) established theability of T. ruralis and other mossesrapidly to recover synthetic metabolismwhen rehydrated. The speed of this recov-ery was dependent upon the rate of priordesiccation: the faster the rate of desicca-tion, the slower the recovery. In addition,although the pattern of protein synthesis inthe first 2 h of rehydration of T. ruralis isdistinctly different from that of hydratedcontrols, novel transcripts were not madein response to desiccation (Oliver andBewley, 1984b; Oliver, 1991). Hence it wassuggested that T. ruralis responds to desic-cation by an alteration in protein synthesisupon rehydration that is in large measurethe result of a change in translational con-trol. Changes in transcriptional activitywere observed for nearly all transcriptsstudied (Scott and Oliver, 1994) but did notresult in a qualitative change in the tran-script population during desiccation orrehydration. It thus appears that T. ruralisrelies more upon the activation of pre-existing repair mechanisms for desiccationtolerance than it does on either pre-estab-lished or activated protection systems.

In a detailed study of the changes inprotein synthesis initiated by rehydration

in T. ruralis, Oliver (1991) demonstratedthat during the first 2 h of hydration thesynthesis of 25 proteins is terminated, orsubstantially decreased, and the synthesisof 74 proteins is initiated, or substantiallyincreased. Controls over changes in synthe-sis of these two groups of proteins, the for-mer termed hydrins and the latterrehydrins, are not mechanistically linked.It takes a certain amount of prior water lossto fully activate the synthesis of rehydrinsupon rehydration. This may in turn indi-cate that there is also a mechanism bywhich the amount of water loss is ‘sensed’and ‘translated’ into a protein syntheticresponse upon rehydration. Such a sce-nario was also proposed for the novel pat-tern of protein synthesis associated withthe drying of S. stapfianus (Kuang et al.,1995). Perhaps this is a strategy that hasevolved to link the amount of energyexpended in repair to the amount of dam-age potentiated by differing extents of dry-ing.

Since rehydrins appear to be synthe-sized from a transcript pool that is qualita-tively unaffected by desiccation orrehydration (Oliver, 1991), it was of inter-est to determine what are the translationalcontrols of gene expression that operateduring the initial phases of recovery in T.ruralis. A partial answer was gained fromthe use of rehydrin cDNA clones isolatedfrom differential screening of a T. ruralisrehydration cDNA library (Scott andOliver, 1994). RNA blots revealed that sev-eral rehydrin transcripts accumulate dur-ing slow drying (Oliver and Wood, 1997;Wood and Oliver, 1999) at a time when it isassumed that transcriptional activity israpidly declining. These transcripts do notaccumulate during rapid desiccation, nor istheir accumulation during slow dryingassociated with an increase in endogenousABA accumulation. ABA is undetectable inthis moss (Bewley et al., 1993; M.J. Oliver,unpublished data), and T. ruralis does notsynthesize specific proteins in response toapplied ABA. The accumulation of thesetranscripts was postulated to be the resultof an increase in mRNA stability broughtabout by the removal of water from the



cells (Scott and Oliver, 1994). Recent stud-ies clearly demonstrate that these tran-scripts are sequestered in the driedgametophytes in messenger ribonucleopro-tein particles (mRNPs) (Wood and Oliver,1999) and that this results in the change intheir stability. The implication from thiswork is that the sequestration of mRNAsrequired for recovery hastens the repair ofdamage induced by desiccation or rehydra-tion and thus minimizes the time neededto restart growth upon rehydration. Thesefindings may also explain the ability of T.ruralis to ‘harden’ during recurring desic-cation events in the absence of inducibledehydrin or sugar responses (Schonbeckand Bewley, 1981a,b).

11.9.1. Rehydrins

Eighteen rehydrin cDNAs, isolated by Scottand Oliver (1994), have been sequenced(Oliver et al., 1997; Wood et al., 1999).Only three exhibit significant sequencehomology to known genes in the Genbankdatabases. Tr155 has a strong sequencesimilarity to an alkyl hydroperoxidaselinked to seed dormancy in barley (Aalenet al., 1994) and Arabidopsis embryos(Haslekas et al., 1998), and in rehydratedbut dormant Bromus secalinas L. seeds(Goldmark et al., 1992). Tr213 exhibits ahigh degree of similarity to polyubiquitinsfrom several plant sources. The finding thatpolyubiquitin is a rehydrin is indicative ofan increased need for protein turnover dur-ing recovery from desiccation, an idea thatis not new (Ingram and Bartels, 1996). InTortula there are three detectable ubiquitintranscripts, two appear to be constitutivelyexpressed but the third is responsive todesiccation and rehydration (O’Mahonyand Oliver, 1999). This is in contrast toSporobolus, where only two classes ofubiquitin transcripts are evident and bothrespond to desiccation and rehydration(O’Mahony and Oliver, 1999). Tr288 has adehydrin-like K box sequence at the C-terminus of the predicted protein but lit-tle other sequence similarity to knowndehydrins. However, the predicted Tr288

protein contains 15 15-amino-acid repeatspredicted to form amphipathic helices(Velten and Oliver, 2001), which is veryreminiscent of the predicted structuralcharacteristics of group 3 LEA proteins(Dure et al., 1989). The interesting aspectof this protein is that it appears to be syn-thesized during the rehydration event andnot at all during drying, indicating thatLEA-like proteins may play a role in dam-age repair as well as cellular protection. Itis also possible that some LEA proteinsplay a role in protecting the cell from dam-age initiated during rehydration.

More recently, Wood et al. (1999)reported the establishment of a smallExpressed Sequence Tag (EST) databasefrom a cDNA library constructed fromslow-dried gametophyte polysomal RNA(in an attempt to target sequencessequestered in mRNPs – see above). Of 152ESTs that were generated and partiallysequenced, only 30% showed significanthomology to previously identified nucleicacid and/or polypeptide sequences.Interestingly, several ESTs showed signifi-cant similarity to unidentified desiccation-tolerance genes isolated from C.plantagineum (Bockel et al., 1998).

11.10. Transgenic Approaches towardsImproving Plant Dehydration/

Desiccation Tolerance

The relevance of desiccation tolerance indetermining productivity under moisture-limited environments is debatable as, agri-culturally, desiccation represents a smallproportion of the total instances of drought(Subbarao et al., 1995). Furthermore, yieldreduction due to water deficit becomesimportant before desiccation occurs.However, improvement in yield in relationto limiting water supply is agronomicallydesirable. In environments where waterdeficits can occur at any stage of growth,knowledge of the mechanisms of desiccationtolerance should play a role in survival ofthe crop until soil moisture levels improve.

Transgenic approaches offer a powerfulmeans of gaining valuable information



towards a better understanding of themechanisms that govern stress tolerance.They also open up new opportunities toimprove stress tolerance by incorporatinggenes involved in stress protection fromany source into agriculturally importantcrop plants. To date, the ‘transgenicapproach’ has been to transfer a single geneinto plants and then observe the pheno-typic and biochemical changes before andafter a specific stress treatment. A limita-tion of this strategy is that the functions ofvery few genes involved in desiccation tol-erance have been established, or notenough is known about the regulatorymechanisms. Molecular marker analysishas been used to study dehydration toler-ance, principally in cereals (Quarrie, 1996).Based on results from this type ofapproach, it is mainly viewed that toler-ance to water deficit is a complex quantita-tive trait, since no single diagnostic markerfor tolerance has been found. However,transgenic plants with improved toleranceto water deficit have been produced usingvarious genes (see Table 11.3) and will bedescribed in the following text.

11.10.1. Compatible solutes or osmolytes

Many plants respond to water deficit byaccumulating organic compounds of lowmolecular weight, known as compatiblesolutes or osmolytes. Therefore, engineer-ing-increased osmolyte content in trans-genic plants is a rational strategy forprotecting plants against dehydrationstress. Transgenic plants harbouring genesencoding enzymes involved in the produc-tion of proline, fructans and trehaloseshow a reduction of dehydration stress.Most of these studies have been carried outin tobacco and Arabidopsis because thetransformation technology is very wellestablished; however, an improvement instress tolerance has also been reported inother species such as rice and sugar beet.

The enzyme �1-pyrroline-5-carboxylatesynthetase (P5CS) catalyses the conversionof glutamate to pyrroline-5-carboxylate,which is then reduced to proline.

Overexpression of a gene encoding formoth bean P5CS in transgenic tobaccoplants resulted in accumulation of prolineup to 10–18-fold over control plants andbetter growth under dehydration stress(Kavi-Kishor et al., 1995). The transgenicplants demonstrated enhanced biomassproduction and flower development, asdetermined by increased root length, rootdry weight, capsule number and seed num-ber per capsule. The same gene was alsointroduced into rice under the control of anABA-responsive promoter (Zhu et al.,1998). The transgenic plants accumulatedup to 2.5-fold more proline than controlplants under stress conditions. This studyshowed that the stress-inducible expres-sion of the p5cs transgene in rice plantsresulted in an increase in biomass asreflected by higher fresh shoot and rootweight under salt- and water-stress condi-tions compared with untransformed plants.

As previously mentioned, trehaloseaccumulates in a large number of organ-isms in response to different stress condi-tions. With the exception of tworesurrection plants (see Section 11.4.2.),trehalose is generally not accumulated inplants. However, genes for trehalose metab-olism have been identified in higher plantsand characterized by expression studiesand functional complementations of corre-sponding yeast mutants (Müller et al.,1999). The data obtained from plants exter-nally supplied with trehalose or fromtransgenic plants expressing trehalosebiosynthesis genes from microorganismspoint towards a role for trehalose in dehy-dration tolerance. For example, the yeasttrehalose 6-phosphate synthetase gene(TPS1) was introduced into tobacco andthe resulting trehalose-accumulating plantsshowed improved dehydration tolerance,although a decrease of 30–50% in growthrate in conditions optimal for growth wasalso reported (Holmström et al., 1996). In asecond study, Pilon-Smits et al. (1998)introduced bacterial trehalose 6-phosphatesynthase (otsA) and trehalose 6-phosphatephosphatase (otsB) into tobacco. The leavesof the transgenic plants were larger andshowed better growth, in terms of dry




Tab

le 1

1.3.

Tran

sgen

ic p

lant

s w

ith a

ltere

d to

lera

nce

to w

ater

defi

cit.

Gen

eO

rigin

Gen

e pr

oduc

tH

ost

Tole

ranc

e ph

enot

ype

Ref

eren

ce

Adc

Oat

Arg

inin

e de

carb

oxyl

ase

Ric

eLo

wer

chl

orop

hyll

loss

Cap

ell e

t al.,

199

8

cdt-

1C

rate

rost

igm

aR

egul

ator

y R

NA

or s

hort

pol

ypep

tide

Cra

tero

stig

ma

Sur

viva

l of c

allu

s w

ithou

t AB

Apr

etre

atm

ent

Fur

ini e

t al.,

199

7

DR

EB

1aA

rabi

dops

isD

RE

-bin

ding

tran

s-fa

ctor

Ara

bido

psis

Hig

her

surv

ival

rat

eK

asug

a et

al.,

199

9

HV

A1

Bar

ley

Gro

up 3

LE

Apr

otei

nR

ice

Hig

her

grow

th r

ate

and

dela

y in

dam

age

Xu

et a

l., 1

996

Whe

atsy

mpt

oms

Siv

aman

i et a

l., 2

000

otsA

Esc

heric

hia

coli

Treh

alos

e-6-

phos

phat

e sy

ntha

seTo

bacc

oH

ighe

r ph

otos

ynth

etic

rat

e an

d in

crea

sed

Pilo

n-S

mits

et a

l., 1

998

otsB

E. c

oli

Treh

alos

e-6-

phos

phat

e ph

osph

atas

eTo

bacc

odr

y w

eigh

t

p5cs

Mot

h be

an�

1 -py

rrol

ine-

5-ca

rbox

ylat

e sy

nthe

tase

Toba

cco

Enh

ance

d ro

ot b

iom

ass

and

flow

er

Kav

i-Kis

hor

et a

l., 1

995

Ric

ede

velo

pmen

tZ

hu e

t al.,

199

8

sacB

Bac

illus

sub

tilis

Fru

ctos

yl tr

ansf

eras

eTo

bacc

oH

ighe

r gr

owth

rat

eP

ilon-

Sm

its e

t al.,

199

5F

ruct

osyl

tran

sfer

ase

Sug

arbe

etP

ilon-

Sm

its e

t al.,

199

9

TP

S1

Sac

char

omyc

es

Treh

alos

e-6-

phos

phat

e sy

nthe

tase

Toba

cco

Incr

ease

in le

af s

urvi

val

Hol

mst

röm

et a

l., 1

996

cere

visi

ae

IMT

1M

esem

brya

nthe

mum

Myo

-inos

itol O

-met

hyltr

ansf

eras

eTo

bacc

oH

ighe

r ph

otos

ynth

etic

rat

eS

heve

leva

et a

l., 1

997

crys

talli

num

Sod

Nic

otia

na

Man

gane

se-s

uper

oxid

e di

smut

ase

Luce

rne

Hig

her

surv

ival

rat

eM

cKer

sie

et a

l., 1

996

plum

bagi

nifo

lia


weight, under dehydration stress. Detachedleaves from young, well-watered transgenicplants showed better capacity to retainwater when air-dried than the wild-typeplants. These transgenic plants also hadmore efficient photosynthetic activity.Although the trehalose protective effect isunclear at the molecular level, correlativeevidence suggests that trehalose stabilizesproteins and membrane structures understress (Colaco et al., 1995; Iwahashi et al.,1995).

Fructans are polyfructose molecules thatare produced by many plants and bacteria.Owing to their high solubility, they mayhelp plants survive periods of osmoticstress. Pilon-Smits et al. (1995) introduceda gene encoding a bacterial fructan syn-thase (sacB) isolated from Bacillus subtilisinto tobacco. Under unstressed conditions,the presence of fructans had no significanteffect on growth rate and yield. The trans-genic plants performed significantly betterunder osmotic stress than wild-typetobacco, and the stress resistance correlatedwith the amount of fructan accumulated.The same sacB gene was introduced intosugarbeet to produce bacterial fructans(Pilon-Smits et al., 1999). The transgenicsugarbeets accumulated fructans to low lev-els in both roots and shoots. Two indepen-dent transgenic lines of fructan-producingsugarbeets showed significantly bettergrowth under dehydration stress than diduntransformed beets. Dehydration-stressedfructan-producing plants attained highertotal dry weights than wild-type sugarbeet,due to higher biomass production ofleaves, storage roots and fibrous roots.Again, no significant differences wereobserved between the transgenic and wild-type plants under well-watered conditions.The introduction of fructan biosynthesis intransgenic plants is therefore a promisingapproach to improving crop productivityunder dehydration stress.

Expression of a cDNA encoding myo-inositol O-methyltransferase (imt1) intobacco during salt and dehydration stressresulted in the accumulation of methylatedinositol D-ononitol (Sheveleva et al., 1997).Photosynthetic carbon dioxide fixation in

transgenic plants was inhibited less duringdehydration and salt stress, and the plantsrecovered faster than wild type.Furthermore, preconditioning of plantsexpressing the imt1 cDNA in low-saltmedia increased D-ononitol amounts andresulted in increased protection when theplants were stressed subsequently with ahigher salt concentration. Unlike solutessuch as proline and sucrose, D-ononitollevels did not show significant diurnalfluctuations This led the authors to suggestthat stress-inducible solute accumulationmay provide better protection underdrought conditions than do strategies usingosmotic adjustment by metabolites that areconstitutively present.

Polyamines are small nitrogenous cellu-lar compounds that have being implicatedin a variety of stress responses in plants.Polyamines accumulate under several abi-otic stress conditions including drought.Cultivars demonstrating a higher degree ofsalt tolerance contain higher levels ofpolyamines. Furthermore, exogenousapplication of polyamines results in pro-tection against osmotic stress. Transgenicrice cell lines and plants have been pro-duced that express an oat arginine decar-boxylase cDNA, the gene product of whichconverts ornithine to the diamineputrescine, under the control of the cauli-flower mosaic virus (CaMV) 35S promoter(Capell et al., 1998). A four- to sevenfoldincrease in arginine decarboxylase activitywas observed in transformed plants com-pared with wild-type controls. Biochemicalanalysis of cellular polyamines indicatedup to a fourfold increase in putrescine lev-els in transgenic plants. Although theplants had improved drought tolerance interms of chlorophyll loss under droughtstress, constitutive expression of this geneslowed down growth severely.

11.10.2. Oxygen-scavenging proteins

One consequence of dehydration and manyother stresses is the production of activatedoxygen molecules that cause cellular injury(see Chapters 9 and 10). The protection of



sensitive metabolic reactions by stabiliza-tion of protein complexes or membranestructures by increasing the capacity forhydroxyl radical scavenging in plants shouldprovoke improved performances under non-lethal stress conditions. Transgenic lucerneexpressing Mn-superoxide dismutase cDNAtended to have reduced injury from water-deficit stress as determined by chlorophyllfluorescence, electrolyte leakage andregrowth from crowns (McKersie et al.,1996). A 3-year field trial indicated thatboth yield and survival of transgenic plantswas significantly improved, supporting thehypothesis that tolerance of oxidativestress is important in adaptation to fieldenvironments.

11.10.3. LEA proteins

A barley group 3 LEA protein termedHVA1 is specifically expressed in the aleu-rone layer and the embryo during the latestages of seed development, correlatingwith the acquisition of seed desiccationtolerance. ABA and several stress condi-tions including dehydration also induceHVA1 expression in young seedlings. Xu etal. (1996) produced transgenic rice plantsexpressing the barley HVA1 gene, drivenby a constitutive promoter. This led to theconstitutive accumulation of HVA1 proteinin both leaves and roots of transgenic riceplants. The second-generation transgenicrice plants showed increased tolerance towater deficit and salinity. In a secondstudy, HVA1 was introduced into springwheat (Sivamani et al., 2000). High levelsof expression of the HVA1 gene, regulatedby a maize ubiquitin promoter, wereobserved in leaves and roots of indepen-dent transgenic wheat plants. Progenies offour selected transgenic wheat lines weretested under greenhouse conditions for tol-erance of soil water deficit. Potted plantswere grown under moderate water deficitand well-watered conditions, respectively.Two homozygous and one heterozygoustransgenic lines expressing the HVA1 genehad significantly higher water-use effi-ciency values as compared with the non-

expressing transgenic and non-transgeniccontrols under moderate water-deficit con-ditions. The two homozygous transgenicplant lines also had significantly greatertotal dry mass, root fresh and dry weights,and shoot dry weight compared with thetwo controls under soil water-deficit condi-tions. As is the case for all LEAs, the pre-cise mode of action of HVA1 under droughtconditions remains unclear.

In contrast, attempts to introduce threelea-like genes from the resurrection plantC. plantagineum into tobacco did notresult in a drought-tolerant phenotype(Iturriaga et al., 1992). However, this resultis perhaps less surprising considering thatdrought stress does induce an array of dif-ferent LEA-related proteins in plants (seeSection 11.4.1). It is also likely that otherfactors are required for the expression oftolerance where LEA-type proteins areinvolved.

11.10.4. Regulatory genes

The machinery leading to the expression ofdehydration-responsive genes is expectedto conform to a general cellular model. Ingeneral, signal transduction cascades canbe divided into the following basic steps:perception of stimulus; processing, includ-ing amplification and integration of the sig-nal; and a response reaction in the form ofde novo gene expression. As mentionedearlier (see Section 11.5), studies of dehy-dration-activated signalling cascades haveresulted in the identification of potentialregulatory genes, such as transcription fac-tors. The transformation of plants usingregulatory genes is an attractive approachfor producing dehydration-tolerant plants.Since the products of these genes regulategene expression and signal transductionunder stress conditions, the overexpressionof these genes can activate the expressionof many stress-tolerance genes simultane-ously.

Expression of many Arabidopsis dehy-dration-responsive and cold-regulated(COR) genes is mediated by a DNA regula-tory element termed the dehydration-



responsive element/C-repeat (DRE/CRT)(Yamaguchi-Shinozaki and Shinozaki,1994). A major breakthrough was madewhen a transcriptional activator, CBF1, thatbinds to the DRE/CRT was identified(Stockinger et al., 1997). In a second reportit was demonstrated that overexpression ofCBF1 in transgenic Arabidopsis plants atnon-acclimatizing temperatures inducesCOR gene expression and increases plantfreezing tolerance (Jaglo-Ottosen et al.,1998). More recently, Kasuga et al. (1999)transformed Arabidopsis with a cDNAencoding DREB1a, a homologue of CBF1,driven by either the constitutive CaMV 35Spromoter or an abiotic stress-inducible pro-moter. The overexpression of this gene acti-vated the expression of many stress-tolerance genes such as lea genes and P5CS.In all cases, the transgenic plants weremore tolerant to drought, salt and freezingstresses. However, the constitutive overex-pression of DREB1a also resulted in severegrowth retardation under normal growthconditions. In contrast, the stress-inducibleexpression of this gene had minimal effectson plant growth and provided greater toler-ance to stress conditions than genes drivenby a strong constitutive promoter.

Although C. plantagineum can tolerateextreme dehydration, in vitro-propagatedcallus derived from this plant has a strictrequirement for exogenously applied ABAin order to survive a severe dehydration.This property has been exploited for isola-tion of dominant mutants by activation tag-ging, in which high expression of residentgenes activated by insertion of a foreignpromoter would confer desiccation toler-ance to the transformed cells without priorABA treatments. One gene was identified(cDT-1), whose high expression did conferthe expected phenotype in calli and led toconstitutive expression of several ABA-and dehydration-inducible genes (Furini etal., 1997). In a second experiment, trans-genic calluses that constitutively express cDT-1 under the control of two differentpromoters were produced. When callusesfrom both lines were dehydrated, transfor-mants from both lines were able to with-stand desiccation in the absence of ABA. In

contrast, calluses transformed with controlvectors did not survive dehydration. Thedesiccation-tolerant transformants accumu-lated anthocyanins and their phenotypewas indistinguishable from the originaltransfer DNA (T-DNA)-tagged mutant line.RNA hybridization analysis confirmed thatthe desiccation-tolerant transformants con-tained high levels of the 0.9 kb transcriptand constitutively expressed the ABA-responsive marker genes. This regulatorygene has a unique structure, but does sharesome features with mammalian retrotrans-posons. The function of the cDT-1 geneproduct is not immediately obviousbecause it encodes a transcript with nolarge open reading frame. It is possible thatthe biologically active product of cDT-1 is aregulatory RNA or a short polypeptide.

11.11. Conclusions and Perspectives

Molecular analyses of desiccation-tolerantsystems use a variety of strategies andinvolve different plant species. Initialresearch has been largely descriptive andmany genes have been isolated that play apotential role in desiccation tolerance.Furthermore, major themes in the molecu-lar response have been established such aschanges in sugar metabolism and theexpression of lea genes. Studies havebegun to examine mechanisms that controlgene expression and regulatory pathwaysare being established. Attempts to under-stand gene function have used transgenicplants, the results of which are of clearbiotechnological importance.

In order to address further the questionof gene function and ultimately to under-stand the molecular basis of desiccationtolerance, other experimental approachesare required. One strategy is the develop-ment of a genetic model system to studydesiccation tolerance in vegetative tissue.Insertional mutagenesis via T-DNA ortransposon tagging could then be employed– both are proven methods to deduce thefunction of genes in genetic model systemssuch as Arabidopsis. Secondly, naturalallelic variation has proved successful for



identifying genes involved in plant devel-opment (Swarup et al., 1999). Quantitativetrait locus (QTL) analysis of plant acces-sions that exhibit extensive variation fordesiccation tolerance may be a means ofidentifying genes in complex regulatorynetworks.

In mosses, where desiccation-toleranttissues are haploid, the possible use ofgene replacement, either directed or byrandom tagging, utilizing efficient homolo-gous recombination techniques, offers anovel and powerful technology for func-tional gene analysis (Puchta, 1998; Reski,1999).

Comparative mapping studies havedemonstrated that closely related plantspecies have highly conserved gene con-tent and chromosomal positions of genesare also maintained, even though chromo-somal rearrangements differ. The similarityin gene content of, for example, grassesindicates that genes are rarely createdwithin individual species and variationbetween species is likely to arise from geneduplication and/or minor sequence modifi-cations of existing genes (Bennetzen andFreeling, 1993). This hypothesis is sup-ported by evidence that major modifica-tions in plant morphology and growth

habit can occur via minor gene alterations(Doebley and Stec, 1991).

The existence of ‘common genomes’suggests that genes required for any path-way are present in all plant species.However, the genes involved in pathwaysmay differ in their spatial expression pat-terns and locations, due to minor changesin regulatory regions. For example, differ-ences in tissue specificity and/or control ofgene expression among members of a tran-scription factor gene family in desiccation-tolerant and desiccation-sensitive speciesmay account for differences in desiccationtolerance. Therefore, it may be possible toalter significantly multigenic traits such asdesiccation/dehydration tolerance, by theidentification and transfer of single genesthat account for the physiological differ-ence between the species.


The work in the laboratory of D. Bartelswas supported by the DFG Schwerpunkt‘Molekulare Analyse der Phytohormon-wirkung’. We thank A. Richter, Vienna, forcommunicating data on sugar analysisbefore publication.


11.13. References

Aalen, R.B., Opsahl-Ferstad, H.G., Linnestad, C. and Olsen, O.A. (1994) Transcripts encoding anoleosin and a dormancy-related protein are present in both the aleurone layer and in the embryoof developing barley (Hordeum vulgare L.). The Plant Journal 5, 385–396.

Albini, F.M., Murelli, C., Patritti, G., Rovati, M., Zienna, P. and Finzi, P.V. (1994) Low-molecularweight substances from the resurrection plant Sporobolus stapfianus. Phytochemistry 37,137–142.

Alpert, P. and Oechel, W.C. (1987) Comparative patterns of net photosynthesis in an assemblage ofmosses with contrasting microdistributions. American Journal of Botany 741, 1787–1796.

Bartels, D. (1999) Late embryogenesis abundant (LEA) proteins: expression and regulation in the res-urrection plant Craterostigma plantagineum. In: Bowles, D.J., Smallwood, M.F. and Calvert,C.M. (eds) Plant Responses to Environmental Stress. BIOS Scientific Publishers, Oxford, UK, pp.153–160.

Bartels, D., Schneider, K., Terstappen, G., Piatkowski, D. and Salamini, F. (1990) Molecular cloningof ABA-modulated genes from the resurrection plant Craterostigma plantagineum which areinduced during desiccation. Planta 181, 27–34.

Bennetzen, J.L. and Freeling, M. (1993) Grasses as a single genetic system – genome composition,collinearity and compatibility. Trends in Genetics 9, 259–261.



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Bewley, J.D. and Oliver, M.J. (1992) Desiccation-tolerance in vegetative plant tissues and seeds: pro-tein synthesis in relation to desiccation and a potential role for protection and repair mecha-nisms. In: Osmond, C.B. and Somero, G. (eds) Water and Life: a Comparative Analysis of WaterRelationships at the Organismic, Cellular and Molecular Levels. Springer-Verlag, Berlin,pp. 141–160.

Bewley, J.D., Halmer, P., Krochko, J.E. and Winner, W.E. (1978) Metabolism of a drought-tolerant anda drought-sensitive moss: respiration, ATP synthesis and carbohydrate status. In: Crowe, J.H.and Clegg, J.S. (eds) Dry Biological Systems. Academic Press, New York, pp. 185–203.

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Bianchi, G., Gamba, A., Limiroli, R., Pozzi, N., Elster, R., Salamini, F. and Bartels, D. (1993) Theunusual sugar composition in leaves of the resurrection plant Myrothamnus flabellifolia.Physiologia Plantarum 87, 223–226.

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oat arginine decarboxylase cDNA in transgenic rice (Oryza sativa l.) affects normal developmentpatterns in vitro and results in putrescine accumulation in transgenic plants. Theoretical andApplied Genetics 97, 246–254.

Clausen, E. (1952) Hepatics and humidity: a study on the occurrence of hepatics in a Danish tractand the influence of relative humidity on their distribution. Dansk Botanisk Arkives 15, 5–80.

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12 Rehydration of Dried Systems:Membranes and the Nuclear Genome

Daphne J. Osborne,1 Ivan Boubriak1 and Olivier Leprince21The Oxford Research Unit, Open University, Foxcombe Hall, Boars Hill OX1 5HR,UK; 2UMR Physiologie Moléculaire des Semences, Institut National d’Horticulture,

16 Bd Lavoisier, F49045 Angers, France

12.1. Introduction 34412.2. The Dangers of Rehydration and Membrane Changes 344

12.2.1. Factors governing imbibitional injury 34412.2.2. Causes for solute leakage as mechanisms of imbibitional

injury: the fate of plasma membranes during rehydration 34612.2.2.1. Conformational properties of plasma membranes 34612.2.2.2. Mechanical stress 347

12.2.3. Protections against imbibitional injury 34712.2.3.1. Seed coat 34712.2.3.2. Protection at the molecular level 349

12.3. Maintaining Integrity of the Genome 35012.3.1. Hydration-determined changes in DNA 35012.3.2. Seeds 350

12.3.2.1. Survival after rehydration reflects the dry state experience 351

12.3.2.2. First events of seed rehydration 35212.3.2.3. Partial rehydration: priming 35312.3.2.4. The recalcitrant seeds 354

12.3.3. Pollen 35412.3.3.1. First events of pollen rehydration 355

12.3.4. Whole plants 35612.3.5. Requirements for successful rehydration of a genome 356

12.4. Acknowledgements 35912.5. References 359



12.1. Introduction

The period of water uptake by a dry anhy-drobiote is one of hazard and sensitivity tostresses. Imbibitional injury can occur in awide range of desiccated organisms includ-ing seeds (Vertucci, 1989; Hoekstra andGolovina, 1999), pollens (Hoekstra et al.,1992, 1999), algae, mosses and ferns(Bewley, 1979), and yeasts (van Stevenickand Ledeboer, 1974).

The embryos of most seeds, certain pol-lens and spores, many lower plants and adiverse, but limited, selection of wholevascular plants – the so-called resurrectionplants (Oliver et al., 1998) – have devel-oped remarkable mechanisms to permitwater loss without irreparable damage totheir cytoplasmic integrity and withoutimperilling their future survival. But thosecell types that successfully pass throughthe hazard of dehydration must undergopreparation for this event ahead of time,and it is critical to the success of dehydra-tion and to the subsequent orderly accom-plishment of rehydration that both cellularcompartmentation and genetic informationare maintained unimpaired. The restora-tion of cytoplasmic organization and func-tion within minutes of water beingreadmitted to these same dehydrated cellsremains to be properly understood, partic-ularly since all dry tissues show some leak-age of cytoplasmic contents on their firstimpact with free water.

Several important crops, particularlythose of tropical and subtropical origins,are known to suffer when the seeds take upwater at chilling temperatures. These cropsinclude soybean, maize, sorghum, cotton,bean, cowpea and rice. Injury may alsooccur at room temperature in species suchas pea and Brassica sp. when the seeds arevery dry before imbibition. Upon imbibi-tional stress, dry anhydrobiotes leaksolutes and macromolecules, leading tofaulty metabolism, loss of cellular integrityand/or infection by opportunisticpathogens, which consequently lead to thedeath of the tissues. Despite its widespreadoccurrence, the impact of imbibitionalstress on the expression of desiccation tol-

erance has not always been fully appreci-ated. For example, Kovach and Bradford(1992) showed that the loss of viability inwild rice was due to imbibitional damagethat had previously been interpreted asdesiccation intolerance. A similar conclu-sion was reached by Ellis et al. (1990) withpea seeds and by Sacandé et al. (1998) withseeds of neem, a tropical recalcitrantspecies, which exhibits an orthodox behav-iour when stored seeds are rehydratedabove 20–25°C. Vegetative anhydrobiotictissues (mosses, lichens and resurrectionplants) also appear to be sensitive to therehydration phase (Oliver et al., 1997,1998; Quartacci et al., 1997).

Essential components for this mainte-nance of cellular integrity throughout theprocesses of drying and rehydration are thestructural organization and composition ofintracellular membranes and the fidelityand conformation of the DNA in nuclearchromatin and probably also that in mito-chondria and plastids.

This chapter addresses what is currentlyknown and speculated upon for these twomajor determining factors in cell survivalunder the temporal and successive condi-tions of changing water availability.

12.2. The Dangers of Rehydration andMembrane Changes

12.2.1. Factors governing imbibitional injury

The sensitivity of seeds and pollen to imbi-bitional stress is controlled by at least threefactors: the initial moisture content of thetissues, the temperature of the imbibitionmedium and the rate of water uptake.Chilling temperatures, very low moisturecontents and rapid water uptake generallyresult in greater injury (Fig. 12.1). Slowingdown the rate of water uptake by soakingseeds in polyethylene glycol solutionsimproves the quality of seedlings of vari-ous species (Woodstock and Taylorson,1981; Vertucci and Leopold, 1983; Priestleyand Leopold, 1986; Chern and Sung, 1991).While the low water potential of dry anhy-drobiotes is the driving force for imbibi-

344 D.J. Osborne et al.


tion, the permeability of the seed regulatesthe rate of water uptake. The permeabilityof seed tissues to water is a complex func-tion of the seed morphology, structure,composition and water content. It is note-worthy that, during ageing, seeds andpollen become increasingly sensitive toimbibition (Woodstock and Taylorson,1981; Bruggink et al., 1991; van Bilsen etal., 1994; Sacandé et al., 1998).

Experimental evidence (Vertucci andLeopold, 1983; Wolk et al., 1989) and theo-retical considerations about water penetra-tion in dry materials (reviewed by Vertucci,1989) suggest that the water uptake occursin two phases: an initial wetting phase anda subsequent hydraulic flow. It has beensuggested that for seeds imbibitional dam-age is imposed by the initial wettingbecause the moisture content at which thisphase is observed corresponds to the mois-

ture content at which the seed is most sen-sitive to imbibition (Vertucci and Leopold,1983). Wolk et al. (1989) suggested that thewettability depends on the water-bindingcharacteristics of the dry tissues. In dry tis-sues a glassy state is presumed to prevail(Chapter 10), which has certain peculiarphysical properties. Whether the conditionof the glassy state plays a role in the initialwettability of the tissues remains to beascertained. Large increases in seed vol-ume are usually observed during imbibi-tion (Vertucci, 1989), and these changes involume have been interpreted as an unfold-ing of biopolymers of unknown nature asthe seed takes up water (Vertucci, 1989).This author calculated that the differencebetween the rate of water uptake and rateof volume change during imbibition isgreatest at the time when the seed is mostsensitive to imbibitional damage, suggest-

Rehydration of Dried Systems 345

Initial water content (% dw)

% G

erm

inat

ed s

eeds

25�C

5�C

100

80

60

40

20

00 5 10 15 20 25

Fig. 12.1. The effect of initial water content of axes on the final percentage of germination of cowpea(Vigna unguiculata). After equilibration at various relative humidities, seeds were soaked in water at 5°C (�)or 25°C (�) for 1 h, then rolled in filter papers and incubated at 20°C for 7 days. At both temperatures, thereis a threshold water content below which seeds suffer from imbibitional injury. Least-squares linearregressions were determined for the data below the threshold water content. Water contents are expressedon a dry weight (dw) basis.


ing that structural factors may contribute toimbibitional damage. It is likely that thevolume increase during imbibition alsointerferes with the wettability of the tissues(Vertucci and Leopold, 1983).

The considerable volume of literature onimbibitional injury points to plasma mem-branes as the target of the rehydrationstress. Indeed, imbibitional injury interfereswith the rapid re-establishment of mem-branes, as has been shown from the exten-sive leakage of cytoplasmic solutes and thedisorganization and ruptured appearance ofmembranes following rehydration(Vertucci, 1989; Bedi and Basra, 1993;Hoekstra et al., 1999). One can argue thatplasma membranes are the first macromole-cular structures to be affected during imbi-bition because solute leakage may occurwithin a few seconds to minutes, which isbefore the resumption of metabolism.

12.2.2. Causes for solute leakage asmechanisms of imbibitional injury: the fate of

plasma membranes during rehydration

In the past, several hypotheses have beenpromoted to explain the mechanisms ofleakage during the imbibition of seeds andpollen. Successive hypotheses (e.g. the dis-ruption of membranes in the dry state, orthe formation of hexagonal phases), whichfirst prevailed and were then abandoned,have been recently reviewed by Crowe etal. (1997), Hoekstra and Golovina (1999)and Hoekstra et al. (1999). Our currentunderstanding points to two causes thatlead to solute leakage during rehydrationas a result of imbibitional injury: the physi-cal and/or conformational properties ofmembranes in the dry state (see Chapter 9;Crowe et al., 1992) and mechanical stressesimposed during rehydration (Spaeth, 1987;Vertucci, 1989; Hoekstra et al., 1999).

12.2.2.1. Conformational properties ofplasma membranes

Imbibitional injury in anhydrobiotes hasbeen linked to the occurrence of mem-branes in gel phase in the dry state. During

rehydration, a transition from a gel to a liquid phase occurs under those conditionsof temperature and moisture content thatpromote solute leakage and loss of viabilityupon imbibition (Hoekstra et al., 1992,1999; Hoekstra and Golovina, 1999). Earlierexperiments on liposomes had shown thatduring a thermotropic phase transitionfrom gel to liquid crystalline, membranesbecome leaky (Crowe et al., 1989). Leakageis thought to occur because of the struc-tural defects at the boundary between thecoexisting phases. Analogous to model sys-tems, it was suggested that the transientlycoexisting phases allow leakage of soluteson the penetration of liquid water in thedry plant tissues (Crowe et al., 1989;Hoekstra et al., 1992). This hypothesisreceived support from pollen experimentsshowing that treatments prior to imbibitionthat reduce leakage (such as exposure tohumid air or an increase in soaking tem-perature) also promote the return of thephospholipid bilayer to the liquid-crystalline state (Crowe et al., 1992;Hoekstra et al., 1992). Impatiens pollen iscomparatively tolerant to imbibition at lowtemperature (Hoekstra and Golovina,1999). It appears that the phase transitiontemperature of plasma membranes in dryImpatiens pollen is very low, suggestingthat a gel phase barely forms, thus prevent-ing leakage during imbibition. Thus, thesedata would imply that the transition tem-perature (Tm) at which membranes undergoa phase change can determine imbibitionalinjury.

Recent evidence, however, now suggeststhat changes in membrane phase per se areinsufficient to explain the permanent loss ofmembrane integrity in rehydrating tissues.In Typha latifolia pollen, the permeabilityafter a membrane-phase change during rehy-dration appears to exhibit both a transientand permanent character (Hoekstra et al.,1999). Using an electron paramagnetic reso-nance (EPR) spin-probe technique that per-mits a detailed analysis of the kinetics ofleakage during imbibition of pollen, it wasshown that plasma membranes are highlypermeable within the first 10 s of imbibi-tion. Furthermore, this permeability persists



in conditions that permit plasma mem-branes to pass through a phase transition(Hoekstra et al., 1999). To explain this obser-vation, another mechanism leading toincreased permeability has been proposed.Golovina et al. (1998) showed that amphi-pathic compounds such as flavonoids,which are present in the cytoplasm, parti-tion into the lipid phase during drying andvice versa during rehydration. Endogenousamphipathic compounds extracted fromvarious dry anhydrobiotes were found tofluidize membranes isolated from theseorganisms and also from prepared lipo-somes (Hoekstra et al., 1997; Golovina et al.,1998). Results on liposomes and in situexperiments on pollen using an amphiphilicspin probe suggest that an anhydrobiotewould leak as long as the amphiphilic com-pounds resided in the plasma membrane.Since the increase in water volume duringimbibition eventually induces the partition-ing back into the cytoplasm (Golovina et al.,1998), leakage should then cease. The tran-sient nature of such leakage was confirmedwith leakage experiments using a fluores-cent dye (Hoekstra et al., 1999). The tran-sient leakage is thought to be responsible forsome loss of germinative vigour of thepollen but not of viability. The above obser-vations lead to the conclusion that the per-manent nature of the leakage that leads toloss of viability must originate from an irre-versible damage that is other than anamphipath partition phenomenon. In con-trast to liposome systems, where the transi-tion from the gel to liquid crystalline phaseduring rehydration is accompanied by atransient leakage, it is possible that a phasetransition in anhydrobiotes may inducemuch more severe and irreversible changesduring rehydration (Hoekstra et al., 1999).Indeed, electron microscope observations offreeze–fracture images of imbibitionallyinjured pollen and seeds show folding irreg-ularities and holes in the plasma mem-branes (Hoekstra et al., 1999; Claessens etal., 2000). Similar damage has been widelyreported in earlier electron microscopestudies in which dry tissues were fixed incold aqueous fixatives (Buttrose, 1973).When anhydrous fixation is used, plasma

membranes of dry mosses, pollen and seedsappear intact (Platt et al., 1994, 1997;Hoekstra et al., 1999; Claessens et al., 2000).

12.2.2.2. Mechanical stress

Whether the ultrastructural damage tomembranes upon imbibition results frommechanical stresses and/or changes inphysical properties of the membrane phaseremains to be fully ascertained. Severallines of evidence suggest that membraneswill undergo considerable mechanicalstress during imbibition. Vertucci (1989)estimated that the rate of volume increaseon imbibition is larger than the rate ofwater uptake. Thus, the resulting cellularexpansion may stretch the plasma mem-brane beyond its extensibility limit andinduce lesions, as demonstrated for cucum-ber cotyledons (Willing and Leopold,1983). Spaeth (1987) suggested that inter-nal pressure during imbibition may be adriving force for membrane damage. Hissuggestion was based on the observationsof proteins and starch grains that wereextruded through blisters formed on thesurface of imbibed cotyledons of bean andpea. The process of extrusion resembled aprocess in which viscous fluids are forcedby internal pressure through irregular ori-fices (Spaeth, 1987). Further evidence sup-porting the role of internal pressure inimbibitional injury comes from studies onthe formation of cracks during imbibition.Cracks in cotyledons originate from tensilestresses in the dry interior of partiallyhydrated tissues (Spaeth, 1987, and refer-ences therein). Since tensile stresses aredue to compressive strains (i.e. a form ofpressure), it implies that there is an inter-nal pressure that is applied on partiallyimbibed tissues. However, this contentionhas not received further attention.

12.2.3. Protections against imbibitionalinjury

12.2.3.1. Seed coat

The seed coat or testa is extremely impor-tant in protecting the seed from imbibi-



tional injury. Differences in vigour and via-bility of seeds have been associated withthe pigmentation of the testa.Characteristically, seeds of the samespecies with a light-coloured or translucenttesta are more sensitive to imbibition thanseeds with a darker testa. The relationshipbetween the presence of a coloured testaand the absence of imbibition injury hasbeen established in a wide range of crops(e.g. pea, dwarf French bean, faba bean,cowpea; Legesse and Powell, 1992; Kantaret al., 1994; Demir, 1996) and weeds (prosomillet, Khan et al., 1996). A cause–effectrelationship between seed-coat pigmenta-tion and imbibition stress has been con-firmed by comparing the imbibitionaldamage in five pairs of isogenic lines inpea, differing only in the A gene for seedcolour (Powell, 1989).

Several lines of evidence suggest that therole of the seed coat against imbibitionalinjury is to act as a physical barrier that reg-ulates water movement both temporallyand spatially. The kinetics of water uptake(Powell, 1989) in pea seeds indicated thatpigmented seeds imbibed more slowly thanthose with completely or partially light-coloured or white testas. Harvesting prac-tices that damage the seed coat such as theformation of epidermal cracks (Duke et al.,1986; McCormac and Keefe, 1990; Brugginket al., 1991) or the actual removing of thetesta (Abdel Samad and Pierce, 1978; Dukeand Kakefuda, 1981; McDonald et al., 1988)resulted in a higher rate of water uptake bythe dry seeds and higher leakage rate dur-ing imbibition. Even imbibitional damage-resistant seeds become sensitive to chillingstress after testa damage (Tully et al., 1981;Prusinski and Borowska, 1996). Theseseeds also showed increased hydrationrates and solute leakage. In the particularcase of Arabidopsis thaliana, the rate ofwater uptake is likely to be mainly con-trolled by a mucilage layer surrounding theseed coat without the interference of theseed coat tannins and anthocyanins (Albertet al., 1997; Debeaujon et al., 2000). Thismay explain the lack of correlation betweenimbibitional stress and testa colour inArabidopsis seeds.

In certain crops, the seed coat semi-per-meability or impermeability to water hasbeen attributed to the presence of waxymaterials embedded in the epidermis(McDonald et al., 1988) and high levels ofphenolics or hydroxyphenolics that areoxidized (Marbach and Mayer, 1974)and/or polymerized into insoluble ligninpolymers (Marbach and Mayer, 1974; Egleyet al., 1983, and references therein). In soy-bean, hydroxyproline-rich glycoproteinsaccumulate in large amounts in the seedcoat during late maturation. They could beinvolved in regulating the water entry inthe embryonic tissues (Cassab et al., 1985).It is not yet understood how these proteinsor polymeric substances interact withwater molecules to influence the rate ofimbibition. According to Powell (1989), thepresence of anthocyanins in coloured seedcoat is thought to decrease the wettabilityof the inner surface of the seed coat.However, in pea, this cannot explain whycoloured seeds suffered less imbibitionaldamage than white seeds. Indeed, the dif-ferences in imbibition kinetics betweencoloured and white seeds were maintainedwhen the seed coat was removed (Powell,1989). In resurrection plants, there is nodirect evidence indicating that protectivelayers may regulate the rehydration ofdried tissues. However, several studiesreport that leaves of Craterostigma sp.,Xerophyta viscosa and Sporobolus stapfi-anus are covered with waxes or cuticularcoatings or hairy structures, which arethought to control the loss of water duringdrying (Dalla Vecchia et al., 1998; Sherwinand Farrant, 1998). Thus, it may be possi-ble that these epicuticular waxes and otherepidermal features may also act to regulatewater entry during rehydration.

Decoated seeds suffer more from imbibi-tional damage than do intact seeds.However, in groundnut, the seed coat doesnot appear to pose a physical barrier torehydration of the embryonic tissues(Abdel Samad and Pierce, 1978). Thisobservation leads to the suggestion that fac-tors other than regulating the water uptakemay be involved in protecting the seed tis-sues from imbibitional stress. Considering



that the seed coat contains phenolics,which are amphiphilic substances(Marbach and Mayer, 1974), and that thesesubstances can alter the conformationalstate of the membranes (Hoekstra et al.,1997; Golovina et al., 1998), it would beinteresting to investigate whetheramphiphilic substances migrate from theseed coat to the surface cells of the embryowith the water front, thereby modifying thephysical properties of membranes con-comitant with the water entry. Theswelling properties of the seed coat duringimbibition may also contribute to the regu-lation of water uptake. For example, thetesta of coloured pea seeds were found toremain closely associated with the cotyle-dons whereas the white seed coats wereloosened and water was held between theseed tissues and the testa (Powell, 1989).

12.2.3.2. Protection at the molecular level

Have anhydrobiotes evolved protectingstrategies at the molecular level against thedangers of rehydration? Studies on pollenmembranes during rehydration havedemonstrated that depressing the phasetransition temperature of dry tissues maybe beneficial in reducing the risk of leakageduring water uptake. This depression isthought to be achieved by altering thephospholipid composition, as in Impatienspollen, and/or synthesis of fluidizingamphipaths that partition into the mem-brane during drying (Hoekstra andGolovina, 1999). From results of in vitroexperiments using liposomes, it has beensuggested that disaccharides (sucrose andtrehalose) can reduce the risk of imbibi-tional injury by suppressing the transitiontemperature of the membrane during dry-ing and increasing fluidity (Crowe et al.,1997). However, the concentrations of sug-ars are not sufficient to protect fully in vivomembranes of dry anhydrobiotes, even ifall sugar molecules would form hydrogenbonds with the phospholipid head groups(Hoekstra et al., 1997).

Another strategy has evolved in mossesand resurrection plants. In these tissues, theextensive damage that occurs during drying

and/or water uptake is repaired by mole-cules that are synthesized during dryingand/or rehydration. Work on Tortula ruralisand two resurrection plants (Craterostigmaplantagineum and Sporobolus stapfianus)has identified a series of transcripts, whichare referred to as rehydrins (Ingram andBartels, 1996; Oliver et al., 1997; seeChapters 1 and 11). So far, three of the rehy-drin transcripts (Tr155, Tr213 and Tr288)have been studied. Tr213 shows a highdegree of similarity to polyubiquitins. Themain function of ubiquitin is to eliminateundesirable proteins that are damaged orbeing recycled. The degradation involvesthe conjugation of ubiquitin with targetedproteins and degradation via a reaction cas-cade involving the proteosome (Jentsch andSchlenker, 1995). The presence of polyubiq-uitins during rehydration points to anincreased removal of proteins during imbi-bitional stress. Thus, repair machineryappears to play an active role during rehy-dration of vegetative tissues in eliminatingdamaged proteins, which are accumulatedduring drying. The mechanisms that regu-late the synthesis of this machinery appearto be complex. Rehydrins can be constitu-tively expressed in the plant or both quali-tatively and quantitatively transcribedduring dehydration and/or rehydration(Ingram and Bartels, 1996; Oliver et al.,1997; O’Mahony and Oliver, 1999). Thequestion remains as to whether a ubiquitin-based mechanism of repair is also essentialto alleviate imbibitional stress in seeds. Inthis respect, Tr288 is an interesting tran-script because it has similarity to a tran-script specifically expressed duringrehydration of dormant embryos of barleyand Bromus secalinas (Oliver et al., 1997).

In addition to repair mechanisms, dryanhydrobiotes appear to be endowed withspecific proteins that protect macromolecu-lar structures from the rapid entry of water,although data supporting this idea arescant. Dry oily seeds contain large amountsof oleosins, the interfacial proteins thatsurround the oil bodies. The main functionof oleosins is thought to be to maintain theintegrity of the oil bodies as discreteorganelles during rehydration (Leprince et



al., 1998). In those recalcitrant seeds thatare devoid of oleosins (cocoa), the oil bod-ies remain relatively stable after slow orfast drying, but, during rehydration, theyfuse to form large droplets, resulting in theloss of cellular integrity. So far, no struc-tural proteins other than the oleosins havebeen detected in dry anhydrobiotes thatcould similarly protect cytoplasmicorganelles during imbibition.

12.3. Maintaining Integrity of theGenome

12.3.1. Hydration-determined changes in DNA

Cells in the dry state are never wholly dry.Molecules of water are closely associatedwith specific chemical groups on thecharged proteins and nucleic acids withinthe cytoplasmic matrix. In the nuclearDNA, water molecules are intrinsic to thephosphate backbone of the fully hydratedB-form DNA that exists in most living cells.Loss of water molecules from the DNAbackbone can, in vitro, successively con-vert B-form to A-form and Z-form confor-mations and there is evidence for suchconformational changes during dehydra-tion in prokaryotes (Setlow, 1992a) andduring differentiation in specializedeukaryote cells (Nordheim et al., 1986). AsSetlow (1992a) has shown for Bacillus subtilis, the A-form is maintained by thebinding of small acidic soluble proteins(SASPs) synthesized during dehydration,and the dry spores, with their A-form DNA,are remarkably resilient to high or low tem-peratures and to chemical stress.Furthermore, the A-form is converted backto the normal B-form when the spores arerehydrated through the action of an SASP-specific protease, at which point the sporeslose their tolerance to desiccation andother stresses (Setlow, 1992b). This indi-cates the critical part played by availablewater and DNA conformation in determin-ing the survival of the bacterial sporethrough both dehydration and rehydrationprocesses.

Currently we have no information as towhether such hydration-driven changesoccur during the successful desiccationand rehydration of any plant cell or howfar chromatin topology might be altered byeven small changes in the overall water sta-tus. However, the variable and decreasinglongevity of seeds held under differentconditions of even small levels of increas-ing humidity tells us at once that the cyto-plasm of embryo cells is in a physicallydynamic state with intracellular molecularinteractions undergoing constant change.Since so little is known of DNA integrity inthe mitochondria or plastids in seeds orpollen, this part of the rehydration chapteris essentially confined to what is known ofnuclear DNA.

12.3.2. Seeds

Perhaps the earliest evidence for nuclearDNA changes in stored dry seeds has comefrom microscopic studies showing theincrease in chromosomal aberrations thatappear in nuclei of embryo cells (Navashin,1933). That embryos could still germinateafter such DNA damage and that cells didnot necessarily perpetuate the initial chro-mosomal aberrations through subsequentmeristematic cell divisions were the firstclear evidence for DNA repair processesoperating early during the biochemicalevents of rehydration (Nichols, 1941).However, the extent of change during stor-age also depends upon the condition of theseed when it is shed from the parent plant.The maternal history during seed develop-ment and maturation is partly determinedgenetically and partly determined by theenvironmental conditions (temperature,humidity, sunlight) that the mother plantexperiences. Thus, when the seed is har-vested or shed, factors such as potentialdormancy, seed-coat restrictions andembryo vigour are already predeterminedvariables. Given the signal inputs that anembryo receives during development, theuncertainties of the maturation and finaldesiccation phases, and the continuousmolecular changes that progress through-



out the period of seed storage, it can beseen that the biochemical processes ofrehydration and germination do not repre-sent a single or simple system.

12.3.2.1. Survival after rehydration reflectsthe dry state experience

Continued storage, even under optimumconditions, leads to a progressive deteriora-tion, and all the events listed timewise forrye (see following section) are reduced inactivity and extended in time whenretested as the period of storage becomeslonger. The progressive lessening of theability to incorporate amino acids into pro-tein in the early hours of imbibition is ameasure of an embryo’s being no longerable to germinate quickly. The time to rootemergence, the first critical round of S-phase DNA synthesis and the first cytoki-nesis can be delayed for hours or even days(Osborne, 1983). Eventually, the embryocan no longer synthesize protein at all onrehydration, but, interestingly, there is alate stage in the detrimental programme ofchange in the dry state when transcriptionof short oligonucleotides can still takeplace in the nucleus; however, neitherstored messages nor these new small tran-scripts are translated by the embryo (Brayand Dasgupta, 1976; Sen and Osborne,1977). Currently, we do not know whatthese small transcripts might code for.They might, of course, just represent themost stable sequences in the heterochro-matin of genomic DNA and do not there-fore code for anything that is critical toembryo survival.

The detrimental changes in an ageingembryo are multiple and much work hasbeen directed towards determining the bio-chemical and physical nature of theseevents. Of all the macromolecules of theliving cell, only one, DNA, is known to beroutinely repaired. A caveat may be addedhere that the post-transcriptional editing ofmRNA has also been considered as a repairprocess as the molecule is not degradedbefore bases are removed and replaced.Such editing was first detected in mito-chondrial transcripts of trypanosomes in

1986 and later in plant mitochondria andplastids (for review, see Benne, 1996) andinvolves the insertion or deletion of uri-dine residues in a complex of severalenzymes (the editosome), or direct conver-sions of cytosine to uridine (Smith et al.,1997). The conversion of L-aspartylresidues in ageing proteins into abnormalL-isoaspartyl groups can also be achievedin many plant and animal tissues by theaction of L-isoaspartyl methyl transferasewithout total protein degradation, and thisenzyme has been shown to be present inseeds (Mudgett and Clarke, 1993). All theother nucleic acids, proteins, lipids andpolysaccharides are subject to synthesisand degradation and are not, as far as weknow, subject to an enzymic, energy-requiring process of molecular repair. Thismakes DNA and the genomic information itcarries of special importance in embryocell survival in the dry state and critical tothe competence for DNA repair when thecells first imbibe water on rehydration.

There is no precise moisture contentthat determines what we call the ‘dry state’of a seed. When embryos reach maturationdehydration to moisture contents below10%, the cytoplasm of the embryo entersinto a glassy state in which molecularmovement is strictly limited (Williams andLeopold, 1989; Sun and Leopold, 1993;Buitink et al., 2000; see Chapter 10). But asdifferent parts of a seed can be at slightlydifferent levels of hydration and the attain-ment of the glassy state in different speciesdepends upon the molecular compositionof the cells (Leopold et al., 1994), the levelsof hydration that provide a glassy state forall the different tissues of a seed can differ.As far as current detection methods canreveal, there is no respiration or ATP gen-eration, no transcription and no translationin dry seeds; synthetic events of all kindsare therefore excluded (Bewley, 1979).Non-energy-requiring processes are not,however, excluded, so free radicals can begenerated as local events and non-energy-requiring enzyme activities such as thoseof nucleases and proteases can occur wherethey are in close molecular juxtapositionwith their substrates. The progressive loss



of DNA integrity to increasing levels of ran-dom-sized small-molecular-weight frag-ments without loss of total DNA (Cheahand Osborne, 1978) and the loss of activityin many enzymes without loss of the actualprotein molecules are evidence of this(Osborne, 1983). But, whereas somehydrolytic enzymic proteins are remark-ably stable (DNases, RNases) and long out-live in function the life span of the embryo(Osborne et al., 1977), others start to loseenzymatic activity soon after harvest andon storage they become progressively lessefficient with time. In this group are theenzymes concerned with DNA repair:nuclear DNA polymerases (Yamaguchi etal., 1978), �-type polymerase (Castroviejoet al., 1990), DNA polymerase-� (Coello-Coutiño et al., 1994) and DNA ligase (Elderet al., 1987), so that, when a fresh or astored seed is rehydrated from the drystate, the extended time required to repairfragmentation lesions in the DNA and tosynthesize new repair enzymes is immedi-ately evident (Elder et al., 1987). This canaccount for the delays in germination thatare observed between freshly harvestedseeds and those that have been stored.

12.3.2.2. First events of seed rehydration

Assuming that seeds have been maturedunder satisfactory conditions on themother plant and that they have not beenheld after harvest in a way that wouldimpair their ability to germinate, then theevents of rehydration from the dry state fol-low an essentially similar pathway ofnuclear reactivation for all the embryosthat have been studied in detail.

On imbibition in water, there is first aphysical hydration of the cytoplasm, whichis usually completed by 60 min(Obroucheva, 1999). This will occur inembryos of dead as well as living seeds(Hallam et al., 1972, 1973) but in livingseeds further uptake then proceeds contin-uously. The transcription of all classes ofRNA within 20–40 min has been demon-strated by electrophoretic fractionation ofnewly synthesized radiolabelled nucleicacids in rye embryos (Sen et al., 1975),

and, using autoradiography, incorporationof tritium(3H)-labelled precursors intonucleoli was evidence of rRNA synthesiswithin 10 min. Incorporation of 3H-thymi-dine into nuclei of embryo cells has shownthat DNA repair (but not replication) is alsoactivated within minutes of imbibition inrye, and incorporation of labelled aminoacids into protein is apparent in the cyto-plasm within 15 min (Osborne et al., 1977).

The initiation of S-phase DNA synthesisand DNA replication from 2C to 4C levels,first evident in nuclei of root-tip meris-tems, occurs late in the process of rehydra-tion and the first cytokinesis may be hoursor days after RNA transcription and proteinsynthesis are fully established. Whetherthe start of replication requires a particularphysical state of hydration of the nucleusis not known. Embryos become highlymetabolically active on imbibition eventhough they may remain dormant; those ofAvena fatua can reach a moisture contentas high as 40% without a sign of growth.These embryos may defer amplification ofnuclear DNA from 2C to 4C levels formonths or years before initiating the entryto cell cycling and attendant germination.The control factors that hold the cells ofimbibed embryos in this homoeostatic butmetabolic state, while also preventing pro-gression into cell cycling, cell expansionand cell division, have still to be deter-mined, and this is still a key question inseed biology. It is of special interest tostudies of desiccation tolerance because,throughout their dormancy, imbibed seedscan be dehydrated back to their originalweight without loss of viability or celldeath. A clue to the survival of dormantembryos is their ability to maintain an activeDNA repair from the time of rehydration andthroughout the duration of imbibition.Although replicative DNA synthesis isblocked, DNA repair synthesis is as effectivein dormant as in germinating embryos. Asexperiments with A. fatua have shown, eventhe repair of �-irradiation-induced single-and double-strand breaks occurs as effi-ciently and in the same time in dormant andnon-dormant imbibed material (Elder andOsborne, 1993).



12.3.2.3. Partial rehydration: priming

It might be concluded that even a shortperiod of imbibition that is then followedby dehydration back to the dry state with-out damage or loss of any of the newly syn-thesized components might lead to animprovement in subsequent seed germina-tion when water is again available. In fact,this is generally true and forms the basis ofpriming seed (Heydecker and Coolbear,1977; van Pijlen et al., 1996). Successfulpriming achieves and conserves both DNArepair and the early events of germination,including a significant level of mitochon-drial DNA synthesis. These are events thatare stable to desiccation and take placebefore the embryo cells pass into the condi-tion of desiccation sensitivity, whenembryo cells will die if dehydrated (Ashrafand Bray, 1993). The conversion from astate of tolerance to cytoplasmic water lossto one of intolerance is still not fullyunderstood, but it coincides with the stagein embryo cells, particularly those of theroot tip, when they approach the firstcytokinesis at G2M and are on the border ofundergoing the first cell division. Provided

visible cell division has not started and theembryo has not been held at G2M for a pro-longed period, imbibed or primed seed canbe dried back safely and the seedling willemerge rapidly on planting (Fig. 12.2).

The secret of successful priming, there-fore, is to establish the stage before the firstcell cycle division when the nucleus hasreinitiated transcription, has fully repairedany DNA damage from the dry state andthe optimal spectrum of newly translatedbut desiccation-stable proteins has beentranslated and they are present in activeform in the cytoplasm.

However, this is not necessarily thewhole story, for, although primed seedswill germinate quickly, they frequentlystore less well (Tarquis and Bradford, 1992;Nascimento and West, 2000), and in somespecies they lose viability more readilythan those of unprimed seeds. The reasonsfor this are uncertain, but would appear tobe linked to the changes that take placeduring priming in the physical state of thenucleus and cytoplasm and to the progres-sion in the stage of the cell cycle at whichthe nuclei are dried back. A low level ofreplicative DNA synthesis continues


90

50

10

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oist

ure

cont

ent

Desiccation-sensitive

Desiccation-sensitive

Desiccation-tolerant

Replicationarrest

G1

G 1 →

G2M Cytokinesis

Seedmaturation

Drystate

Rehydration

Fig. 12.2. Progression of hydration-linked cell cycle events in a seed embryo during water restriction atmaturation and the renewal of free water at rehydration, showing the relation with desiccation tolerance.


throughout priming, progressing from 2Ctowards 4C though not to cytokinesis.There is evidence that cells will accumu-late at G2M and this may represent a topol-ogy for nuclear DNA that is particularlyaccessible to nuclease fragmentation. Notall imbibed or primed seeds from differentspecies will reach this same condition ofcytoplasmic or nuclear organization afterthe same duration of rehydration, andtherefore on drying back they will notreach similar stages of vulnerability to stor-age. A comparison of primed seeds (ach-enes) of Ranunculus sceleratus andRanunculus arvensis is one such example.With R. sceleratus, longevity of the dried-back seeds is increased, whilst that of R.arvensis is reduced (Probert et al., 1991).

12.3.2.4. The recalcitrant seeds

Many seeds, particular those of tropicalspecies, and many large-seeded species oftemperate climates maintain a high per-centage of water in both axes and cotyle-dons. Although they can accommodate asmall amount of water loss without harm,desiccation to levels of 10–26% are lethaland embryos do not restore synthetic activ-ity when rehydrated. A feature that hasnow been explored is whether or not theDNA repair function is lost on dehydrationof these seeds below a certain critical level.In Avicennia marina, a mangrove speciesindigenous to the tropics and subtropics, awater loss from the seed that exceeds20–30% normally leads to seed death.Experimental samples of the excised axeswere dehydrated under a cool air stream todifferent levels of water loss, then irradi-ated from a �-source to introduce a similarlevel of single- and double-strand breaks ineach, and then rehydrated for a period of2 h (Boubriak et al., 2000). Results haveshown that fully hydrated embryos (Fig.12.3a and b) permit almost full recovery offragmented DNA to levels of that in unirra-diated controls. However, generating a 22%dehydration of the axes prior to irradiationled to the complete failure of DNA repair(Fig. 12.3c) and, by 46% dehydration, thefragmentation of DNA continued into fur-

ther low molecular weight DNA disintegra-tion (Fig. 12.3d).

12.3.3. Pollen

Certain pollens can stay dry and alive forprolonged periods of time, depending onthe ambient relative humidity. The level ofmoisture in dry pollen can vary and is low-est (c. 8%) for wind-dispersed pollen.Damage to these dry pollen cells can bemore severe than that for the embryos ofseeds. This is because the individual wind-blown pollen cells are unprotected fromultraviolet light (UV) and other environ-mental stresses from the moment they areshed from the anther. Since these pollencells are haploid and lack the resource thatdoubled (diploid) genetic information pro-vides, damage to the single genome canresult in serious genetic consequences tothe next generation. In these special cir-cumstances, DNA repair in pollen plays acrucial role and both photoreactivation anddark DNA repair systems operate togetherin a germinating pollen grain (Ikenaga andMabuchi, 1966; Jackson and Linskens,1978). Incorporation of 3H-thymidine ingerminating pollen grains of Petunia startsimmediately water becomes available andcontinued incorporation in the presence ofhydroxyurea (which blocks replication)confirmed active DNA repair synthesis ingerminating pollen. Hydration of birchpollen at 100% humidity is not of itselfsufficient to permit full excision repair,although the germination rate is improved,but, on transfer to free water, fully imbibedpollen will then complete repair within 2 h(Grodzinsky and Bubryak, 1985). Duringrehydration, the excision DNA repair sys-tem of pollen can remove a number of dif-ferently induced lesions incurred in thedry state, including those of chemicalmutagenesis (Jackson and Linskens, 1978,1979), heavy metal damage and �-irradia-tion (Jackson and Linskens, 1982; Bubryaket al., 1991). Efficiency of such repair candiffer in pollens depending upon the dif-ferent levels of environmental stress underwhich they were formed, i.e. lowland or



high-altitude mountains (Bubryak andGrodzinsky, 1985; Grodzinsky andBubryak, 1988).

12.3.3.1. First events of pollen rehydration

Efficient DNA repair during the imbibitionof pollen grains is an essential physiologi-cal event only for two-nuclei pollen grains(e.g. birch) where, at shedding, eachnucleus is held at G2 (2C DNA values).During germination, the generative nucleusof the pollen grain divides to produce twohaploid sperm cells, so it is at this G2checkpoint that DNA repair is absolutely

essential to retain genetic fidelity. This isnot so if pollen is shed as a three-nucleicell in which the generative nucleus hasalready divided to produce the two haploidsperm cells (as in maize). For a long time ithas been puzzling that there was no evi-dence for excision repair during rehydra-tion of maize pollen even though this isalso wind-distributed and can therefore bedamaged by UV. Maize pollen is viable fora few days only, so it is possible that noDNA repair system is maintained in thesehaploid sperm cells since they havealready passed their last G2 checkpoint.

An efficient DNA repair system is not


1.2

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750 Gy (fully hydrated)

750 Gy (22% dehydrated)2 h Rehydrated

750 Gy (fully hydrated)2 h Imbibed

750 Gy (46% dehydrated)2 h Rehydrated

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Migration

Abs

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(a)

(c)

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(d)

Low Mol. Wt. High Mol. Wt.

Fig. 12.3. Loss of DNA integrity with loss of DNA repair capability in embryo axes of Avicennia marinaseeds following different levels of dehydration, shown by competence to repair a �-irradiation damage of750 Gy during 2 h rehydration. Molecular weight profiles of scans of DNA fractionated by electrophoresison neutral agarose gels: (a) fully hydrated axes after �-irradiation ((Low Mol. Wt.) DNA strand breaksinduced); (b) fully hydrated axes, �-irradiated, then rehydrated 2 h ((Low Mol. Wt.) DNA repaired); (c) 22%dehydrated axes, �-irradiated, then rehydrated 2 h (no DNA repair); and (d) 46% dehydrated axes, �-irradiated, then rehydrated 2 h (further DNA disintegration).


confined to wind-pollinating pollens buthas been shown to occur also in the two-cell pollens of certain insect-pollinatedplants such as Petunia and Amaryllis.These pollens can repair induced UV and�-irradiation damage despite the fact thatthey rarely receive a high radiation doseduring the pollination process. Althoughsuch repair can occur, it is less efficientthan in wind-pollinated species (Bubryakand Grodzinsky, 1985).

It is quite possible that, in pollen, check-point repair (in G2) for generative nucleirequires time and, therefore, a repair capa-bility to withstand DNA damaging factors inthe environment is maintained for longer.This may include desiccation tolerance ofthe two-nuclear pollen both for wind-polli-nated and insect-pollinated species. Beforeshedding from the hydrated enclosure of thecatkin, pollen is killed if it is dehydrated,but at shedding (when the moisture contentis reduced to 8–14%) pollen becomes stableto dry storage for long periods of time (yearsfor hazel and birch). Such pollen can behydrated in moist air and dehydrated backseveral times without losing the capabilityfor excision repair and for germination.However, if exposed to liquid water,hydrated pollen grains germinate and then,like the embryos of seeds, the germinatedpollen loses tolerance to drying andbecomes, once again, desiccation-sensitive(Osborne and Boubriak, 1994). What we donot know is the topological organization ofpollen nuclear DNA throughout thesechanging levels in water status.

12.3.4. Whole plants

Much interest is currently directed towardsthe survival mechanisms of vascular plantsunder drought conditions, and a relativelysmall group of plants including mono-cotyledon grasses, dicotyledon ‘resurrec-tion plants’, many liverworts, mosses andferns are sufficiently drought-tolerant tosurvive a 95% water loss for months oryears, then recover and become metaboli-cally active when free water is again avail-able (Bewley, 1979; Gaff, 1980). Much is

now known of the genes induced duringdehydration processes in these desiccation-tolerant plants (Neale et al., 2000; seeChapter 11) and of mechanisms involvedin transcription and translation (Oliver etal., 1997, 1998). However, we have littleevidence yet that links the successful rehy-dration of these plants to their competenceeither to sustain fidelity of the nuclear,mitochondrial or plastid genomes on dehy-dration or, perhaps more importantly, tothe restoration the overall fidelity of thedifferent genomes on rehydration.

Current knowledge indicates that pro-gressive changing states of gene expressionoccur at distinct thresholds of hydrationthroughout a dehydration process (Neale etal., 2000; Chapter 11). The possibility thenarises that there could be alterations in theconformational state of DNA as it becomesprogressively dehydrated, so perhaps thisarea should be investigated for evidence ofa transition to A-form DNA. What occurson rehydration is even less sure, and howfar cell survival depends upon the mainte-nance of genomic fidelity during the periodof desiccation or upon the repair of DNAlesions at rehydration, or both, remains tobe discovered.

At least it now seems sure that waterdeficits can lead to a rise in abscisic acid(ABA) levels and thence to the induction ofABA-directed new gene expressions. It isalso now sure, however, that desiccationtolerance is not always controlled by ABAin all plants. In certain mosses and ferns,for example, ABA is either not detected orthe levels may actually fall during desicca-tion (Reynolds and Bewley, 1993).Considering the diverse groups of plantsand tissues that have acquired desiccationtolerance, it seems likely that more thanone survival mechanism will have evolved.For all, however, preservation of geneticinformation will be paramount.

12.3.5. Requirements for successfulrehydration of a genome

There would appear to be two aspects ofthe rehydration of a dry but living cell that



are essential for the successful re-establish-ment of organized metabolic activity. Oneis the physical rehydration of dryorganelles and the rehydration of a drycytoplasm. This involves the molecularreorganization of the membranes definingeach region of compartmentation, the rehy-dration of the protein–polysaccharidestructures of the cell walls and the free-water access to folded macromolecules ofproteins and nucleic acids (see Section12.2). Before information exchange cantake place between nucleus and cytoplasm,the nuclear chromatin must itself be rehy-drated and the DNA made available for reg-ulated transcription. For accurate transferof genomic information to the cytoplasm,any damage present in DNA must first bereplaced. Only if this second and metabolicaspect of rehydration is achieved can a cellre-establish directed metabolic activitywith an opportunity for renewed cellgrowth and development. Only if the mito-chondrial genome is also fully restored canan integrated informational exchange prop-erly operate.

Another aspect of DNA degradation, therecovery from which could be consideredas a DNA repair operation, is the loss ofrepetitive telomeric sequences from the ter-mini of DNA molecules within the doublehelix. Ageing human fibroblasts show ashortening of these telomeric regions(Harley et al., 1990) and convincing exten-sions of the life span in retinal pigmentepithelial cells has been achieved by trans-fecting cells obtained from telomerase-negative cell types with vectors encodingthe human telomerase catalytic subunit(Bodnar et al., 1998); telomere-expressingclones have elongated telomeres, contin-ued cell divisions and an absence of senes-cence.

Telomeres and telomerases are presenton the chromosomes in nearly all plantcells (Adams et al., 2000; Leitch, 2000).The role of telomerases in seeds is there-fore of considerable interest with respect toageing and the events of early rehydration.In studies of DNA repair in embryos of rye(Vazquez-Ramos and Osborne, 1986), ofwheat (Marciniak et al., 1987) and of maize

(Zarain et al., 1987), an early synthesis ofDNA that did not correspond in characterto either repair or replication was found.One possibility was that this representedmitochondrial DNA synthesis (Vazquez-Ramos and Osborne, 1986) but furtherstudies by Bucholc and Buchowicz (1992)using oligonucleotide-hybridizing tech-niques showed that part of the DNA repairsynthesis that occurs after imbibition of theembryo can be attributed to a new synthe-sis of telomeric DNA. Furthermore, theyfound that one of the degradative changesin DNA of stored wheat embryos is the pro-gressive cleavage and removal of the telom-eric repeats.

This shortening of telomeric DNA cap-ping repeats on storage of seed embryos is,like the loss that occurs in mammaliancells, a measure of their age and may repre-sent a commonality in the progress tosenescence and death. Delay or failure toreinitiate these DNA end groupings maytherefore be another critical factor in deter-mining success in the rehydration that pre-cedes germination. The stability of thetelomere polymerase may be found to playa key role in restoring the overall integrityof the embryo genome.

Although dry seeds appear to accumu-late most DNA damage in the form of single-strand DNA breaks, presumably fromenzymatic endonuclease cleavage or possi-bly also from free-radical attack and byspontaneous base loss (Dandoy et al.,1987), the effects of the accumulation of alltypes of damage before rehydration can belinked to the progressive failure of DNArepair functions when water is again avail-able. Such events have led to the proposi-tion that loss of DNA repair is a majorfactor in determining the loss of genomicintegrity and poor survival of an embryo onrehydration (Elder and Osborne, 1993;Osborne and Boubriak, 1994; Boubriak etal., 1997).

Evidence that competent DNA repair isindeed an essential factor for successfulrehydration comes from experiments withthe embryos of rye (Boubriak et al., 1997).If isolated embryos are �-irradiated from acaesium source (750 Gy) whilst in the dry



state in order to introduce DNA damage,(Fig. 12.4a,b) subsequent rehydration inwater for 2.5 h leads to complete restora-tion of the fragmented DNA to high molec-ular weight (Fig. 12.4c). These embryossurvive. However, if DNA repair is blockedby inhibitors of both �- and �-polymerase(aphidicolin and dideoxythymidine tri-phosphate, respectively), DNA integrity isnot restored, DNA becomes even more frag-mented over 2.5 h and the embryos die (seeDNA scans in Fig. 12.4d). The inhibition ofDNA repair has prevented the successfulrehydration and survival of these DNA-damaged embryos, even though they were

still in the desiccation-tolerant periodwhen rehydrated (Boubriak et al., 1997). Itwould be of considerable interest now toassess if there is a similar requirement forfunctional DNA repair during the rehydra-tion of other desiccation-tolerant and leafyspecies such as mosses and resurrectionplants.

Although nuclear transcription in theembryos of fresh, dry seeds starts almostimmediately on imbibition, it is unclear atwhat level of hydration the mitochondriafirst become active and ATP first becomesavailable for synthetic processes (Attucci etal., 1991). It may be as low as 14% (apple


Control (dry)

750 Gy (rehydrated 2.5 h)

750 Gy (dry)

750 Gy (rehydrated 2.5 hin AP + dTTP)

Abs

orba

nce

at 2

60 n

m (

rela

tive

units

)

1.5

1.0

(a) (b)

(c) (d)

1.6

1.1

1.5

1.1

1.5

1.0

MigrationLow Mol. Wt. High Mol. Wt.

Fig. 12.4. Restoration of DNA integrity on rehydration requires DNA repair, shown by the effect of blockingDNA repair in �-irradiated (750 Gy) embryos of rye, Secale cereale. Molecular weight profiles of scans of DNAfractionated by electrophoresis on neutral agarose gels from: (a) untreated dry embryos (controls); (b) dryembryos �-irradiated ((Low Mol. Wt.) DNA strand breaks induced); (c) irradiated embryos rehydrated for 2.5 h(DNA repaired); and (d) irradiated embryos rehydrated for 2.5 h in presence of aphidicolin (AP) �-polymeraseinhibitor and dideoxythymidine triphosphate (dTTP) β-polymerase inhibitor (DNA repair blocked).


embryos) or as high as 25% in pea seeds(Leopold and Vertucci, 1989). Because of thelow levels of early ATP synthesis at rehydra-tion, an alternative non-mitochondrialcytosolic source has been sought to fuel theearliest synthetic events through a glycolicfermentation of starch or lipid (Raymond etal., 1985; Perl, 1986). How important thismight be and at what levels of hydration itmight operate are not yet known. Certainly,continued mitochondrial ATP generationhas a critical function in providing theessential energy generation for new proteinsynthesis, including that for new DNArepair enzymes and for the mitochondrialdehydrogenases, both of which lose activitywith time in a stored seed (Throneberry andSmith, 1955; Elder et al., 1987). The extentof the DNA repair function when an embryofirst becomes hydrated from the dry statedepends not only upon the remaining activ-ity of the repair enzymes stored within thedry cells but also upon the available ATP forthis repair to take place in restoring integrityto fragmented nuclear DNA molecules andto the DNA of the mitochondria. Only onthe re-establishment of intact codingsequences in both nucleus and mitochon-dria can new repair-enzyme mRNAs be tran-scribed in either organelle.

These experiments raise intriguing ques-tions in seed and pollen survival, whichremain to be properly resolved. How stableare the circles of mitochondrial DNA? Arethe DNA sequences that code for the DNArepair enzymes specially labile to nucleaseor free-radical assault? Is the conforma-tional folding of DNA, the state of methyla-tion or acetylation and the nature ofDNA-binding proteins at the specific DNArepair sites on these genomes critical tosuccessful DNA repair on rehydration?

Whether or not the sites of DNA cleav-age are specific hypersensitive sites, religa-

tion of these positions and re-establishmentof a fully functional genome are early andnecessary events upon rehydration of bothembryo and pollen cells. The speed andfidelity at which these processes areaccomplished dictate the lag period to theinitiation of the first cell cycle after the drystate and hence determine the eventualsuccess of germination. Not only does DNArepair in seeds have the essential role ofrestoring fidelity to DNA fragmented dur-ing dry storage, it also plays a critical partin maintaining desiccation tolerancethroughout the early hours of rehydration.

Efficient DNA repair is thus a criticalcomponent of the cell machinery, whichmay act immediately on rehydration of alldesiccation-tolerant dry cells, and theextent of the success of DNA repair can beseen as a major factor determining the fateof plant cells following a dehydration/rehydration cycle. It will therefore be ofmuch interest to discover the facts that willbe revealed when resurrection plants arescrutinized for the status of genomic, plas-tid or mitochondrial DNAs following desic-cation and rehydration, and to learnwhether telomere lengths are subject tochange in similar circumstances.


O. Leprince is grateful to the NetherlandsOrganization for Scientific Research, theNetherlands Technological Foundation forScientific Research and the French Ministryof Agriculture and Fisheries for support. DrF.A. Hoekstra is acknowledged for his con-structive comments on Section 12.2.

D.J. Osborne and I. Boubriak acknowl-edge support from Hortlink MAFF (ScottishOffice), Framework IV FAIR5–3711 and theRoyal Society, London.


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Quartacci, M.F., Forti, M., Rascio, N., Vecchia, F.D., Bochicchio, A. and Navari-Izzo, F. (1997)Desiccation-tolerant Sporobolus stapfianus: lipid composition and cellular ultrastructure duringdehydration and rehydration. Journal of Experimental Botany 48, 1269–1279.

Raymond, P., Al-Ani, A. and Pradet, A. (1985) ATP production by respiration and fermentation andenergy charge during aerobis and anaerobis in twelve fatty and starchy seeds. Plant Physiology79, 879–884.

Reynolds, T.L. and Bewley, J.D. (1993) Characterization of protein synthetic changes in a desiccationtolerant fern, Polypodium virginianum – comparison of the effects of drying, rehydration andabscisic acid. Journal of Experimental Botany 44, 921–928.

Sacandé, M., Hoekstra, F.A., van Pijlen, J.G. and Groot, S.P.C. (1998) A multifactorial study of condi-tions influencing longevity of neem (Azadirachta indica) seeds. Seed Science Research 8,473–482.

Sen, S. and Osborne, D.J. (1977) Decline in ribonucleic acid and protein synthesis with loss of viabil-ity during the early hours of imbibition of rye (Secale cereale L.) embryos. Biochemical Journal166, 33–38.

Sen, S., Payne, P.I. and Osborne, D.J. (1975) Early ribonucleic acid synthesis during germination ofrye (Secale cereale) embryos and the relationship to early protein synthesis. BiochemicalJournal 148, 381–387.

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dons during imbibition. Plant Physiology 85, 217–223.Sun, W.Q. and Leopold, A.C. (1993) The glassy state and accelerated aging of soybeans. Physiologia

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Woodstock, L.W. and Taylorson, R.B. (1981) Soaking injury and its reversal with polyethylene glycolin relation to respiratory metabolism in high and low vigor soybean seeds. PhysiologiaPlantarum 53, 263–268.

Yamaguchi, H., Naito, T. and Tatar, A. (1978) Decreased activity of DNA polymerase in seeds of bar-ley during storage. Japan Journal of Genetics 53, 133–135.

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Part V

Retrospect and Prospect



13 Damage and Tolerance in Retrospect and Prospect

Michael Black,1 Ralph L. Obendorf2 and Hugh W. Pritchard31Division of Life Sciences, King’s College, Franklin Wilkins Building, 150 Stamford

Street, London SE1 6NN, UK; 2Seed Biology, Department of Crop and Soil Sciences,Cornell University, Ithaca, New York, USA; 3Seed Conservation Department, Royal

Botanic Gardens Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK

The preceding chapters have provided awide perspective on desiccation in plants.In this brief, concluding chapter we willselect some information from theseaccounts, which point to generalizationsthat can be made. This is not intended as acomprehensive overview or evaluation butrather an attempt to identify some areasthat offer prospects for further research andincrease in our understanding.

The ability to tolerate desiccation obvi-ously lies at the heart of the desiccation andsurvival scenario. But there are problemsimplicit in this statement: how do we defineand measure tolerance and survival? We cansee from foregoing chapters in this book thattolerance, as considered by authorities, cov-ers a wide spectrum of properties from, forexample, a rigorously defined, single bio-physical event (Chapters 4 and 10), such asthe partitioning of an amphiphile into amembrane, to the ability of a system (suchas a seed or a whole plant) to resume nor-mally the whole gamut of its growth andmetabolic abilities. Between these twoextremes there is a range of parameterstaken by researchers to indicate tolerance,and, in some cases, where a high degree oftolerance exists, some measure of intoler-ance nevertheless remains. For example, the

ability to complete germination is oftenemployed as the indicator of desiccation tol-erance in seeds. Since the completion ofgermination, i.e. expansion of the axis torupture the enclosing structures, normallyoccurs before cells begin to divide, germina-tion as an index of tolerance may not reflectthe integrity of cell division. Some authori-ties therefore insist that unequivocal desic-cation tolerance is shown only by seeds thatgerminate to produce normal seedlings,where the capacity for cell division has notbeen compromised. Moreover, since germi-nation in the strict sense is a response spe-cific to the axis, the elongation ability of thepreviously desiccated axis may not neces-sarily indicate the level of tolerance in theremainder of the embryo, i.e. the cotyledonsor scutellum. Considerations of a similartype might also apply to recalcitrance,which is often taken as the ‘archetype’ ofdesiccation sensitivity in seeds. Yet suchseeds possess or exhibit many features oftolerant seeds, such as the occurrence ofabscisic acid (ABA) and late embryogenesisabundant (LEA) proteins, and degrees ofsubcellular integrity maintained after dehy-dration. The characteristics that are criticalto intolerance in recalcitrant seeds indeedprove difficult to identify.



Similar complications exist in respect ofvegetative tissues. For example, some resur-rection plants (the homoiochlorophylloustypes) exhibit a high degree of desiccationtolerance in that the chloroplasts retaintheir organization and chlorophyll afterdehydration, whereas the poikilochloro-phyllous types, though in almost all otherrespects desiccation-tolerant, are intolerantso far as chloroplast and chlorophyllintegrity are concerned. These few exam-ples illustrate that desiccation tolerancemust be carefully defined and that indica-tors of tolerance and intolerance can existside by side in a single biological system.

This complexity might arise partlybecause of the multiplicity of damagingevents that occur as cells dry out (Chapter 9)and the relative readiness for different kindsof damage to be repaired (Chapter 12). Inrespect of the former, it is clear that differentlevels of arrest or damage occur as �decreases, though the biophysical or molecu-lar reasons for this are not clear in all cases.It is interesting to speculate that a similarscale of response to � might also occur in therepair processes during rehydration butmore information on this is needed.

Another factor that might contribute tothe coexistence of tolerance and intoler-ance indicators as mentioned above is thesimultaneous occurrence, as dehydrationprogresses, of protective mechanisms (i.e.conferring tolerance) as well as damageprocesses. The inception of the protectivemechanisms occurs in response to initialdehydration and develops to completionprovided that water loss is not too precipi-tous, while damage increases progressivelyas � falls The rate of drying (Chapter 3)therefore determines the relative progressof protective and damaging events, i.e. thedevelopment of both tolerance and intoler-ance characteristics and their presence inthe final desiccated system.

One important point that has beentouched on in the preceding chapters (e.g.Chapter 10) is the relationship betweendesiccation tolerance and longevity in thedry state: are the same or similar regulatorymechanisms involved? We are far fromresolving this question, which clearly

requires further study. An interesting find-ing is that the majority of desiccation-toler-ant bryophytes age most rapidly in therange �9 to �22 MPa and have greatestlongevity at between �150 and �300 MPa(Chapter 7); this is also generally true fororthodox seeds and pollen (Chapters 5 and6). Thus, the water relations of survival andlongevity appear to be similar in materialthat is physiologically, biochemically andmorphologically diverse. Moreover, develop-ing seeds that are detached from the parentplant readily become desiccation-tolerantwhen held at the upper � range, whereashere most recalcitrant seeds succumb toirreversible damage. These approximatewater-potential ranges thus appear to becrucial for tolerance and longevity, andresearch aimed at understanding theseprocesses should, perhaps, be focused there.At the same time, we must address the prob-lem in mechanistic terms of why seeds ofvarious species show such differences inlongevity around two orders of magnitudeunder identical storage conditions. Thepractical benefits with regard to storage andconservation arising from such knowledgeare obviously extremely important.

The multiplicity of factors participatingin desiccation tolerance have been exten-sively discussed in several chapters. As bio-physical, biochemical, cell biological andmolecular technologies have become moresophisticated and adapted for use in seeds,pollen, spores and vegetative tissues, ourunderstanding of the processes involved intolerance has gathered momentum. Butuncertainty (verging on controversy) hasdeveloped in respect of some topics. Forexample, while the evidence shows thatglass formation is an important element inthe protective mechanisms in ‘dry’ cells, it isnot clear what the components of the vitrifi-cation system are (Chapter 10). Sugars, espe-cially sucrose, are certainly involved, andpossibly also proteins (e.g. LEA proteins) butthe role of oligosaccharides, which onceexercised a strong claim for participation (in longevity also), has been questioned(Chapter 10; Bentsink et al., 2000). Most of the evidence for the participation ofoligosaccharides in tolerance and longevity

368 M. Black et al.


comes from studies of correlations betweenthe oligosaccharide content and the appro-priate physiological property. But, since des-iccation tolerance and presumably longevityare likely to depend on multi-componentaction (e.g. carbohydrates, LEA proteins,antioxidants, free-radical removal, etc.), cor-relative evidence may be difficult to inter-pret if one or more components other than,for example, oligosaccharides become limit-ing: in such a case, the oligosaccharide con-tent would be scarcely relevant. In additionto the raffinose-series oligosaccharides(galactosides of sucrose), other galactosides –of cyclitols – are commonly present in seeds.Maturing seeds of some species accumulatefagopyritols, for example, which have beensuggested to substitute for the role ofoligosaccharides, so knowledge restricted tothe content of the latter might not provide afull picture (e.g. Steadman et al., 1996).

But, although there is persuasive evi-dence against a determinative role foroligosaccharides in desiccation toleranceand longevity, including on genetic grounds(Bentsink et al., 2000), the accumulation ofthese compounds during seed maturation,apparently in response to the incipient dry-ing signal (Blackman et al., 1992; Black etal., 1999) (and similarly stimulated inhypocotyls (Brenac et al., 2002)), demandsan explanation. After its maturation, twomajor developmental steps await the seed –quiescence and then germination. For thecompletion of germination, easily utilizablereserves should be immediately available inthe extending organ itself, usually the radi-cle, and oligosaccharides such as raffinoseoften fulfil this need (see review byPeterbauer and Richter, 2001). It might be,therefore, that the maturing seed uses thesame signal, incipient drying, to register theimminence of two different processes, thefirst being quiescence in the ‘dry’ state, forwhich desiccation tolerance is necessary,and the second being germination, forwhich readily utilizable reserves (relativelylow-molecular-weight carbohydrates suchas the raffinose-series oligosaccharides)must be prepared. The coincidencebetween oligosaccharide accumulation andthe onset of desiccation toleration may

therefore have a spurious appearance of acausal relationship. Longevity is measuredby the ability to germinate, which mightalso reflect reserve oligosaccharide content.

Both drought stress and desiccation inplants have been widely studied at a molec-ular level, especially in vegetative tissues.In the former, numerous genes have beenidentified whose expression is regulated bywater stress. Much has also been learnedabout signal transduction mechanismsoperating in the stress syndrome, and, inthe case of many, but not all genes, ABAparticipates at an early stage in signalling.In the desiccation phenomenon too, expres-sion of many genes occurs in response toearly water loss from vegetative tissues,some of which are, again, ABA-regulated(Chapter 11). But, in contrast, our under-standing of molecular events involved inseed desiccation is less advanced.Expression of several genes in developingseeds is certainly affected by drying, themost intensively studied being those forseveral reserve proteins in which the capac-ity for expression is ‘switched off’ by thedehydration experience (Jiang et al., 1995),an event that is unlikely to contribute todesiccation tolerance. There is some evi-dence, though, that certain reserve proteinsor close relatives might be involved in celldesiccation processes (Chapter 5). Severalpositive effects of drying on gene expres-sion or enzyme production have beenrecorded; but many of these are connectedwith reserve mobilization (e.g. Cornford etal., 1986) and are therefore unlikely to beinvolved in desiccation tolerance. The pro-motive effects of incipient dehydration onoligosaccharide accumulation in maturingseeds (see above) may possibly operatethrough positive action on expression ofgenes involved in their biosynthesis (seereview by Peterbauer and Richter, 2001),such as galactinol synthase or stachyosesynthase, but this has not been determined.The loss of water can also convert a seedfrom the developmental to the germinativemode in respect of patterns of synthesizedproteins, an effect that has been confirmedas operating at gene level (for review, seeKermode, 1995), but again it is not likely

Damage and Tolerance in Retrospect and Prospect 369


that these observed changes are related todesiccation tolerance. The essential pointis, however, that gene expression in devel-oping seeds can be modified by water status,so this effect could participate in the estab-lishment of desiccation tolerance. Themajor relevant genes that might be affectedby drying are those encoding the LEA pro-teins and certain heat-shock proteins, bothof which may aid in the protection againstsome of the damage that desiccation wouldinflict. None the less, it is not certain thatexpression of these genes is universally up-regulated by the hydration state of develop-ing seeds; on the contrary, certain lea genesdo not appear to respond to desiccationwhile others are down-regulated, at least indeveloping seeds that have been dried pre-maturely (Han et al., 1996).

ABA and LEA proteins are almost cer-tainly involved in the medium to long termin the desiccation scenario in seeds and veg-etative tissues. In the latter, concentrationsof hormone increase in response to droughtstress and impending desiccation. In seeds,however, the highest concentrations of ABAare normally reached in mid-developmentand are declining at the time when dehydra-tion commences; there is no evidence thatloss of water generally provokes ABA syn-thesis, as it does in leaves, for example.None the less, application of ABA can con-fer desiccation tolerance on developingembryos (Chapter 5), so it is conceivablethat the hormone participates in the inplanta situation. In vegetative tissues, ABAup-regulates the expression of several leagenes, including the dehydrins. There is cir-cumstantial evidence that some LEA pro-teins are also up-regulated by ABA indeveloping seeds but the association amongdehydration, ABA and LEA proteins at thestage of seed development when desiccationtolerance is initiated is actually fairlyobscure. Though the implication of LEAproteins and ABA in the desiccation phe-nomena of seeds may be undeniable, theprecise details of their involvement remainunresolved. One confusing point is that thedeveloping embryos of several types ofrecalcitrant seeds are relatively rich in bothLEA proteins and ABA and yet they are, of

course, desiccation-sensitive (Chapter 5).In general, then, there is a paucity of

information about gene expression associ-ated with desiccation in seeds, especiallyas compared with what is known in respectof vegetative parts. Another aspect of thedesiccation scenario for which we lack abalanced perspective concerns post-dryingrehydration. During this process a discreteset of genes and proteins is expressed inmosses that are likely to be involved in var-ious repair and protective phenomena. Weknow little about this so far as seeds (andvegetative tissues) of angiosperms are con-cerned, and it seems important to explorethis area if we are to advance our knowl-edge of survival mechanisms.

One problem inherent in the study ofgene activity during seed desiccation is thatseveral processes are occurring during mat-uration/drying when tolerance is expressed(induced) in planta. Some of these havebeen mentioned above and in addition thereare those associated with the establishmentof dormancy and with the alterations in pro-duction, destruction and sensitivity to hor-mones, for example ABA. It may be difficultto identify those changes in gene expressionthat are specifically associated with the syn-drome of desiccation damage and/or toler-ance but, none the less, experimentalprotocols could be designed to minimizesuch extraneous complications. For exam-ple, one way to separate operationally thespecific desiccation-related processes fromothers is to use very young embryos (priorto dormancy inception and reserve accumu-lation), as was done in early experimentswith barley (Bartels et al., 1988) and morerecently in the examination of amphiphilepartitioning in wheat (Golovina et al., 2001).

There is mounting evidence that theresponses to drying and the early events inthe desiccation scenario in seeds and vegeta-tive tissues are initiated by the first changesin water content at the beginning of dehydra-tion (see above). But what actually fires thestarting signal of the dehydration transduc-tion chain remains to be clarified. A plausi-ble assumption is that the detection of earlywater loss in vegetative tissues is the samewhether only relatively mild water stress is

370 M. Black et al.


suffered or whether the loss continues intosevere desiccation. Detection of changes inturgor are likely to be responsible, involvingtrans-membrane sensors such as the putativehistidine kinase osmosensor in Arabidopsis(Urao et al., 1999). An intriguing point, how-ever, is that the release of the initial signalsets in motion, in one case, a series of eventsthat culminates in protection againstextreme dehydration (e.g. in resurrectionplants) but, in others, only the ability to copewith, in comparison, mild drought stress.

One of the earliest events in droughtstress is the generation of transient calciumsignals (for review, see Knight and Knight,2001), and these are also likely to occur evenwhen water loss continues to desiccation.Whether or not calcium signalling partici-pates in all the multiple aspects of the desic-cation syndrome has to be resolved. In seedsand pollen almost nothing is known aboutthe early signalling events set in motion bythe incipient dehydration that initiates theacquisition of desiccation tolerance. Is turgorloss by the embryo the first perception ofdrying and is this, too, followed shortly bytransient calcium signalling?

Recent research indicates that in pollenand seeds an early detectable intracellularresponse to dehydration is the partitioningof amphiphiles from the aqueous cytoplasmto the membrane lipid phase (Chapter 10).This has been suggested to occur in theestablishment of desiccation tolerance, pos-sibly exerting a protective effect on mem-branes by virtue of the properties (e.g.antioxidant) of certain endogenousamphiphiles. It is important to determinewhat the endogenous amphiphiles are and ifthe same partitioning phenomenon occursin desiccation-tolerant vegetative tissues.

Investigation of the cell and molecularbiology of signal perception and transduc-tion in seeds and the early events thatclosely follow will require the utilizationof experimental systems that are rigorouslycontrollable. It is doubtful if the methodsthat have been employed hitherto – dryingwhole seeds, either in planta or in vitro atdifferent rates and relative humidities, etc.– will be satisfactory. The use of isolatedembryos (or parts thereof) under condi-

tions where water loss can be strictly regu-lated (such as by osmotic means) might bean approach that will allow the applicationof advanced biological techniques, but cau-tion must be exercised to minimizemechanical damage by rapid dehydrationor rehydration of tissues not protected by atesta or pericarp.

In summary, then, there is still much tobe learned about the extent to which thevery early, medium- and longer-term eventsare shared by seeds, pollen and vegetativetissues.

How do we take our understanding for-ward? Clearly, identifying marker moleculesfor desiccation stress tolerance is highly desir-able; and quantifying the products of geneexpression and how they interact or comple-ment each other’s effect needs to beaddressed. As has been noted in Chapter 11, aquantitative comparison of LEA proteins intolerant and sensitive tissues would be partic-ularly informative; in the same context, heat-shock proteins also qualify for furtherinvestigation. Ultimately, we should know theintracellular location of these proteins andexamine the possibility of a redistribution ofthese molecules and others as part of the tol-erance scenario (Chapter 10). Quantificationand identification of free radicals in biologicalsystems is a similar challenge, as short half-lives means that determination by electronparamagnetic resonance is difficult. There is aneed to identify new techniques and methodsin pursuit of the causes of desiccation sensi-tivity as, until then, we may well run the riskof measuring mainly the consequences of des-iccation stress. The other side of the coin is, ofcourse, recovery from such stress upon rehy-dration and the repair processes involvedtherein. This must surely be an area meritinggreat attention. Desiccation damage, protec-tion against it, and repair all involve a multi-plicity of components but we might ask if allplay equally important roles or if the effectsand actions of some are more critical thanothers.

Work on lower plants (Chapters 1, 7 and11) and angiosperm seeds (Chapters 5 and8) has revealed some interesting generalassociations with habitat, i.e. likely adapta-tions to local conditions. Continuing to

Damage and Tolerance in Retrospect and Prospect 371


screen biodiversity will undoubtedly sug-gest some interesting paradigms on which towork. As the power of geographical infor-mation systems increases, in terms of layersof information that can be analysed and theprecision of the data, it will be easier in thefuture to consider biological data in an eco-logical context. Such a holistic approach tothe issue of desiccation sensitivity will beimportant if we are to capitalize on theeffort made since the 1980s in genomics andto gain the most from emerging work in thisarea. Biomolecules contribute to the ‘bodyplan’ of organisms, and form has an impacton function in specific desiccating environ-ments. Proteomics will have an importantpart to play in this context.

Looking to the not-too-distant future,one can see the use of our knowledge tomanipulate desiccation tolerance and seedlongevity. Rapid advances in genomics andrelated sciences will increasingly provideopportunities for the development ofunique tools and reporter systems tounderstand and regulate the mechanismsby which seeds and other organs respondto internal and external signals. The appli-cation of our knowledge to enhance or con-fer tolerance of desiccation and to increaselongevity has important potential in agri-culture for the improvement of the quantityand quality of the food supply and will addto the success with which we can con-tribute to plant conservation.

372 M. Black et al.

References

Bartels, D., Singh, M. and Salamini, F. (1988) Onset of desiccation tolerance during development ofthe barley embryo. Planta 175, 485–492.

Bentsink, L., Alonso-Bianco, C., Vreugdenhil, D., Tesnier, K., Groot, S.P.C. and Koornneef, M. (2000)Genetic analysis of seed-soluble oligosaccharides in relation to seed storability of Arabidopsis.Plant Physiology 124, 1595–1604.

Black, M., Corbineau, F., Gee, H. and Côme, D. (1999) Water content, raffinose, and dehydrins in theinduction of desiccation tolerance in immature wheat embryos. Plant Physiology 120, 463–471.


Brenac, P., Horbowicz, M., Dickerman, A.M., Miseray, F., Smith, M.E. and Obendorf, R.L. (2002) Up-regulation of raffinose and stachyose accumulation in buckwheat (Fagopyrum esculentumMoench) seedling hypocotyls during drying. Planta (submitted).

Cornford, C.A., Black, M., Chapman, J.M. and Baulcombe, D.C. (1986) Expression of �-amylase and othergibberellin-regulated genes in aleurone tissue of developing wheat grains. Planta 169, 420–428.

Golovina, E., Hoekstra, F.A. and van Aelst, A.C. (2001) The competence to acquire cellular desicca-tion tolerance is not dependent on seed morphological development. Journal of ExperimentalBotany 52. 1015–1027.

Han, B., Hughes, D.W., Galau, G.A., Bewley, J.D. and Kermode, A.R. (1996) Changes in late-embryo-genesis abundant (LEA) messenger RNAs and dehydrins during maturation and premature dry-ing of Ricinus communis L. seeds. Planta 201, 27–35.

Jiang, L., Downing, W.L., Baszczynski, C.L. and Kermode, A.R. (1995) The 5� flanking regions ofvicilin and napin storage protein genes are down-regulated by desiccation in transgenic tobacco.Plant Physiology 107, 1439–1449.

Kermode, A.R. (1995) Regulatory mechanisms in the transition from seed development to germina-tion: interactions between the embryo and the seed environment. In: Kigel, J. and Galili, G. (eds)Seed Development and Germination. Marcel Dekker, New York, pp. 273–332.

Knight, H. and Knight, M.R. (2001) Abiotic stress signalling pathways: specificity and cross talk.Trends in Plant Science 6, 262–267

Peterbauer, T. and Richter, A. (2001) Biochemistry and physiology of raffinose family oligosaccha-rides and galactosyl cyclitols in seeds. Seed Science Research 11, 185–197.


Urao, T., Yakubov, B., Satoh, R., Yamaguchi-Shinozaki, K., Seki, M., Hiroyami, T. and Shinozaki, K.(1999) A transmembrane hybrid-type histidine kinase in Arabidopsis functions as an osmosen-sor. Plant Cell 11, 1743–1754.


Glossary

ABA: Abscisic acid a sesquiterpenoid plant hormone.adsorption: The attraction of gas or liquid molecules to active surfaces. The rehydration

of dry biological tissues in humidified atmosphere is through water adsorption.ageing: Time-dependent deterioration of cells and biomolecules. Ageing reactions are

often water-content-dependent. algae: An informal term covering photosynthetic organisms (largely aquatic) other than

the green land plants (bryophytes, pteridophytes, gymnosperms, flowering plants).Algae embrace red, green and brown seaweeds and unicellular, colonial and filamen-tous organisms from a taxonomically diverse range of groups, variously defined bydifferent authors.

amphiphilic: Having an affinity for both aqueous and non-aqueous phases. Typically usedto describe the behaviour of a molecule with a polar group that can interact with thecytoplasm and a non-polar, hydrophobic group that interacts with the membrane.Molecules with two such different groups are amphipaths.

angiosperms: Flowering plants, consisting of two groups, the monocotyledons and thedicotyledons, characterized by having one or two seedling leaves, respectively.

anhydrobiosis: Suspended life in the dried state.anthophyte hypothesis: This hypothesis refers to the evolutionary relationships of seed

plants. It proposed that the gnetophytes are the sister group (closest living relatives) tothe angiosperms and thus they shared a common ancestor. Though formerly stronglysupported, recent molecular data have generated considerable doubt about the idea, andthere is strong evidence for the gnetophytes being most closely related to the conifers.

antioxidants: Molecules that remove activated oxygen species from cells by serving as cat-alysts (enzymes such as catalase, superoxide dismutase and glutathione reductase) orsubstrates (ascorbate or tocopherol) in reactions that donate unpaired electrons.

ascospore: A meiospore borne in an ascus (saclike structure formed by the Ascomycota).bicellular (also binucleate): Refers to the developmental stage of the male gametophyte of

higher plants that has undergone one nuclear division after meiosis. One cell (thevegetative cell) is involved in directing pollen tube growth; the other cell laterdivides into two sperm cells and is located inside the vegetative cell. After division,the tricellular (or trinucleate) pollen consist of one vegetative cell with two spermembedded in it. Depending on whether anther dehiscence occurs after the first or thesecond mitosis, mature pollen is designated bicellular or tricellular.

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bitegmic: Of ovules (cf. unitegmic or ategmic). Ovules having both outer and inner integu-ments prior to fertilization and seed development. Where both persist in the matureseed, the inner integument becomes the tegmen, and the outer integument becomesthe testa sensu stricto.

blastospore: A spore arising by budding such as a conidium arising from a narrow regionof the conidiogenous cell with elongation and swelling and then delimitation of theconidium by a septum.

Boltzman distribution law: The expression for the ratio of populations of molecules atany two levels of energy.

bound water: A concept to describe hydration of macromolecules based on interactions ofwater molecules with a macromolecular surface and with other water molecules.Bound or vicinal water has sufficient interactions with the macromolecular surface tocause changes in its thermodynamic properties and molecular mobility comparedwith water in a dilute solution. The concept of bound water was originally based onsimple sorption theory, but has evolved to consider adsorption sites with differentcharacteristics and relationships between bound water and the structure and activityof macromolecules.

brachycytes: Spherical, thick-walled cells that are formed on moss protonemata afterextended periods of culture.

Bryophyta (bryophytes): Green land plants with an alternation of haploid and diploid gen-erations, in which the diploid sporophyte (capsule) remains dependent on the haploidgametophyte for water and at least in part for nutrition (mosses, liverworts – q.v.).

C3 plant: Plant in which the first detectable photosynthetic products are three-carbonmolecules, e.g. 3-phosphoglyceric acid. Includes most plants of wet or mesic habitatsat all latitudes.

C4 plant: Plant in which the first detectable photosynthetic products are four-carbonorganic acids, e.g. oxaloacetic acid, malic acid. Most characteristically plants of semi-arid situations in warm climates.

callose: � 1-4 glucan; substance often formed in pollen upon stress.carbon balance: The cumulative net uptake of carbon by a plant; cumulative gross photo-

sythesis minus respiration.cavitate : To form an air-filled space in water-filled xylem.chalaza: The chalaza is the tissue at the base of the ovule, from which the integuments

arise, and is probably involved in nutrient transfer from the funicle to the developingembryo and endosperm. In some species it becomes heavily developed, surroundingand overtopping the integuments, a state known as pachychalazy.

chaperones: Proteins that modify folding of other (newly formed) proteins and assistassembly of protein oligomers.

chemical shift in NMR: The shift in the position of the resonance line of the nucleusbecause of the local electronic structure, which makes the local magnetic fieldslightly different from the external static field.

chlorophyll fluorescence: Excitation energy absorbed by chlorophyll may: (i) bring aboutphotochemical processes; (ii) be dissipated as heat; or (iii) be re-emitted as red fluo-rescence, measurement of which provides a basis for non-invasive measurements ofvarious aspects of photosynthesis.

compatible solutes (osmolytes): Osmotically active, non-toxic, low-molecular-mass mole-cules (such as quaternary amines, amino acids or sugar alcohols) which may accumu-late in cells in response to water or freezing stress.

conidium: In fungi a non-motile asexual spore usually formed at tips or sides of a sporoge-nous cell; in some cases a pre-existing hyphal cell may be converted into a conidium.

constitutive: Occurring constantly rather than being induced in response to a particularstimulus.

374 Glossary


continuous-wave NMR: A field-swept technique, when the field is swept through the res-onance frequency and a frequency domain absorption spectrum is obtained.

cooperative stress: Stress that is not associated with water loss per se, but exacerbates thedamaging effects of water loss, possibly because it has a similar mechanism of dam-age.

crassinucellate: Of ovules (cf. tenuinucellate). Ovules in which the nucellus is massivelydeveloped, rather than scantily so. The nucellus is the megasporangial tissue sur-rounding the embryo sac, and it persists only relatively rarely in the mature seed,where it forms the perisperm.

critical minimum surface area: The minimum membrane surface area that can be lostfrom the plasmalemma or tonoplast upon contraction during water stress withoutcausing cells to burst upon rehydration.

critical minimum volume: The minimum volume to which cells or vacuoles can contractduring water stress without bursting upon rehydration. Most non-acclimatized cellscan contract no more than to 50% of their original volume.

critical water content: The minimum water content to which cells or macromolecules canbe dried without imposing irreparable damage. This water content reflects the criticalwater potential.

critical water potential: The minimum water potential to which cells or macromoleculescan be dried without imposing irreparable damage. This water potential correspondsto the critical water content.

cryopreservation: The technique to preserve living organisms or cells at subzero tempera-tures, usually below –100°C (e.g. in liquid nitrogen –196°C), which includes pretreat-ment in cryoprotective solutes (if required), cooling to below 0°C, storage at lowtemperature, thawing and preparation for resumption of growth.

dehiscence: Opening of anthers in higher plants, which allows pollen to be dispersed: or,of some fruits, to allow seeds to be dispersed.

dehydrins: Group 2 LEA proteins synthesized in association with dehydration, character-ized by a lysine-rich 15-amino-acid motif (K-segment), contiguous serine residuesand the consensus sequence DEYGNP.

demixing: Rearrangement and consolidation of similar-type molecules that increasespacking efficiency in severely dehydrated systems. Demixing will disturb the originalconfiguration of proteins and lipids within bilayers of membranes and aggregation ofmolecules can result in irreversible structural changes.

desiccation: The extreme form of water loss, in which most of the protoplasmic water islost and a very low amount of tightly bound water remains in the cell.

desiccation avoidance: A protective strategy against drought stress where cells remainhydrated using adaptive structures that scavenge water. This strategy prevents waterloss. See also desiccation resistance.

desiccation damage (sensu stricto): Mechanical or structural damage to cells and cellularconstituents directly resulting from water removal.

desiccation resistance: A protective strategy against drought stress where cells reduce therate of water loss by using adaptive structures that form barriers to water loss or byaccumulating solutes that lower the water potential difference between the cell andthe environment. This strategy prevents water loss.

desiccation tolerance: The ability to recover biological functions after drying to a point atwhich no liquid phase remains in the cells (e.g. water content down to 5% or less ofdry weight, in equilibrium with water potential down to �200 MPa or less).

desorption: Opposite of adsorption; the dehydration of biological tissues in dry atmos-phere is water desorption.

diaspore: Any spore or other plant part able to form a new organism, e.g. the haploidstructure formed out of a part of the moss gametophyte.

Glossary 375


diffusional correlation time: The average time between jumps in position for water mole-cules in the system.

Dollo’s law: Dollo’s law or rule suggests that, for complex characters or traits, parallel ormultiple origin is unlikely, but that reversal or loss may be easy. The assumption isthat many genes must change to create a morphological structure or physiologicaltrait, but only one of them needs to change in order to lose it.

dormancy: An endogenous mechanism that prevents viable hydrated plant parts (e.g.seeds, spores, buds) from resuming growth and full metabolic activity.

drought: In plants, the partial limitation of water content, often for a prolonged period;usually when cell water potentials (�w) are ≤ �3 MPa in non-transpiring cells. Watercontents may reach 20–25% (fresh weight basis), 0.25–0.33 g water g�1 dry weight.

drought tolerance: The ability to survive drought.dry: To remove water (verb) or without water (adjective). Since achieving and measuring

truly dry material is logistically difficult, ‘dry’ is often used as a relative term todescribe material that is drier than its undried counterpart.

electron paramagnetic resonance (EPR): The absorption of electromagnetic energy duringtransition of electrons between two energy levels of Zeeman splitting (q.v.).

endocytotic vesicles: Invaginations of plasma membrane into the cytoplasm observed dur-ing osmotic contraction of protoplasts from non-acclimatized plants. These arebelieved to be deleted from the membrane surface area.

enthalpy (H): A defined thermodynamic variable of state that consists of internal energyof the system (E), specified pressure (P) and volume (V). H = E +PV, where the PVunits are converted to calories, ergs or joules. Different enthalpy, �H, describes thechange of energy status.

entomopathogenic: Pathogens (e.g. fungi, bacteria) that feed on insects.entropy (S): A thermodynamic term that quantifies the randomness or disorder of the sys-

tem. epilithic: Growing on the surface of rocks.EPR imaging: Visualizing the distribution of paramagnetic centres in a sample.eukaryotic: Of cells having a nucleus.exocytotic extrusions: Folding of the plasma membrane to the cell exterior observed dur-

ing osmotic contraction of protoplasts from acclimatized plants. These are reincorpo-rated into the plasma membrane upon expansion and so are believed to be amechanism to retain overall membrane surface area in contracting cells.

ferns: The largest group of pteridophytes, having leaves with branching veins (mega-phylls).

flash drying: Drying by rapid flow of dry air over excised embryonic axes, somaticembryos or small tissue pieces.

Fourier self-deconvolution: Transformation of the absorption bands in the Fourier trans-form infrared spectrum to line shapes with narrower peaks (resolution enhancementtechnique).

Fourier transformation: The mathematical method to convert the time dependence of theNMR signal (FID) into the frequency dependence of the NMR signal.

free energy (Gibbs free energy, G): A quantity that is used to describe the energetics ofchemical, physical and biological processes. Gibbs free energy in a system is the max-imum amount of energy for work. It decreases for a spontaneous process such asrehydration of dry tissues, in which free energy of water decreases.

free induction (FID) signal: The measure of the NMR signal over time.freeze–fracture: A method of preparing specimens for electron microscopy by freezing

and then splitting them.freezing point depression: One of the four colligative properties of solutions in which the

freezing point is depressed below that of the pure solvent.frequency: Number of cycles per second.

376 Glossary


gametophyte: The structure that forms the haploid, gamete-forming part of a plant’s lifecycle.

gas exchange: Usually refers in plant-physiological contexts primarily to uptake or outputof CO2 and O2 in the course of photosynthesis and respiration.

Gauss: The unit of strength of the magnetic field.g-factor: The ratio of the magnetic moment to the spin angular moment of an electron

(determines the position of the EPR spectrum).glass (glassy state): A fluid that is so viscous that it acquires mechanical properties

(strength) of a solid. The viscosity results from numerous intermolecular interactionsin a random array forming an irregular matrix of pores. Because there is no regularpattern to the arrangement of molecules in this fluid (similar to a liquid), the struc-ture is considered amorphous relative to the defined arrangement of molecules in acrystalline solid. Because molecular mobility of molecules within the glass isrestricted, physical and chemical reactions are slowed but not stopped. Thus, theglass is considered kinetically, but not thermodynamically, stable. Glass transitionsare considered second-order state changes (as opposed to phase transitions, whichare first-order) because of a continuous change in enthalpy, entropy and volume (firstderivative of chemical potential) throughout the transition but a discontinuouschange in heat capacity (second derivative of chemical potential).

glass formation: A change from a fluid to a semi-solid state in the protoplasm of desiccat-ing cells, also known as vitrification. Carbohydrates and proteins are the main com-pounds that can contribute to glass formation in the cytoplasm.

heat-shock proteins: Proteins generally synthesized in response specifically to relativelyhigh temperatures (e.g. 40°C): often act as chaperones (q.v.) and protein protectants.

Höfler diagram: The plot of cellular water potential components against protoplast vol-ume.

homoiochlorophyllous: Retaining most or all chlorophyll through a drying–rewetting cycle.homoiohydrous: Maintaining a high water potential and active metabolism during times

of low water availability.hydration force explanation: A hypothesis used to describe how compression of macro-

molecules can lead to their deformation. Compression of cell volume by waterremoval can lead to repulsive forces as molecules with similar charges come withinclose proximity. The repulsion between surfaces can lead to lateral tensions withinstructures that cause phase changes or deformations. The hydration force explanationinvokes non-specific mechanisms for the protection of macromolecules by carbohy-drates (as opposed to the water replacement hypothesis, which invokes specific inter-actions), first as osmotica that resist water loss, and then as glass formers that providemechanical resistance to the compression.

hydration levels: Ranges of water contents or water potentials that define different struc-tures/functions of molecules or physiological activities of cells. Also, ranges of watercontents or water potentials that define changes in thermodynamic or motional prop-erties of water.

hydraulic conductivity: A measure of the diffusional resistance of a water transport path-way within the tissue.

hyperfine interaction: The interaction between nuclei and unpaired electrons in EPR.hyperfine splitting constant: The distance between lines of an EPR spectrum originating

from hyperfine splitting.hyperfine splitting of the EPR spectrum: Separation between lines of an EPR spectrum

due to the hyperfine interaction.hysteresis: The difference in the equilibrium water between desorption and adsorption

isotherms.

Glossary 377


378 Glossary

image contrast: The differences in signal intensity in NMR imaging between differentregions of the sample.

imbibitional stress: Stress imposed on a dehydrated organism by imbibition of water. Theinjury that ensues usually encompasses the loss of plasma membrane integrity. Theinjury is severe when imbibition occurs at low temperature and when theorganism/cell is very dry: avoidance can be achieved by prehydration in humid airand warm imbibition.

in vitro: Literally ‘in glass’, referring to tissues cultured in a sterile container on an artifi-cial, sterile nutrient medium.

inhomogeneous broadening: The broadening of the EPR lines because of unresolvedhyperfine structure.

integrated intensity: The area beneath an absorption peak.interfacial region: The boundary between two phases. intermediate seeds: Seeds that tolerate considerable (at least to 30% RH) but not complete

drying. Life spans of stored seeds progressively increase as the storage RH isdecreased to about 50% and then a reversed trend is observed with storage RH <50%. Seeds with intermediate characteristics cannot be stored using standard recom-mended storage protocols: though they appear to survive low water contents, they donot survive the added stress of exposure to �18°C. See orthodox, recalcitrant.

IRGA (infrared gas analysis): Methods using gas-phase absorption bands in the nearinfrared to measure concentration changes in CO2 (or other gases).

isotropic motion: Uniform tumbling of a spin probe in all directions; the isotropic EPRspectrum originates from the averaging of the spectral anisotropy.

LEA (late embryogenesis abundant) proteins: A broad family of universal plant proteinswith conserved amino acid motifs which accumulate to high levels during late stagesof embryo development or in response to osmotic stress in vegetative tissues. Theyare very hydrophilic, remain soluble at T � 90°C, and are believed to protect cellsduring water stress through an, as yet, unknown mechanism. See also dehydrins.

lichenized fungus: A fungus that has formed a lichen in association with an alga.line width: The width at half-height of absorption peak.liverworts: Bryophytes (q.v.) with leafy or (less commonly) thalloid, usually dorsiventral

gametophytes and short-lived sporophytes (Hepaticae; Hepaticopsida).magic angle: The angle (54° 55�) of the axis of mechanical rotation of the sample in high-

resolution NMR to eliminate line broadening.magnetic susceptibility: The ratio between magnetization and magnetic field strength.magnetogyric ratio: The ratio of magnetic dipole moment to the spin angular moment of a

specific nucleus.manometric methods: Techniques using change in pressure (Warburg) or volume (e.g.

Gilson) in a gas space over the reaction mixture or material to follow a metabolicprocess (e.g. respiration or photosynthesis).

minimum critical volume: See critical minimum volume.monolayer hydration: The level of hydration at which only polar sites are bounded by

water.mosses: Bryophytes (q.v.) typically with (+ or �) radial leafy shoots, and sporophyte cap-

sules borne on a long-lived seta (Musci, Bryopsida).NMR imaging: Imaging of spatial distribution of water or water properties based on pro-

ton magnetic resonance.nuclear magnetic resonance (NMR): The absorption of electromagnetic energy during the

transition of a nucleus between two discrete energy levels of Zeeman splitting.oligosaccharide: A carbohydrate whose molecules are composed of a few (< 20) generally

mixed (e.g. glucose, fructose, galactose) monosaccharide units: three, four and five inraffinose, stachyose, verbascose, respectively.


order parameter: Quantity calculated from the shape of the EPR spectrum, which indi-cates the degree of motional anisotropy.

orthodox seeds: Seeds that tolerate the immediate effects of severe water loss (i.e. desicca-tion-tolerant). Life spans of stored seeds progressively increase as the storage RH isdecreased to about 20% and then a reversed trend may be observed with storage RH< 20% (decreasing life span with decreasing RH). Seeds with orthodox characteristicscan be stored using standard recommended storage protocols of drying to 0.05 ± 0.02 gwater g�1 dry mass and storing in a freezer at about �18°C. Storage longevity can bepredicted from temperature and seed water content. See intermediate, recalcitrant.

osmolytes: See compatible solutes. osmotic adjustment: The net accumulation of solutes after the plant tissue has been

exposed to a predetermined rate of water deficit.osmotic excursions: Reversible shrinking and swelling of cells and protoplasts during

exposure to cycles of low and high water potentials. osmotically inactive: Apoplastic water present in very small pores and strong water-

binding sites of biological surfaces in plant tissues. osmotically unresponsive: Membrane vesicles that fail to swell when water stress is

relieved because the lumen lacks osmotically active constituents. This most probablyoccurs when different membrane systems compress together during dehydration andfuse, excluding formerly contained constituents.

ovule: The female (mega) spore and gametophyte of higher plants: becomes the seed afterfertilization.

pachychalazy: See chalaza.paramagnetic: Atom or molecule containing an unpaired electron.partitioning: Distribution of molecules, e.g. between lipid and aqueous phases.permanent wilting point: Minimum water potential tolerated by non-transpiring cells.

Similar in concept to critical water potential.phase separation: A consequence of demixing. The aggregation of similar-type molecules

into enriched domains leads to higher localized chemical potentials and greater like-lihood of phase changes.

phase transition: Change of state between solid, liquid and vapour phases. Phase changesin lipids are complex because of the diverse crystalline states of pure lipid and lipidmixtures. For polar lipids, phase changes occur when a fluid gel converts to a liquidcrystalline or hexagonal phase. Phase changes in polar lipids can be induced by alter-ing the water status (drying favours gel and hexagonal phases) or temperature (lowtemperatures favour gel phases). Phase changes are termed first-order transitionsbecause of an abrupt, discontinuous change in the enthalpy, entropy and volume(first derivatives of chemical potential) and a consequent infinite heat capacity (sec-ond derivative of chemical potential).

phycobiont: In lichens, the alga that is associated with the fungus, or mycobiont.phylogenetic classification: Phylogenetic classifications attempt to generate systems that

reflect as closely as possible the evolutionary relationships and history of a group oforganisms. They recognize only those groupings of species that are monophyletic, i.e.all the members of the group are likely to be descended from a single common ances-tor.

phytochrome: Chromoproteins that undergo a reversible conformational change maxi-mally upon absorption of red or far-red light. They regulate many aspects of plantfunction.

plasma membrane: The membrane that envelops a cell protoplast.plasmolysis: Withdrawal of the cytoplasm from the cell wall when the cell is placed in a

solution of lower osmotic potential than the cell sap.poikilochlorophylly: The ability to reversibly lose chlorophyll during desiccation.

Glossary 379


poikilohydrous: Of plants whose water content closely follows fluctuations of humidity intheir environments (in contrast to homoiohydrous plants), typically suspendingmetabolism during periods of drought.

pollen: Male gametophyte of higher plants, which functions to deliver its haploid spermcells to the ovules in order to bring about fertilization.

population (of molecules): The number of molecules occupying a particular energy level.prothallus: Gametophyte (haploid) in ferns, horsetails, Selaginella and Lycopodium that is

formed from spore germination and which produces gametes. After fertilization thediploid sporophyte grows out of the prothallus.

protonema: The structure (immature gametophyte) that develops from spore germination,e.g. in mosses.

Pteridophyta (pteridophytes): Green plants with an alternation of haploid (gametophyte,prothallus) and diploid (sporophyte) generations, both (at least potentially) capable ofliving independently, the sporophyte being the dominant plant (ferns, horsetails, club-mosses, spikemosses).

pulsed NMR: NMR technique based on the recording of NMR signals with time after excit-ing nuclei by a short intense pulse of radio-frequency radiation.

PV curve: The relationship between the reciprocal of water potential and relative watercontent of a tissue.

radical adduct: The product of the interaction of a primary free radical with a spin-trapmolecule.

reactive oxygen species (ROS): Molecules containing oxygen with an unpaired electron ora pair of electrons with parallel spin (singlet oxygen). These molecules, often resultingfrom free-radical reactions, seek an additional electron and so are highly reactive withbiomolecules which are electron-rich. Reacting with ROS, biomolecules are peroxi-dized to become reactive themselves, initiating a cascade of degradative reactions.

recalcitrant seeds: Mature seeds that do not survive if desiccated to water potentials lessthan about �15 MPa (about 90% RH) and hence cannot be stored in the ‘dry’ state.Hydrated storage at either cryogenic or supra-freezing temperatures appears to be thebest storage option.

receptivity of the isotope: The product of the sensitivity of the isotope for NMR experi-ments and its natural abundance.

rehydrins: Proteins synthesized specifically in association with rehydration in desiccation-tolerant plants.

relative humidity: Water activity multiplied by 100.relative water content (RWC): Tissue water content relative to (fraction or percentage)

water content at full turgor.relaxation process: The return of magnetization to thermal equilibrium.resurrection plants: A small group of poikilohydrous higher plants which tolerate almost

complete water loss in their vegetative tissues and resume normal functional activityafter rehydration: occur in specific ecological niches with seasonal water availability.

RH (relative humidity): Water content of air expressed as percentage (or fraction) of satu-rated water content at the same temperature.

rotational correlation time: The time it takes for a molecule to rotate one radian around itsaxis.

rubber: A fluid that is more viscous than a syrup but less viscous than a glass. There arefewer intermolecular interactions and larger pore sizes in rubbers compared withglasses and this allows for greater elasticity in the structure.

RWC: Relative water content.second-derivative analysis: Mathematical procedure used for increasing the resolution of

an FTIR spectrum.self-incompatibility: In higher plants the phenomenon of pollen being unable to establish

fertilization within the same plant or some individuals of the same plant species.

380 Glossary


sensitivity (spectral): The minimal number of spins (electrons, nuclei) which can be mea-sured by a method (EPR, NMR, respectively).

sensitivity of the isotope for NMR experiments: The quantity depending on magnetogyricratio (�) and spin quantum number (I).

spatial resolution: The precision in the determination of the location of a signal source(or the measure of the minimal distance between two signal sources within the sam-ple which still allows them to be distinguished).

spectral anisotropy: The dependence of EPR spectra on the orientation of the spin probes,originating from the anisotropy of the interaction of an electron with the externallyapplied magnetic field.

spectral resolution: Quantity that expresses how the lines in a spectrum are separatedfrom one another.

spectroscopy: The measurement of the energy differences between discrete energetic lev-els of atoms or molecules.

spin label (spin probe): Stable free radical that contains a nitroxide fragment with anunpaired electron.

spin trap: Diamagnetic molecule forming a nitroxide radical when interacting with a pri-mary free radical.

spin-echo technique: Pulse NMR based on applying a second pulse after a set of delaytimes to eliminate the effect of the inhomogeneity of the magnet.

spin-lattice (or longitudinal) relaxation time (T1): The time constant of magnetizationdecay because of the interaction of nuclear/electron magnetic moments (spin) withthe environment (lattice).

spin–spin (or transverse) relaxation time (T2 ): The time constant of magnetization decaybecause of the interaction of nuclear/electron magnetic moments (spin) with eachother.

sporangia: Spore-producing structures, e.g. in pteridophytes.spore: In mosses, ferns, horsetails, Lycopodium and Selaginella, a haploid, stress-resistant

cell formed by meiosis; in fungi, spores may not necessarily be the result of meiosisand also may be diploid.

spore bank: Layer of accumulated spores in the soil.sporocarp: Structure in certain ferns that contains the sporangia.sporophyte: The diploid (asexual) phase of the alternation of generations in plants.structural water: Water required to maintain the configuration of macromolecules nor-

mally observed under aqueous conditions, and so is most often identified in dryingsystems as the minimum amount of water required to prevent a conformationalchange. Water, at water contents where conformational changes in macromoleculesoccur, has unusual thermodynamic properties or restricted mobility. Consequently,structural water is a component of bound or vicinal water in hydration models usingthe bound-water concept. In alternative solution-based models of hydration, struc-tural water is a likely component of the super-viscous solutions with rubbery orglassy characteristics.

sucrose: A disaccharide; molecules each contain one glucose and one fructose unit.symbiosis: A regular association between two organisms characterized by mutual benefit

and interdependence.Tg: Glass–liquid transition temperature.Tm: Gel–liquid crystalline temperature of membranes.teliospore: A thick-walled resting spore of fungi belonging to the rusts and the smuts, in

which karyogamy occurs.trehalose: A disaccharide in which each molecule contains two glucose units with an �

1→1 linkage: often associated with desiccation tolerance in animals and some plants.tricellular (or trinucleate): See bicellular.turgid: Swollen or firm because of the pressure of water within the cell or tissue.

Glossary 381


turgor: Cellular pressure generated from the movement of water into a cell. Pressureexerted by the cell wall, balancing the difference between the osmotic potential of thecell contents and the water potential of the surroundings.

urediniospore: A binucleate spore of Uredinales (rust fungi).vascular plants: Green land plants which possess vascular tissues for water conduction,

comprising pteridophytes and seed plants (gymnosperms and angiosperms).viability: Potential ability of a cell, tissue, organ or plant part to resume full metabolism

and growth under favourable conditions (e.g. of seeds).vitrification: A change from a fluid to a semi-solid state in the protoplasm of desiccating

cells, also known as glass formation.volumetric elasticity module: An expression to quantify the relationship between the

change in volume and the applied pressure.water activity: Proportion of water available compared with pure water. Water activity is

usually measured in the vapour above a condensed phase and ranges from 0 (nowater in the condensed phase) to 1 (pure water in the condensed phase). At equilib-rium, the water activities of the vapour and condensed phases are the same. The nat-ural log of water activity directly relates to the chemical potential of water. See waterpotential.

water content: A measure of the concentration of water that is usually expressed by massratios on either an absolute scale of 0 to ∞ (no water to pure water) by dividing massof water by mass of dry material, or on a relative scale of 0–1 (no water to full hydra-tion). The denominator for the relative expression is either total mass or mass ofwater in fully hydrated tissues, an empirically derived value that varies with species,tissue and tissue development (a.k.a. relative water content or RWC).

water potential: A measure of availability of water in terms of pressure that decreasesfrom 0 (pure water) to �∞ as water content decreases. Units are usually expressed asMPa. The related parameter, chemical potential of water, which describes the avail-ability of water or its potential for effecting reactions in energy terms (units are usu-ally joules), is the difference between the molar free energy of pure water at standardtemperature and pressure (STP) and the water potential times the molar volume ofpure water at STP (18 cm3 mol�1). Chemical potential of water inversely correlateswith chemical potentials of solutes, which in turn are components of the free-energydifference that drive reactions.

water replacement hypothesis: A mechanism of protecting macromolecules from struc-tural changes during dehydration by inserting hydrophilic groups of specific solutes(usually carbohydrates) on to hydrophilic sites of macromolecules. This substitutionprevents aggregation of molecules by van der Waals forces and the separation pre-vents deleterious interactions.

water sorption isotherm: A plot that describes the relationship between water contentand RH (RH ≤ 93%) or water content and water potential (�w ≥ �20 MPa) for a par-ticular material at a particular temperature.

water-clustering function: A volumetric analysis of water relation for the formation ofwater clusters (e.g. water–water self-association).

wave number (spectroscopy): The number of waves per centimetre.xeromorphic: Having morphological characteristics particularly adapted to conserving

water under dry conditions.Zeeman splitting: The splitting of the energy levels of the electron/nucleus due to the

interaction of its magnetic moment with the external static magnetic field.zygospore: A resting spore that results from the fusion of two gametangia in

Zygomycotina (fungi).

(Terms included do not necessarily reflect the collective views of the authors)

382 Glossary


Taxonomic Index

Authorities for names are not included and synonymy is not rationalized, but can be cross-checkedvia the relevant chapters.

Acanthaceaevegetative desiccation tolerance 222

Acer platanoides

cell cycle 174oligosaccharides 172respiration 173seed anatomy 252seed development 156–157seed drying time 159see also Norway maple

pseudoplatanuscell cycle 174desiccation tolerance, and seed drying

time 159oligosaccharides 172respiration 173 seed anatomy 252seed development 156–157seed dormancy 158see also sycamore

sp. free-radical scavenging 174variation in desiccation tolerance 155,

244Aceraceae

angiosperm phylogeny 242recalcitrant seeds 247seed storage classification 248

Acinetobacter radioresistanssurvival at 31% RH 10

Acoralesseed storage classification 247

Actiniopterisdesiccation tolerant species

dimorpha 218radiata 218

Adiantaceaedesiccation tolerant species 218

Adiantumdesiccation tolerant species

incisum 218gametophyte cryopreservation

tenerum 225trapeziforme 225

spore desiccation tolerance capillus-veneris 192

Aesculus hippocastanumaxis water stress 267critical water potential and water content 50seed development 159seed dormancy 158, 245 water loss curve 68water and seed longevity 103

Afrotrilepis pilosain situ/natural habitat 12, 224longevity when dry 225specialized structures/velamen 224 vegetative desiccation tolerance 220

Agathis robusta

seed storage classification 241sp.

orthodox and recalcitrant seeds 244–245seed weight 245

Aglaonema sp. desiccation sensitive pollen 187

Agrostemma githagoseed development 153

Alismatalesseed storage classification 247

Allium sp.vegetative propagules 228

383

Dessication Taxonomic Index 3/4/02 2:30 pm Page 383

Aloina aloidesABA-induced tolerance 216

Alternariadesiccation tolerant spores 195

porri 196Amaranthaceae

angiosperm phylogeny 242seed storage classification 248

Amaryllis sp. pollen DNA repair 356

Amborellaceaeangiosperm phylogeny 246

Amomyrtus lumaseed storage classification 241

Anacardiaceaerecalcitrant seeds 247

Anacystis sp.desiccation-tolerant cysts 17

Anamodon viticulosus chlorophyll fluorescence 212desiccation tolerance 209, 216photosynthesis rate 210recovery processes 214

Andira inermiscritical water potential and water content

50Andreaea

rothiiCO2 uptake 214desiccation tolerance 213

sp. desiccation tolerance 209

Andreaealesdesiccation tolerance 209

Anemia phyllitidis

spore desiccation tolerance 192tomentosa

desiccation tolerant species 220Anemone coronaria

tuber desiccation tolerance 228angiosperms

vegetative desiccation tolerance 220Annonaceae

seed storage classification 247–248Anomodon viticulosus

dark respiration 215in situ, desiccated and hydrated 14photosynthesis and water potential 229

Anthuriumseed storage classification 240

Apiaceaeseed storage classification 248see also Umbelliferae

Apialesseed storage classification 247

Apocynaceaeseed storage classification 248

appleorthodox seed 252TBARS assay 119see also Malus

Aquifolialesseed storage classification 247

Arabidopsissp.

aba, abi-3 and other mutants 164–165abi-3 and other gene products 163, 332alkyl hydroperoxidase 330AtPer1 expression 170desiccation intolerant mutants 25, 253,

310, 324‘dormancy’ gene 29HSP 165, 309osmosensor 371protein denaturation 169transgenic plants 326, 331–332, 334–335

thaliana1H-NMR of seeds for betaine 132EST collections 30homologues of lea cDNAs 162lea genes 164 protein–sugar glass 306seed mucilage 348seed storage stability 304trehalose synthesis 168

Araceaedesiccation sensitive pollen 9, 187seed storage classification 248

Araucariaangustifolia

recalcitrant seeds 245seed respiration 173

araucanarecalcitrant seeds 245

bidwilliirecalcitrant seeds 245seed size and weight 246

brownii seed size 246

cunninghamiiseed weight 245

heterophyllaseed weight 245

hunsteiniirecalcitrant seeds 245seed weight 246water and seed longevity 103

mirabilisseed size 246

Section of Araucariaceae 245–246sp.

orthodox and recalcitrant seeds 244sphaerocarpa

seed size 246

384 Taxonomic Index


Araucariaceae habitat and seed storage 252seed desiccation sensitivity 244–245

Archidium alternifoliumspore germination 194

Arecaceaehabitat and seed storage 252recalcitrant seeds 247seed anatomy 252 seed storage classification 248–249, 253see also Palmae

Arecalesnumber of desiccation sensitive seeded

species 246–247Artemia

desiccation tolerant cysts 281 water difussion coefficient in cysts 130

Arthropteris orientalisdesiccation tolerant species 220

Artocarpus heterophyllusaxis drying curve 99

Asclepiadaceaeseed storage classification 248

Asparagalesseed storage classification 247

Aspergillus japonicusdesiccation tolerant spores 196

Aspleniaceaedesiccation tolerant species 219

Asplenium desiccation tolerant species

aethiopicum 219bourgaei 219pringlei 219ruta-muraria 220rutifolium var. bipinnatum 219sandersoni 219trichomanes 220

vegetative desiccation tolerance septentrionale 220

Asteraceaeseed storage classification 248tricellular pollen 188see also Compositae

Asteralesseed storage classification 247

Athyrium filix-feminaspore bank 193spore storage 193

Atrichum androgynumpartial dehydration 216

Austrobaileyaceaeangiosperm phylogeny 246

Avenafatua

seed DNA repair 353see also oat

sp.desiccation sensitive pollen 187

Avicennia marinaABA and seed development 171axis drying curve 99cell vacuolation 174desiccation sensitivity 159, 268DNA repair 174, 354–355respiration 172seed desiccation 153seed development 157stachyose 172sub-cellular de-differentiation 173viviparous germination 158

Aylthonia blackiivegetative desiccation tolerance 222

Azadirachta indicaaxis drying curve 99EPR of chilling stress 122 imbibitional damage 102seed storage classification 241variation in desiccation tolerance 157, 266see also neem

Azollaspore dormancy 192sporocarp storage

filiculoides 193

Bacillus subtilisDNA form 350gene product 332–333

Balsaminaceaepollen storage life 190

Barbacenia vegetative desiccation tolerance

flava 222longifolia 222riedeliana 222sellovii 222

Barbaceniopsisvegetative desiccation tolerance

boliviensis 222humahuagensis 222

Barbula sp. temperature and survival 213

barleyalkyl hydroperoxidase 330chemical shift imaging of seeds 131‘dormancy’ gene 29, 349gene product 332lea gene 25LEA proteins 24, 334PER1 protein 170pulsed (spin-echo) NMR of developing

seeds 130

Taxonomic Index 385


barley continuedseed development 153see also Hordeum

Bauhiniaseed anatomy 252

beanaxis water stress 267cell contraction 270chilling damage 344DSC data 306glass formation in axes 302, state diagram 303, 305seed imbibition damage 334

Beauveriadesiccation tolerant spores

bassiana 196brongniarti 196sp. 195

bell pepperoligosaccharides 307

birchDNA repair in pollen 354, 355

Blechnum spicantspore storage 193

Blossfeldia liliputanavegetative desiccation tolerance 222

Boea hygroscopica

carbohydrates 325forest understorey 17non-xeromorphic 10predrying 226vegetative desiccation tolerance 222water content and survival 101

sp.single desiccation tolerant species 217

Bombacaceaeseed storage classification 248

Boraginaceaeseed storage classification 248

Boryainopinata

vegetative desiccation tolerance 221nitida

chloroplasts 272predrying 226tolerant and sensitive individuals 10vegetative desiccation tolerance 221xeromorphic characteristics 224

septentrionalisvegetative desiccation tolerance 221

sp.in situ 12leaf desiccation tolerance 9

Brachyachne patentifloravegetative desiccation tolerance 221

Brassica sp.seed imbibition damage 344

Brassicaceaeseed storage classification 248species coverage 246see also Cruciferae

Brassicalesseed storage classification 247

Bromeliaceaeseed storage classification 248

Bromus secalinas‘dormancy’ gene 29, 330, 349

Bryaceaeinduced desiccation tolerance unexplored

216Bryum

predryingcaespiticium 216capillare 216pseudotriquetrum 216

buckwheatfagopyritol 168see also Fagopyrum esculentum

BunyaSection of Araucariaceae 245–246

cabbageTBARS assay 119

Cactaceaeseed storage classification 248

Calophyllum sp. orthodox and recalcitrant seeds 244

Caltha palustrisseed storage classification 241, 244, 253

Camellia sinensisseed drying curve 99variable seed desiccation tolerance 266see also tea

Capparaceaeseed storage classification 248

Cardamine sp.vegetative propagules 228

Carex physodesvegetative desiccation tolerance 217, 220

carrot somatic embryos and LEA proteins 309

Caryophyllaceaeseed storage classification 248tricellular pollen 188

Caryophyllalesseed storage classification 247

Castanea sativa

electron transport chain 173HSP 166, 171, 310

sp.desiccation sensitive seeds 249

Castaneoideaesubfamily phylogeny 249

386 Taxonomic Index


Castanopsisorthodox and recalcitrant seeds 244, 249seed weight 249

Castanospermumaustrale

axis drying curve 99sp.

seed anatomy 252cattail

pollen membrane Tm 299, 301Celastraceae

seed storage classification 248Ceratodon purpureus

predrying 216spore storage 194

Ceratophyllalesorthodox seeds 246seed storage classification 247

Ceratophyllum demersumorthodox seeds 246

Ceterach desiccation tolerant species

cordatum 220officinarum 220

Chamaegigas intrepidusdrying in situ 226leaf desiccation tolerance 9morphology 10natural habitat 224vegetative desiccation tolerance 223

Cheilanthesdesiccation tolerant species

albomarginata 218bonariensis 218buchtiennii 218capensis 218depauperata 218dinteri 218distans 219eckloniana 218farinosa 218fragillima 219glauca 218hirta 218inaequalis 218integerrima 218lasiophylla 219lendigera 219marginata 218marlothii 218multifida 218myriophylla 218parviloba 218paucijuga 219pringlei 219sieberi 219sp. 10, 217tenuifolia 219

vellea 219wrightii 219

Cheilothela chloropusdesiccation tolerance 209

Chenopodiaceaeangiosperm phylogeny 242seed storage classification 248tricellular pollen 188

Chenopodium quinoaseed storage classification 241

Cibotum glaucumgametophyte cryopreservation 225

Citrus limon

variable seed desiccation tolerance 266sp.

orthodox and recalcitrant seeds 240, 244see also lemon

Cladonia dark respiration

convoluta 215furcata 215

Clusiaceaeseed storage classification 247–248

cocoaoil body fusion 350oligosaccharide:sucrose ratio 172see also Theobroma cacao

Cocos nuciferaseed anatomy 252

Coffea arabica

development and desiccation tolerance159

effect of drying 266seed water sorption 64

sp.compilation of desiccation sensitive seeds

240habitat and seed storage 252seed moisture content at dispersal 252variation in desiccation tolerance 155,

253, 266water and physiological activity 52

Colletotrichum gloeosporioidesdesiccation tolerant spores 196

Coleochloa pallidior

vegetative desiccation tolerance 221setifera

longevity when dry 225vegetative desiccation tolerance 221velamen 224

Commelinalesseed storage classification 247

Compositaepollen germination time 188see also Asteraceae

Taxonomic Index 387

Dessication Taxonomic Index 9/4/02 9:34 am Page 387

388 Taxonomic Index

Commelinaceaepollen storage life 190

Convolvulaceaeseed storage classification 248

Coprosma sp. orthodox and recalcitrant seeds 244

Cordia alliodoraseed storage classification 241

Cornalesseed storage classification 247

Corylus avellanaseed storage classification 241

cottonlea mRNAs 171LEA proteins 24seed imbibition damage 344

cowpeaseed imbibition damage 344–345, 348

Craterostigma hirsutum

carbohydrates 325lanceolatum

carbohydrates 325monroi

vegetative desiccation tolerance 223nanum

tolerance of rapid desiccation 224vegetative desiccation tolerance 223

plantagineumABA 25carbohydrates 28, 325desiccation tolerance 217drying in situ 226gene expression 30–31, 326, 330glass formation 27, 302homologues to TIPs and PIPs 166HSP 26, 165, 307, 3092-octulose 26LEA proteins 25, 162lipoxygenase inhibitor 296molecular studies 321water uptake 227–228rehydrins 349RNA 323tolerance of rapid desiccation 224transgenic calli 335vegetative desiccation tolerance 223

sp. control of water loss 348gene product 332molecular studies 322 transgenic plants 332vegetative desiccation tolerance 321, 327

wilmsiianthocyanin levels 224, 296ascorbate peroxidase activity 296desiccated and hydrated state 11folded cell walls 25

post-germination response 254 tolerance of rapid desiccation 223vegetative desiccation tolerance 60

Cruciferaetricellular pollen 188see also Brassicaceae

Ctenopteris heterophylladesiccation tolerant species 220

cucumberaxes CO2 production 295metabolic imbalance 169plasma membrane lesions 347

Cucurbitadesiccation sensitive pollen 187pollen proline content 190

Cucurbitaceaedesiccation sensitive pollen 9, 187pollen storage life 190seed storage classification 248

Cucurbitalesseed storage classification 247

Cupressus macrocarpaseed storage classification 241

Cyatheaspore storage

delgadii 193spinulosa 193

Cyperaceaein situ 188seed storage classification 248vegetative desiccation tolerance 220, 243

Cyperus bellisvegetative desiccation tolerance 221

Dactylis glomeratapollen storage 192

Davallia fejeensisgametophyte cryopreservation 225

Davalliaceaedesiccation tolerant species 220

Dendrographa minorphotosynthesis and water potential 229

dicotyledonsvegetative desiccation tolerance 222

Dicranalesdesiccation tolerance 209

Dicranoweisia cirrata

desiccation tolerance 209crispula

spore storage 194Dicranum elongatum

temperature and photosynthesis 213Diffenbachia

desiccation sensitive pollen 187Dioscoreales

seed storage classification 247


Diospyros sp. orthodox and recalcitrant seeds 244

Diphyscium foliosumABA-induced tolerance 216

Dipsacalesseed storage classification 247

Dipterocarpaceaehabitat and seed storage 252recalcitrant seeds 247

Dipterocarpusseed anatomy

alatus 252tuberculatus 252

variation in seed desiccation tolerance sp. 155

Doryopterisdesiccation tolerant species

concolor 219kitchingii 219pedata 219triphylla 219

Dovyalis hebecarpaseed storage classification 241

Drymaria quercifoliagametophyte cryopreservation 225

Dryopteris filix-mas

spore desiccation tolerance 192paleacea

chlorophyll fluorescence and germination192

spore desiccation tolerance 192Dumortiera hirsuta

negative turgor pressure 57dwarf French bean

seed imbibition damage 348

Ekebergia capensisseed drying curve 99

Elaeis guineensisseed storage classification 241see also oilpalm

Encalypta sp.

desiccation tolerance 209streptocarpa (contorta)

predrying 216Encalyptales

desiccation tolerance 209Equisetum

arvensespore germination 194

hyemalespore longevity 194

Eragrostiellavegetative desiccation tolerance

bifaria 221

brachyphylla 221nardioides 221

Eragrostisvegetative desiccation tolerance

hispida 221invalida 221nindensis 221paradoxa 221

post-germination responsenindensis 254

Ericaceaeseed storage classification 248

Ericalesnumber of desiccation sensitive seeded

species 246–247Erythrina caffra

respiratory enzymes 173Escherichia coli

gene product 332LEA-like proteins 162

Euphorbiaceaeseed storage classification 248

Eurhynchium pulchellumvegetative desiccation tolerance 209

EutactaSection of Araucariaceae 245–246

Exormotheca holstiiABA-induced vegetative desiccation

tolerance 216

faba beanseed imbibition damage 348

Fabaceaeseed anatomy 252seed storage classification 248see also Leguminosae

Fabalesseed storage classification 247

Fagaceaeseed storage classification 246, 248–249,

253species coverage 246

Fagalesseed storage classification 247

Fagoideaesubfamily phylogeny 249

Fagopyrum esculentumseed storage classification 241see also buckwheat

Fagus sp.desiccation tolerant seeds 249seed weight 249

Fimbristylisvegetative desiccation tolerance

dichotoma 221sp. 221

Taxonomic Index 389


Fissidens adiantoidespredrying 216

Flacourtia indicaseed storage classification 241

Fragraea fragransseed storage classification 241

Funaria hygrometricaABA-induced tolerance 216

Garcinia sp. orthodox and recalcitrant seeds 244

Garryalesseed storage classification 247

Gentianaceaeseed storage classification 248

Gentianalesseed storage classification 247

Geranialesseed storage classification 247

Gesneriaceaecarbohydrates 325desiccation tolerant species 10vegetative desiccation tolerance 220, 243,

321Gramineae

cryogenic storage of pollen 191desiccation sensitive pollen 187–188desiccation tolerance and seed

development 155pollen germination time 188pollen shape 188tricellular pollen 188vegetative desiccation tolerance 192see also Poaceae

Grammitidaceaedesiccation tolerant species 220

Grimmiaapocarpa

in situ 13desiccation tolerance 209

laevigatadesiccation tolerance 213in situ 13longevity 209survival after storage 7

pulvinatachlorophyll fluorescence 212CO2 uptake 214predrying 216recovery of carbon fixation 230

temperature and survival 213Grimmiales

desiccation tolerance 209groundnut

seed coat and rehydration 348Guifoylia monostylis

respiratory enzymes 173

Gymnocarpium dryopterisspore bank 193

Gymnospermdesiccation tolerance 220pollen germination time 188

Haberlearhodopensis

carbohydrates 325vegetative desiccation tolerance 222

sp.desiccation tolerant species 10

Hamamelidalesvegetative desiccation tolerance 243

hazelnutseed storage classification 241

Hedera helixseed storage classification 241

Hedwigia ciliata (albicans)

predrying 216sp.

desiccation tolerance 209Hedwigiales

desiccation tolerance 209Helminthosporium

oryzae 196sp. 195

Hippocastanaceaeseed storage classification 248

Homalothecium lutescensphotosynthesis and water potential 229

Hookeria lucenschlorophyll fluorescence 211

Hookerialesdesiccation sensitivity 209

Hopea sp.variation in seed desiccation tolerance 155

Hordeum sp.desiccation sensitive pollen 187see also barley

Hymenophyllaceaedesiccation tolerant species 220

Hymenophyllumdesiccation tolerant species

tunbridgense 220wilsonii 220

vegetative desiccation tolerancesanguinolentum 220

Hypnalesdesiccation tolerance 209

Hypnobryalesdesiccation studies needed 216

Hypnum sp.desiccation tolerance 209

390 Taxonomic Index


Illicalesangiosperm phylogeny 246

Ilysanthesvegetative desiccation tolerance

purpurascens 223wilmsii 223

Impatiens sp. desiccation tolerant pollen 346oligosaccharides 307pollen membrane phase transition 349thermal events in seeds 136

Indian wild riceseed storage classification 241see also Zizania

Inga sp.seed anatomy 252

IntermediaSection of Araucariaceae 245–246

Iridaceaeseed storage classification 248

Isoetaceaedesiccation tolerant species 218

Isoetes australisdesiccation tolerant species 218

Juglans sp.compilation of desiccation sensitive seeds

240Juncaceae

tricellular pollen 188

Kyllinga albavegetative desiccation tolerance 221

Labiataevegetative desiccation tolerance 223, 243

Lamiaceaeseed storage classification 248

Lamialesseed storage classification 247

Landolphia kirkiidevelopment and desiccation tolerance 160seed drying curve 99

Lauraceaerecalcitrant seeds 246seed storage classification 248

Lauralesnumber of desiccation sensitive seeded

species 246–247Leguminosae

seed anatomy 252see also Fabaceae

Lemnaceaeseed storage classification 248

lemonvariable seed desiccation tolerance 266see also Citrus

lettuceEPR imaging of seeds 126

Leucondon sciuroidesdesiccation tolerance 209

Liliaceaevegetative desiccation tolerance 10, 221, 243

Lilialesseed storage classification 247

Limosella gradiflora

desiccation tolerant corms 9sp.

vegetative desiccation tolerance 223Lindernia

carbohydratesacecularis 325brevidens 325

vegetative desiccation tolerance sp. 223, 321

Litchi chinensisdesiccation tolerance and seed

development 159Lobaria pulmonaria

high-light damage to tissue 229lucerne

transgenic plants 332, 334Lunularia cruciata

lunularic acid 216Lycopsida (clubmosses)

desiccation tolerant species 218Lygodium japonicum

spore desiccation tolerance 192

Magnolia sp.orthodox and recalcitrant seeds 244

Magnolialesorthodox and recalcitrant seeds 246–247

maizeABA-deficient mutants 163–164antioxidants and desiccation tolerance 296chemical shift imaging of kernels 131chromatin 278, 299dehydrin 328DNA repair 357LEA proteins 24, 308, 334pollen DNA repair 355pollen proline content 190pollen shape 189pollen storage 192protein secondary structure 280seed imbibition damage 344sugars and vitreous state 305see also Zea mays

Taxonomic Index 391


Malpighialesseed storage classification 247

Malus sp. compilation of desiccation sensitive seeds

240see also apple

Malvaceaeseed storage classification 248

Malvalesnumber of desiccation sensitive seeded

species 246–247Mariscus capensis

vegetative desiccation tolerance 221Mauritia sp.

compilation of desiccation sensitive seeds240

Melastomataceaeseed storage classification 248species coverage 246

Meliaceaehabitat and seed storage 252multiple criteria 250recalcitrant seeds 247

Mesembryanthemum crystallinumgene product 332

Metarhiziumdesiccation tolerant spores

anisopliae 196sp. 195

flavoviridedesiccation tolerant spores 195–196, 242drying rate and survival 195spore imbibitional injury 197

Michelia champacaseed storage classification 241

Micrairavegetative desiccation tolerance

adamsii 221spinifera 221subulifolia 221tenuis 221

Microchloavegetative desiccation tolerance

caffra 221indica 221kunthii 221

Microdracoides squamosavegetative desiccation tolerance 221

Mielichhoferia elongatapredrying 216

Millettia sp.seed anatomy 252

Mimordicadesiccation sensitive pollen 187

Mniaceaedesiccation tolerance studies needed 216

Mnium hornum

recovery time 230marginatum

predrying 216Mohria caffrorum

desiccation tolerant species 220monocotyledons

vegetative desiccation tolerance 220Moraceae

seed storage classification 247–248moth bean

gene product 332mung bean

chemical shift imaging of seeds 1311H-NMR spectra of water 77

Muntingia calaburaseed storage classification 241

Myrothamnaceaecarbohydrates 325vegetative desiccation tolerance 10, 223,

321Myrothamnus

flabellifolius (flabellifolia)anthocyanin levels 227carbohydrates (flabellifolia) 325desiccated and hydrated state 11desiccation tolerance 217natural habitat 224negative turgor pressure 57rehydration 227trehalose (flabellifolia) 168, 324vegetative desiccation tolerance

(flabellifolia) 220, 223moschata

vegetative desiccation tolerance 223Myrtaceae

seed storage classification 247–248Myrtales


Najas flexilisseed desiccation tolerance 253

Nanuza plicatavegetative desiccation tolerance 222

neemEPR of axis chilling stress 122imbibitional stress 121, 344seed storage classification 241thermal events in seeds 136variable seed desiccation tolerance 266see also Azadirachta indica

nematodestrehalose 168

392 Taxonomic Index


Neosartorya fischeridesiccation tolerant spores 196heat resistant spores 197

Nicotiana plumbaginifoliagene product 332

Norway mapleseed development 156see also Acer platanoides

Nostoc sp.desiccation-tolerant cysts 17water and physiological activity 52

Nothochlaena marantae

vegetative desiccation tolerance 217, 219parryi

temperature and photosynthesis 224vegetative desiccation tolerance 219

Nothofagus sp.desiccation tolerant seeds 249seed weight 249

Nymphaeagigantea

orthodox seeds 249sp.

angiosperm phylogeny 246Nymphaeaceae

recalcitrant seeds 247Nymphaeales

angiosperm phylogeny 246Nyssa aquatica


oakstate diagram for cotyledon 305see also Fagaceae and Quercus

oatgene product 332plasmalemma fusion in leaves 274see also Avena fatua

oilpalmseed storage classification 241see also Elaeis guineensis

Oligotrichum hercynicumspore storage 194

Onoclea sensibilisspore desiccation tolerance 192

Orchidaceaeseed storage classification 248

Oropetiumcarbohydrates

thomaeum 325vegetative desiccation tolerance

capense 221roxburghianum 221thomaeum 221

Orthotrichalesdesiccation tolerance 209

Orthotrichumanomalum

in situ, desiccated and hydrated 14desiccation tolerance 209

Osmunda japonicaspore proline and arginine content 193

Oxalidalesseed storage classification 247

Oxalis sp.bulbils/vegetative propagules 228seed storage classification 240

Paecilomyces desiccation tolerant spores

farinosus 195–196fumosoroseus 196

Palmaerecalcitrant seeds 247 seed anatomy 252see also Arecareae

Pandanalesseed storage classification 247

Papaveraceaepollen shape 188

Papaver dubium

pollen development 189rhoeas

pollen shape 189papaya

intermediate seed 268Paraceterach muelleri

desiccation tolerant species 219pea

axis water stress 267chemical shift imaging of seeds 131carbon dioxide production in axes 294desiccation tolerance 280glass formation in axes 303lipid peroxidation in axes 118metabolic imbalance 169mitochondrial activity on seed

rehydration 359seed imbibition damage 344, 348sHSP 309TBARS assay 119 see also Pisum

Pellaeadesiccation tolerant species

atropurpurea 219boivinii 219calomelanos 219falcata 219glabella 219hastata 219

Taxonomic Index 393


Pellaea continueddesiccation tolerant species continued

longimucronata 219ovata 219quadripinnata 219rotundifolia 219sagittata var. cordata 219ternifolia 219viridis 219

Penicillium bilajidesiccation sensitive conidia 195spore storage 195

Pennisetumdesiccation sensitive pollen

purpureum 188sp. 187

desiccation tolerant pollenamericanum 188typhoides 188

Pentaclethra sp.seed anatomy 252

Pentagramma triangularisforest understorey 17in situ, desiccated and hydrated 13

Petunia sp.pollen DNA repair 354, 356

Phacelia tanacetifolia31P NMR and seed metabolism 132

Phaseolus vulgarisABA and seed development 171NMR imaging of seeds 131premature drying 161seed development 152–153, 159

Phegopteris connectilisspore bank 193

Philonotis seriatapredrying 216

Phycomyces blakesleeanusdesiccation tolerant spores 196

Phyllisis scolopendriumspore storage 193

Pilotrichella ampullacearecovery time on remoistening 214

Piper hispidumseed storage classification 241

Piperaceaeseed storage classification 248

Piperalesorthodox seeds 246–247

Pisum sp.seed anatomy 252see also pea

Pithecellobium sp.seed anatomy 252

Pittosporum sp. orthodox and recalcitrant seeds 244

Plagiomnium rostratumpredrying 216

Plagiothecium undulatumpredrying 216water potential and survival 211

Platycerium stemariavegetative desiccation tolerance 217, 220

Platyhypnidium rusciformepredrying 216

Pleurochaete squarrosachlorophyll fluorescence 212

Pleurosorus rutifoliusdesiccation tolerant species 220

Pleurostima sp.vegetative desiccation tolerance 222

Pleurozium schreiberipredrying 216

Poa bulbosavegetative desiccation tolerance 221

Poaceaecarbohydrates 325desiccation sensitive pollen 9desiccation tolerant species richness 10seed storage classification 248vegetative desiccation tolerance 221, 243see also Gramineae

Poalesseed storage classification 247

Podocarpaceaeseed desiccation sensitivity 244

Podocarpus henkelii

radical tip and axis drying curves 99usambarensis

orthodox seed 244Pohlia elongata

predrying 216Polygonaceae

seed storage classification 248Polypodiaceae

desiccation tolerant species 220Polypodium

desiccation tolerant speciescambricum 220vulgare 220

polypodioidesilluminated drying 226specialized structures 224vegetative desiccation tolerance 217, 220

virginianumdesiccation tolerant species 220repair processes 28

Polystichum setiferumspore storage 193

Polytrichalesdesiccation tolerance 209

394 Taxonomic Index


Polytrichum formosum

recovery time 230piliferum

desiccation tolerance 209Porphyra sp.

large scale drying 97Pottiales

desiccation tolerance 209proso millet

testa colour and imbibition 348Proteaceae

seed storage classification 248Proteales

seed storage classification 247Pseudopezicula

desiccation tolerant spores 195Pteridaceae

desiccation tolerant species richness 10Pteropsida (ferns)

desiccation tolerant species 218Puccinia

desiccation tolerant sporesgraminis 196recondita 196

Quercus robur

ABA and seed development 171critical water content 66critical water potential 50cytoskeleton 174, 273matrix-bound water 53protectant against oxidative stress 174seed development and desiccation

tolerance 158–9soluble sugars 172volatiles and unregulated respiration 173

rubracritical water potential or water content

50pressure–volume curve 58sorption isotherm of cotyledon tissue 67soluble sugars 172water hydration sites 66

sp.desiccation sensitive seeds 249variation in desiccation tolerance 155see also oak

Racomitrium aciculare

spore storage 194lanuginosum

chlorophyll fluorescence 212

desiccation tolerance 213, 216recovery processes 215temperature and photosynthesis 213water potential and survival 211

sp.desiccation tolerance 209survival pattern 213

Ramalina maciformiswater storage and gas difussion 20wetting and drying 18

Rammondia sp.desiccation tolerant species 10

Ramondacarbohydrates

myconi 325vegetative desiccation tolerance

myconi 223nathaliae 223pyrenaica 223serbica 223

Ranunculaceaeseed storage classification 248

Ranunculalesseed storage classification 247

Ranunculusseed priming

arvensis 354sceleratus 354

tuber desiccation toleranceasiaticus 228

vegetative propagulesficaria 228

red rice13C labelling and seed metabolism 132

Rhizocarpon geographicumadaptation to climate 230

Rhizophoraceaerecalcitrant seeds 247

Rhynchostegium riparioidespredrying 216

Rhytidiadelphusloreus

chlorophyll fluorescence 211dark respiration 215recovery time 230

sp.predrying 216

Riccia fluitans

effect of ABA 216macrocarpa

gametophyte longevity 209survival after storage 7

ricelea gene 25non-detection of Tg by DSC 136seed chilling injury 344transgenic plants 331–334

Taxonomic Index 395


Ricinus communisdesiccation intolerance 155seed development 152–153

Rosaceaeseed storage classification 248

Rosalesseed storage classification 247

Roystoneaseed storage classification 240

Rubiaceaeseed storage classification 248, 253

Rutaceaeseed storage classification 247–248

ryeDNA repair in embryos 357–358pollen cryogenic storage 192transcription in embryos 352see also Secale cereale

Sabal sp.seed storage classification 240

Saccharomyces cerevisiae

desiccation tolerant cells 196HSP 310LEA-like proteins 162, 308mutants 196see also yeast

uvarumdesiccation tolerant cells 196

Saccharum sp.desiccation sensitive pollen 187

Salix sp.seed storage classification 240

Santalalesseed storage classification 247

Santalum albumseed storage classification 241

Sapindaceaeangiosperm phylogeny 242seed storage classification 248

Sapindalesseed storage classification 247

Sapotaceaeseed storage classification 247–248

Satureja gilliesiidesiccation tolerant organ/tissue 9, 223

Saxifraga sp.vegetative propagules 228

Saxifragalesseed storage classification 247

Schistidium rivularespore storage 194

Schizaea sp.desiccation tolerant species 220

Schizaeaceaedesiccation tolerant species 220

Schizophyllum communesurvival after storage 8

Scleropodium tourretiidesiccation tolerance 209

Sclerotinia sclerotinumdesiccation tolerant spores 196

Scrophulariaceaecarbohydrates 325seed storage classification 248vegetative desiccation tolerance 223, 243,

321Secale

cerealeDNA repair 358see also rye

sp.desiccation sensitive pollen 187

Selaginelladesiccation tolerant species

caffrorum 218convoluta 218digitata 218imbricata 218njam-njamensis 217–218peruviana 218pilifera 218sartorii 218

lepidophylladesiccation tolerant species 218drying rate 6folded cell walls 60illuminated drying 226membrane organization 22predrying 226resurrection 217

sellowiidesiccation tolerant species 218in situ 12

sp.desiccation tolerant species richness 10,

217evolution of desiccation tolerance 243heat tolerance 8 photosynthesis 17, 227trehalose 324

Selaginellaceaedesiccation tolerant species 218

Septoria nodorumhydrated storage of spores 197

Shorearobusta

free-radical scavenging 174sp.

variation in seed desiccation tolerance155

Solanaceaepollen shape 188seed storage classification 248

396 Taxonomic Index


Solanalesseed storage classification 247

Sordariadesiccation tolerant spores

macrospora 196survival of cavitation 57

sorghumdesiccation sensitive pollen 187seed imbibition damage 344

soybeanantioxidant in seed membranes 296glass composition in axes 305glycoproteins in seed coat 348LEAs in axes 308lipid-soluble antioxidants 16913C NMR and seed metabolism 132seed development and desiccation

tolerance 153seed imbibition 155seed imbibition damage 344water clustering in axes 66

Sphagnum sp.net photosynthesis 17

Spondias sp.orthodox and recalcitrant seeds 244

Sporobolusvegetative desiccation tolerance

atrovirens 221elongatus 221festivus 221fimbriatus 222lampranthus 222pellucidus 222

sp.desiccation tolerance 327gene expression 330molecular studies 322

stapfianuscarbohydrates 325control of water loss 348desiccated and hydrated 15EST collections 30LEA proteins 307molecular studies 321protein synthesis 329rehydrins 349repair processes 28trehalose 168, 324vegetative desiccation tolerance 220xeromorphic characteristics 224

Stagonospora convolvulidesiccation tolerant spores 196spore longevity 197

Sterculiaceaeseed storage classification 248

Streptocarpus sp.desiccation tolerant vegetative tissue 223

sugarbeettransgenic plants 332–333

sunflowerABA-deficient mutants 164sHSP 309

Swieteniaseed storage classification 240

sycamoreseed development 156see also Acer pseudoplatanus

Syntrichiadesiccation tolerance 209

Syzigium guinienseaxis drying curve 99

Talaromyces flavus desiccation tolerant spores 196heat resistant spores 197

Talbotia elegansvegetative desiccation tolerance 222

Taxus brevifoliaorthodox seed 252

teaaxis viability loss 102metabolic imbalances in seeds 280variable seed desiccation tolerance 266see also Camellia sinensis

Telphairia occidentalisviviparous germination 158

Theobroma cacaoABA and seed development 171drying curves of axes 69–71, 99free-radical scavenging 174see also cocoa

Thrinax sp. seed storage classification 240

Thuidium delicatulumprotein analysis 328

Tiliaceaeseed storage classification 248

Timmia austriacapredrying 216

tobacconon-detection of Tg by DSC 136transcription factors 326transgenic plants 326, 331–334

Todea barbaraspore storage 193

tomatoABA-deficient mutants 163fruit shedding 252

Tortelladesiccation tolerance 209

Tortulalatifolia

in situ, desiccated and hydrated 14(ruralis subspecies) ruraliformis

Taxonomic Index 397


Tortula continued(ruralis subspecies) ruraliformis continued

sucrose 26CO2 uptake 214

(syn Syntrichia) ruralisABA 216cellular integrity 327cellular protection and dehydrins 25chlorophyll fluorescence 211–212chloroplast 328dark respiration 215desiccation and hydration in situ 15desiccation tolerance 216drying rate 216EST collections 30–31growth in dark 229leaf longevity 209metabolism and protein synthesis

328–329metabolism on de- and rehydration 29molecular studies 321, 327, 329–330phosphorus and potassium content and

nitrate reductase activity 19predrying 216rapid adaptation 230recovery time on remoistening 214–215rehydrins 349sucrose 27TEM of leaf cells 16temperature and survival 213water potential and longevity 211

sp.desiccation tolerance 209

Trichilia dregeanaaxis drying curve 99 axis viability loss 102cytoskeleton 273water and seed longevity 103

Trichoderma harzianumdesiccation tolerant spores 196

Trilepis sp.vegetative desiccation tolerance 221

Trimeniaceaeangiosperm phylogeny 246

Tripogonvegetative desiccation tolerance

capillaris 222curvatus 222filiformis 222jacquemontii 222lolioformis 222lisboae 222minimus 222polyanthus 222spicatus 222

Triticum sp.desiccation sensitive pollen 187see also wheat

Typha latifolia

membrane permeability 34631P NMR and phospholipids 133pollen imbibitional leakage and EPR

spectra 123pollen storage life 191

sp.pollen monolayer hydration 65

Ulota crispa

chlorophyll fluorescence 212sp.

desiccation tolerance 209Umbelliferae

tricellular pollen 188see also Apiaceae

Uromyces appendiculatusdesiccation tolerant spores 195–196

Urticaceaeseed storage classification 248

Ustilago scitamineadesiccation tolerant spores 195–196spore longevity 197

Vellozia sp.vegetative desiccation tolerance 222

Velloziaceaecarbohydrates 325desiccation tolerant species 222desiccation tolerant species richness 10in situ 12vegetative desiccation tolerance 243

Venturia inaequalisdesiccation tolerant spores 195–196

Vicia narbonensisADP-glucose pyrophosphorylase activity

112Vitellaria paradoxa

habitat and seed storage 252Vitex sp.

orthodox and recalcitrant seeds 244Vochysia honurensis


walnutserotonin accumulation 170

Washingtonia sp.seed anatomy 252

Welwitschia mirabilisfoliage desiccation tolerance 217, 220, 225

Welwitschiaceaedesiccation tolerant species 220

398 Taxonomic Index


wheatamphiphile partitioning 370dehydrins in embryos 309DNA repair 357EM protein 25EPR imaging of kernels 126EPR of seed tissues 124LEA transgene 332, 334NMR imaging of kernels 131proembryo desiccation tolerance and EPR

121seed development 152 T2 water relaxation 129see also Triticum

Wollemia nobilisdesiccation tolerant seeds 245 seed size 245

Woodsia ilvensidesiccation tolerant species 220

Xerophyta humilis

chloroplast 272pinnifolia

velamen 224retinervis

desiccated and hydrated state 11scabrida

CO2 and photosynthesis 19rehydration and respiration 18

sp.adaptation to habitat 230desiccation tolerant species richness 217vegetative desiccation tolerance 222

squarrosalongevity when dry 225

villosacarbohydrates 325molecular studies 321

viscosaanthocyanin levels 227ascorbate peroxidase activity 296control of water loss 348desiccated and hydrated state 11

yeasttrehalose 168, 324see also Saccharomyces cerevisiae

Zea maysdesiccation sensitive pollen 187–188LEA proteins 307see also maize

Zingiber sp. desiccation sensitive pollen 187

Zingiberaceaedesiccation sensitive pollen 187–188seed storage classification 248

Zingiberalesseed storage classification 247

Zizania aquatica

seed storage classification 241palustris

drying temperature 98 imbibitional damage 102, 344post-germination response 266tetrazolium test 104see also Indian wild rice

Zygodondesiccation tolerance 209

Taxonomic Index 399



Subject Index

ABA see Abscisic acidABRE see Abscisic acidAbscisic acid (ABA) 24 et seq, 154 et seq, 190,

194, 216, 226, 308, 309, 323 et seq, 334,356, 367 et seq,

analog 164mutants 25, 163 et seq, 310, 326 et seq, 335response element (ABRE) 164

Abscission 172Accelerated ageing 113Acetaldehyde 114, 115, 169, 173, 295Activity, water 50 et seqADP 294ADP-glucose pyrophosphorylase 112Adsorption 53Ageing 229, 304, 311, 345, 351, 357Aldehydes 114Algae 4 et seq, 320Aleurone layer 135, 163, 170, 334Alkanes 114Alkenes 114 Alkones 114Alkyl hydroperoxidase 33Amino acids 190, 198, 229, 277Ammonia 170Amphipaths 280, 296, 308, 347 et seqAmphiphiles 22 et seq, 113 et seq, 294, 296 et

seq, 311, 347, 370antioxidant 296 et seqendogenous 297, 371

�-Amylase 308Angiosperms 7 et seq, 150 et seq, 220 et seqAnhydrobiotes (anhydrobiosis) 116, 186, 198,

293 et seq, 349 et seqAnnuals, desiccation tolerant 10

Anoxia 8Antarctica 17, 18Anthesis 189Anthocyanins 18, 118, 165, 227, 265, 296, 348Antioxidants 167, 169, 228, 264, 294 et seq,

311, 371Antisense 112Aquaporins 166Aquatic species 241, 249, 250, 253Arthropods 7 Ascopore 57, 196, 197Ascorbate 296Ascorbate peroxidase 296Ascorbic acid 174, 280Arginine 190, 195, 196Arginine decarboxylase 333Aspartyl protein methyl transferase 28, 170, 351Aspartyl residues 28, 170, 351ATP 351, 358, 359Axes see Embryo

Basal meristems, desiccation tolerance 9Betaine 132Bilayers 133

compression 281Boreal zone 17Bovine serum albumin 308Broad leaved forests 249Broadening agents 120 et seqBrowning 113, 303Brunauer-Emmet-Teller (BET) model 60 et seqBryophytes 6 et seq, 207 et seq, 320 et seq, 368Bulbils 228Bulbs 228

401

Dessication Subject Index 3/4/02 2:30 pm Page 401

C3 plants 117, 213C4 plants, desiccation tolerant 12Calcium 168, 278, 371Callus 25, 26, 165, 335Calorimetry 74, 112 et seq, 305Calvin cycle 117Capillary action 227Carbohydrates 296, 321 et seqCarbon 18

balance 18, 20, 208, 215, 327gain 19loss 211, 217

Carbon dioxide 169, 210, 294exchange 114

Carnitine 298Carotenoids 18, 23Catalase 295Cavitation 12, 20Cell

compartmentation 129compartments 132contraction 270cycle 215, 352, 359damage 328 et seqdivision 122, 150, 215, 279, 352, 367enlargement 122expansion 150, 264, 347, 352integrity 303, 350pH 126, 132recovery 328 et seqshrinkage 271size 269ultrastructure 16, 22 et seq, 136volume 112, 226, 265 et seq

Cell walls 21convolution 227, 270elasticity 59folding 270

Chalaza 252 Chaparral 17Chaperonin 26, 167, 310Chemical potential 51 et seqChlorophyll 18, 23, 165, 208 et seq, 223, 265,

320, 333fluorescence 104, 116, 192, 209 et seq, 334,

368Chloroplast 16, 23 et seq, 223, 227, 271 et seq,

307, 328, 368Chromatin 166, 308, 344, 357Chromium oxalate 120cis-Acting elements 326Cladistics 242Cladogram 253Classification 244 et seq

molecular data 246Climbers 250Cold tolerance 8

Compaction, of molecules 273Compartmentation 274, 344Compatible solutes 190, 198, 264, 276, 294, 298,

301, 308, 311, 331Compensation point 17Competition 20Conidia 195, 196, 243Conservation 240 et seqCorms 9 Cotyledons 150, 152, 166, 170, 245, 294, 347,

354, 367Crassulacean acid metabolism 10Critical water activity 65Critical water content 157, 303Critical water potential 241, 268Crustacea 207Cryopreservation 160, 225Cryoprotection 196, 197Crystallization 54, 347, 303, 311Cuticle 217Cyanobacteria 4, 115Cyclitols 369Cytokinesis 351, 353Cytosine 351Cytoskeleton 273Cytosol 307

Damage 21 et seq, 28, 65, 151, 159 et seq, 263 etseq, 328 et seq, 344 et seq

desiccation induced 113, 263 et seq, 294free radical 114 et seq, 321and metabolism 295

D’Arcy–Watt model 61De-esterification 294 Dehydrins see LEAsDehydroascorbic acid 296Dephosphorylation 322Deserts 17Desiccation-sensitive plants 116Desiccation tolerance 150 et seq

animals 7, 207and bacterial infections 10constitutive 208 et seq, 226, 327continuum 151, 242, 246definition 4, 320developmental programme 9vs. drought tolerance 5, 207, 230, 320and drying rates 100ecology 9, 13 et seq, 224, 320, 327environmental induction 9evolution 10, 12, 20, 171, 208, 240 et seq,

321genes 31, 161 et seq, 243, 321 et seqgeographic range 8, 10, 320and germination 9, 37glasses and 303

402 Subject Index


habitats 8, 17, 208, 209, 217 et seqheat-shock proteins 306, 309 et seqhigher plant vs. bryophyte 321induced 216 et seq, 226injury 150 et seq, 263 et seqLEAs and 24 et seq, 161 et seq, 307 et seqlevel 242and longevity 368 et seqmetabolism and 5, 7molecular responses 320 et seq morphological types 10mutants 163 et seq, 310, 324and nutrient availability 10, 18oligosaccharides and 27, 168, 306, 368physiological types 10and productivity 9provenance and 157quantitative 268seeds 150 et seq, 239 et seqsensitivity 239 et seqtaxonomic range 8 et seq, 207 et seq, 321and temperature 213, 224, 280, 327timing 100water potential 157, 368

Devonian–Mississippian 250Dew 13, 18, 20Dew-point depression 53Diaspores 193Dicotyledons

desiccation sensitivity 249 desiccation tolerance 10, 321LEAs 323

Dictysomes 272Dielectric relaxation 74Differential respirometer 114Differential scanning calorimetry 54, 74 et seqDiffusion 113Diffusional correlation time 72Disaccharides 294 et seq, 300 et seqDispersal

fruit 254pollen 186seed 254

DNA 350 et seqamounts 249binding proteins 299breaks 357, 358conformation 299, 344, 356damage 52, 174dehydration 350forms 350free radical damage 117, 357, 359hypersensitive sites 359levels 352ligase 352mitochondrial 117, 353, 357nuclear 117, 350nuclease 351, 352, 357

phosphate 350polymerase 352, 358repair 28, 174, 215, 350 et seqreplication 352sequence data 242stability 278synthesis 351and water 350

Dormancy 9, 29, 150, 173, 186 et seq, 224, 253,330, 349

Drought 298avoiders 230 definition 5evaders 230hardening 209, 215stress 30, 298, 335, 369

Drought tolerance 4, 186, 207, 264 vs. desiccation tolerance 5, 207, 230, 320

Dry matteraccumulation 113and cell shrinkage 271

Drying 113 et seq, 263 et seq, 294 et seq, 368 etseq

in air 4, 94air movement 94 et seqboundary layer 94 et seqcurves 68 et seqcycles 6, 9, 14, 17 et seq, 224equilibration to low humidities 7excised axes 95 et seqfast 68, 208 et seq, 216flash 95 et seqfree radicals 116gene expression 369in light 6methods 96 et seqrapid 6, 19, 195, 225, 328rate 6, 29, 68 et seq, 79, 94 et seq, 321, 327,

328seed shape 98seed size 98seeds 152 et seqin shade 94 et seqsilica gel 95, 152slow 29, 195, 208 et seq, 216, 328and sucrose 27in sun 94 et seqsurface/volume ratio 94 et seqtemperature 94, 98, 157time 71, 96, 112, 226tissues 94 et seq

Editosome 351Electron microscopy 22Electron paramagnetic resonance (EPR) 112 et

seq, 297, 304 346

Subject Index 403

Dessication Subject Index 9/4/02 9:32 am Page 403

Electron spin resonance (ESR) 112 et seq, 297,304

Electron transport 116, 173, 294Em

gene 163protein 25 et seq

Embryo 150, 245, 252, 270 et seq, 299, 334, 344et seq

axes 78, 95 et seq, 150 et seq, 166, 169,294, 295, 303, 305, 354 et seq

drying 95 et seq, 150 et seqsomatic 169, 295, 306

Embryogenesis 164, 266, 278Endoplasmic reticulum 16, 271, 272, 278Endosperm 121, 135, 252Enthalpy 61, 74Entropy 61Enzymes 5

activities 113lability 277repair 263stabilization 169, 303

Eocene 245Ethanol 114, 115, 125, 173, 195, 196, 295Ethylene 115, 131Eudicots 247Evolution, desiccation tolerance 10, 12, 20, 171,

208, 240 et seq, 264Exotherm 54Expressed sequence tags (ESTs) 30Extraction of metabolites 113Extracts 116Extrusion

protein 347starch 347

Fagopyritol 168, 369Fatty acids 167, 294

diunsaturated 271and free radicals 117polyunsaturated 188spin labelled 123

Fermentation 114, 169Ferns 7 et seq, 217 et seq, 320, 344Ferricyanide 120Fixatives 270, 347Flavonoids 296, 297, 347Fluidizing compounds 310, 347 et seqFluorescein 196Fluorescence spectroscopy 114Fog 13Forbs 10, 250Forest tree species 250Fourier transformation 128, 161Fourier transformation infra-red spectroscopy

(FTIR) 112 et seq, 134, 161, 297, 299, 301

Free energy 51, 61and drying 94Gibbs 50

Free radicals 5, 65, 114 et seq, 116 et seq, 173,227, 265, 277, 279, 293 et seq, 351

attack 169desiccation tolerance 116 et seq, 293 et

seq, 321effects on cells 117generators 119processing 296scavenging 174, 265, 279, 295 et seq, 311

Freezing 53, 225, 265, 270, 295, 298, 335Freezing point depression 53 et seqFreezing stress 48Freezing tolerance 7, 8, 59Fructan synthase 333Fructans 298Fructose 322

phosphate 322Fruit structure 243, 244, 252Fungal spores 115Fungi 4

infection 103Funiculus 152

Galactinol 369Galactinol synthase 369Galactopinitol 168Galactosyl cyclitols 306Gametophytes 7 et seq, 29, 186, 209, 245, 253Gas analysis 209Gas diffusion 20Gases 20, 114GCMS 114Genes

ABA responsive 25, 163ABI-3 326desiccation sensitivity 253desiccation tolerance 31, 175, 254, 321 et

seq, 356Em 163enzymatic antioxidants 296expression 157, 320 et seq, 329 et seq, 369,

370fus3 163LEA 24, 161 et seq, 309, 326, 335, 336lec1 165osem 163promoters 326Rab2 28, 163replacement 336Vp 163 et seq

Genetic engineering 210Genome fidelity 356Genomics 372

404 Subject Index


Germinability 152 et seqGermination 122, 162, 253, 266, 306, 309, 353

and desiccation tolerance 9, 174, 367and drying 152 et seqprecocious 154, 164and repair 28test 103, 242

Germplasm preservation 30Gibberellins 164, 193

biosynthesis 165, 193Glass 72, 78, 135, 169, 191, 269, 281 298, 301 et

seq, 311, 324, 351, 345composition 302definition 302in desiccation tolerance 303formation 113, 298, 301 et seqand longevity 303maltodextrin in 306and membranes 303proteins in 306and sucrose 26, 27, 303 et seqand sugars 27, 306temperature 302, 303transitions 27, 75, 136, 301 et seqwater content 302

Globulin 163, 166Glucose 303Glucose-glycerol 325�-Glucuronidase 165Glutamate 190, 196, 298, 331Glutathione 296 Glutathione reductase 174, 295Glyceraldehyde-3-phosphate 322

dehydrogenase 322Glycerol solutions 55, 195, 196Glycine-betaine 298Glycolysis 117Graminoids 10, 250Grana 16, 227, 272Grasses 20, 336Gravitational potential 51 et seqGrowth, effects of desiccation 14

cell 52Growth, g.rate 14, 19, 20GTP – binding protein 28Guanidine-HCl 298Guggenheim-Anderson-de Boer (GAB) model 60

et seqGymnosperms 8, 9, 321, 323

Hairs 19Hardening 216Headspace analysis 114Heat-shock proteins 26, 167, 265, 294, 309 et

seq, 371

in desiccation tolerance 165 et seq, 309 etseq

in seeds 26, 165 et seqtranscription factor 326in vegetative tissues 26

Heat tolerance 8Helices, amphipathic 330�-Helix 135, 161, 166, 277, 323Herbs desiccation tolerant 10Hexagonal phase 274, 346Histidine kinase 371Histodifferentiation 150, 268Homoiochlorophylly 227, 230, 368Homoiohydry 217, 228, 243, 252Hornworts 10HPLC 114Human cells, desiccation tolerance 12Humic substances 126Hydration 50, 52, 66, 72, 114, 155, 267

levels 173, 268, 294Hydraulic conductivity 59Hydraulic flow 345Hydrins 29, 215, 329 et seqHydrogen bonds 72, 135, 169, 277, 278, 298,

300, 301, 307, 311, 324, 349Hydrometer 53Hydroperoxidase 29Hydroperoxides 296Hydrophilins 162Hydrophilly 162Hydrostatic pressure 51 et seq, 60, 68Hydroxyl groups 26, 167, 168, 300Hygrometry 51Hysteresis 60, 105

Imbibition 344 et seq, 351Imbibitional injury (stress) 136, 191, 194, 197,

344 et seqphase change 346temperature 344

Iminonitroxides 126Infra-red spectroscopy 74Inositol D-ononitol 333Insect larvae 7Insertional mutagenesis 335Intermediate seeds 150, 172, 198, 241, 266International Plant Genetic Resources Institute

241International Seed Testing Association 48Intracellular gas 57Invasive techniques 112 et seqIons

distribution 131leakage 215NMR 131sequestration 26, 308

Subject Index 405


406 Subject Index

Isopleth 64Isotherm

curves 51hysteresis 60, 105sorption 60 et seq, 79, 105, 277

K-segment 25, 323Kernel development 121Kinetics

non-equilibrium 70water loss 114

Lactate dehydrogenase 277, 308Late embryogenesis abundant proteins see LEAsLate Palaeozoic 245Late Precambrian 263Leakage 18, 21, 153, 160, 191, 214, 216, 223,

297, 300 et seq, 308, 334, 344 et seq, 348damage 104, 328viability 104vigour 104vital dyes 104

Leathers 268, 280LEAs 24 et seq, 254, 264, 294, 322 et seq, 367 et

seqactions 308binding properties 308dehydrins 24 et seq, 161 et seq, 309, 370and desiccation tolerance 307 et seqgenes 24 et seq, 161 et seq, 309, 326, 335,

336in glasses 306, 309groups 25, 161 et seq, 307, 323HVA1 25, 31, 334hydration 308nuclear 309phosphorylation 309pollen 190proteins 24 et seqRAB17 308structure 25, 323synthesis 153 et seqTAS14 308transcripts 24 et seq, 161 et seq

Leavescarbohydrates in 325curling 226cuticle 348desiccation tolerance 9 et seq, 18, 320 et

seqdrying 101, 223growth 320hairs 348LEAs in 307rolling 265

surfaces 320water content 49 et seqwater potential 53waxes 348

Leeuwenhoek, Anthony von 6Leucine zipper 165, 322, 326Lichens 6 et seq, 114, 136, 320Light 192, 193, 211, 224, 272

damage by 18, 226, 272limiting 20

Lipid bilayer 22oxidation 65peroxidation 117 et seq

Lipid bodies 307Lipids 188, 193Liposomes 26 et seq, 274 et seq, 310, 346

membranes 169, 297 et seqLipoxygenase 296Liquid helium 213Liquid nitrogen 126, 192Longevity 27, 297 et seq, 303 et seq, 368 et seq

oligosaccharides and 305 et seq, 368 et seqpollen 27seeds 27, 241 et seq, 303 et seq

Lyophilization 192Lysine 196, 323Lysozyme 52, 72

Macromolecules 228, 273hydration 298integrity 321stabilization 294, 298, 303, 307

Maillard reaction 172Malonyldialdehyde 117, 118Maltose 301Mannitol 298Marker molecules 371Marsh species 253Matric potential 53 et seq

forces 59 Maturation drying 150 et seq, 296, 370Megagametophyte 150Meiosis 192Membranes

and amphiphiles 296 et seqconformation 346convolution 227damage to 5, 21 et seq, 104, 155 et seq, 173,

328disruption 59, 155 et seqdynamics 133effects of water loss 271 et seqfatty acid domains 273fluidity 124, 134, 191, 279folding 271 et seqfusion 168, 303, 311hydration 73


integrity 121, 160, 169, 214, 228, 276, 293,310, 346 et seq

isolated 124liposomal 26nuclear 227packing 273partitioning into 113, 296 et seqphase 265, 274 et seq, 299 et seqphospholipid 133, 167physical properties 123, 347plastid 227preservation 298protection 26 et seqrehydration 274repair 155, 167rigidity 274, 297sarcoplasmic reticulum 168stability 160, 168stucture 303and sugars 168tearing 166tonoplast 227transition temperature 169, 195, 299vesicles 28, 168

Metabolic activitiesand drying 112and water 114

Metabolism, regulation 294 et seqMetabolites

flux 115NMR 131seeds 115

Microsomes 296Microtubules 215Minimum critical volume 270Mitochondria 16, 116, 161, 189, 227, 271, 273,

279, 296, 297, 344dehydrogenases 359DNA 117, 353, 357genome 357

Mitotic division 150, 264Modulus of elasticity 55, 59Moist forests 17Moist tropics 250Moisture content (MC) 158, 197, 241 et seqMolecular marker analysis 331Molecular movement (mobility) 27, 72, 191,

229, 269, 280, 302, 306 351and ageing 304

Molecular spin probes 78Monocotyledons

desiccation sensitivity 246, 249desiccation tolerance 10, 18, 321LEAs 323

Monosaccharides 172, 303Mosses 29 et seq, 207 et seq, 319 et seq, 344 et

seq

Mucilage 348Multigenic traits 336Mutants 25, 112, 163 et seq, 196, 253, 310, 324

et seqMyo-inositol O-methyltransferase 333

Nematodes 7, 168, 207Neoteny 9Nitrate 192Nitrate reductase 19Nitroxide 120 et seqNMR spectroscopy 74 et seqNon-invasive techniques 112 et seqNuclear magnetic resonance (NMR) 112 et seqNucleases 351, 352, 357Nucleic acids

dehydration 278hydration 52integrity 321synthesis 150and water 350

Nucleolus 170, 309Nucleus 16, 170, 307, 309Nutrient availability 10, 18Nutrient capture 208

2-Octulose 26, 28, 324, 325Oil

bodies 124, 130, 349NMR signals 128in seeds 115 et seq, 242

Oleosins 349Oligosaccharides 27, 118, 167, 170, 189, 294 et

seq, 303, 368 et seqand desiccation tolerance 306, 368 et seqand longevity 305 et seq, 368

Ordovician 243Ornithine 333Orthodox seeds see SeedsOsmole 54Osmolytes 298, 331Osmometer 54Osmosensor 371Osmotic potential 52 et seq, 68 et seq, 264Ovule 186, 252Oxidation 5Oxidative stress 30, 114, 116, 192, 296

damage 293, 297, 311 Oxygen

availability 169exchange 114protection against 198, 333reactive species (ROS) 116, 170, 265, 279,

294scavenging 333

Subject Index 407


Oxygen continuedsolubility 113tolerance of low 8uptake 5, 211, 294

Paramagnetism 120Partitioning

of amphiphiles, amphipaths 22 et seq, 294et seq, 311, 347

within cells 120into membranes 113, 280, 371

Pathogens 229, 344Pectic substances 152Perennating structures 9Perennials, desiccation tolerant 10Permafrost 194Permanent wilting point 270Permo-Carboniferous 245Peroxidases 295Peroxidation 189, 198, 279, 294Peroxide 295Peroxiredoxin 170, 296pH 126, 132Phase transition 22, 134, 167, 274 et seq, 280,

281, 297, 299 et seq, 346 et seqand sugars 300

Phenolics 296, 297Phenols 113 Phloem 323Phosphatase 322, 326Phosphatidylcholine 299Phosphofructokinase 168, 301Phosphoglycerate 322Phospholipase D 322, 326Phospholipid 123, 167, 188, 271 et seq, 294

bilayers 133, 293, 298, 300, 346composition 349hexagonal phase 133and sugars 281, 300 et seq, 307vesicles 169, 188see also Polar lipids

Phospholipid:sterol ratio 270Phosphorus 19Phosphorylation 309, 322Photo-oxidation 5, 264Photoprotection 227Photosynthesis 5, 17 et seq, 29, 52, 114, 209 et

seq, 268, 327, 333Photosystem II 29, 104, 116, 194, 211 et seq,

226, 272, 322Phytochrome 192Pioneers 224Plasma membrane 21 et seq, 153, 189, 196, 226,

270 et seq, 327, 345 et seqfolding 347permeability 122, 346

phase transitions 346rehydration stress 346

Plasmadesmata 21, 270 et seqPlasmalemma 129, 270 et seq, 281Plasmolysis 56, 209, 270Plastids 271, 279, 344Plastoglobuli 23, 271Pleiotropic effects 112Poikilochlorophylly 18, 23, 104, 208, 227, 230,

368Poikilohydry 320Polar lipids, effects of drying 273Pollen 7 et seq, 20, 22, 24, 27, 112 et seq, 133,

150, 186 et seq, 297 et seq, 344 et seq,354 et seq, 368

ABA 190ageing 113, 187amino acids 190bicellular 188compatible solutes 190dispersal 186dormancy 186germination 186 et seqglasses 191hydration 186LEAs 190longevity 186mitochondria 272molecular mobility 72recalcitrant 187shape 188sperm cells 186 et seq, 355storage 191, 198sucrose 189tricellular 188tube 186 et seqviability 186 et seq, 347vigour 347water in 48, 65

Pollination 186Pollinia 191Polyamines 333Polyethylene glycol solutions 55, 104, 295, 344Poly-L-lysine 305Polyols 296, 298Polypeptides 277Polyphosphates 132Poly(ribo)somes 161, 272, 278, 327Polyubiquitin 29, 330, 349Potassium 19Preferential exclusion 298Pressure chamber 53Pressure–volume analysis 49 et seq, 106Priming 353 et seqProductivity

crop 333and desiccation tolerance 9, 20

408 Subject Index


Proembryonic cells 121Proline 190, 195, 331, 333Promoter analysis 326Promoters 326Proteases 167, 351Protectants 165, 228, 264 et seq, 282, 294, 303Protection 268, 321, 322, 327 et seq, 347 et seq,

368 et seqProtein 24 et seq

ABI 165, 322bodies 161, 307channel 166conformation 113, 135, 277, 301, 303, 322denatured 167, 169, 311, 320desiccation related 161early light inducible 322elF1 322Em 25 et seqextraction 113extrusion 347and free radicals 117folding 293, 298FUS3 165heat shock 26, 167, 265, 294, 309 et seq,

371heat stable 190, 194, 198hydration 52, 72, 73, 276, 349hydrins 29, 329 et seqintegrity 321interaction with sugars 135, 301, 311kinase 322, 326labile 293LEC1 165L-isoaspartyl residues 28LEA see LEAsmajor intrinsic (MIPs) 166, 322myb 322, 326phosphatase 326preservation 298protective 151Rab2, 17, 28, 29, 163rehydrins 24, 29, 215, 329 et seq, 349repair 28, 167secondary structure 135, 277, 301stabilization 298, 333storage 164, 165, 193synthesis 20, 150, 154, 161, 189, 213, 215,

272, 278, 327 et seq, 351Vp1 322

Proteomics 175, 372Proteosome 349Prothalli 192Proton exchanges 277Protonema(ata) 193, 194, 209, 216Protoplasts 271Psychrometer 53Pteridophytes 9 et seq, 217 et seq

Putrescine 333Pyrolline carboxylate 331Pyrolline carboxylate synthetase 331

Quantitative trait locus (QTL) 336Quasi elastic neutron scattering 75Quiescence 150, 245, 369Quinones 296

Rab2 protein 28RAB17 163Rachis 152Raffinose 27, 167, 168, 172, 300 et seq, 369Rain 13, 20Raman spectroscopy 74Random coil 135Recalcitrant seeds see SeedsRecovery 228, 328 et seqRegeneration niche 250Rehydration 17 et seq, 22 et seq, 28, 113, 153,

166, 169, 209 et seq, 227, 242, 269 etseq, 294, 321 et seq, 344 et seq, 368 etseq

damage 344 et seqRehydrins 29, 215, 329 et seq, 349Relative humidity 53 et seq, 188, 216, 266 et

seq, 320 et seqair 4, 5and drying 97 et seqequilibrium 51 et seq, 190, 225, 252, 265tolerance of 5

Relative water content (RWC) 49 et seq, 106,207 et seq, 225

Relaxation times 127Repair 5, 21, 28 et seq, 151, 173, 215, 229, 263

et seq, 294, 321, 327, 350 et seqReserve deposition 150, 166, 172Reserve mobilization 369Respiration 19, 52, 114, 150, 173, 209 et seq,

268, 279, 294, 298, 351Resurrection plants 48, 114, 162, 166, 170, 217,

225, 226, 244, 268, 281drying 101, 296 et seq, 320 et seq, 344 et

seq, 367 et seqRetrotransposons 335Ribonucleoprotein

messenger (mRNP) 29, 320 et seq, 330RNA (mRNA) 329, 330, 351Rock pools 226Root pressure 227 Roots 163, 264, 331Rosette plants, desiccation tolerant 10Rotifers 7, 207Rubbers 269, 280Rutin 297

Subject Index 409


Salinity stress (see salt stress) 25, 48Salinity tolerance 8, 334Salt solutions 53, 55, 97Salt stress 25, 333, 335Savannah 252Scales 19, 224Scutellum 367Seed ferns 250Seedling 254, 367Seeds 7 et seq, 112 et seq, 149 et seq, 239 et seq,

320, 344 et seq, 367 et seqageing 113banks 241, 253chromosomal aberrations 350coat (see testa) 22, 347 et seqcolour 348desiccation sensitive 239 et seqdesiccation tolerance 150 et seq, 239 et seqdevelopment 24, 31, 122, 123, 150 et seq,

334, 350dormant 150, 253, 330, 349dry weight 151drying 152 et seqexpansion 166filling 112free radical damage 117fresh weight 151gene expression 369, 370germinating 115, 132germination 152, 253imbibition 166, 344 et seq, 351imbibitional injury 136, 191, 194, 197, 344

et seqintermediate 150, 172, 241, 266longevity 27, 241 et seqmaturation 24, 115, 150 et seq, 253, 266,

294, 295, 307, 350, 369maturity 155, 254metabolites 115moisture content 158, 241 et seq, 250, 252non-endospermic 252oils 115 et seq, 135orthodox 9, 24, 52, 65, 95 et seq, 150 et seq,

172, 240 et seq, 266 et seq, 344 et seq,368 et seq

production 30recalcitrant 9, 50, 53, 66, 95 et seq, 105,

151 et seq, 172, 240 et seq, 266 et seq,294 et seq, 344 et seq, 367 et seq

reserves 157size 98, 244, 250shape 98, 250storage behaviour 155 et seq, 241, 250structure 243sugars 115tree 9volume 345

water in 48 et seq, 151 et seqwater loss 151viability 27, 28, 66, 102, 241, 278weight 249

Selection pressure 159, 253Self incompatibility 186Semi-arid grasslands 18Senescence 152Serine residues 25, 308, 323Serotonin 170Shade plants 17Shedding 155, 157, 172�-Sheet 135Signalling pathways 326, 334, 369 et seqSilica gel 95, 152, 225Silurian 243, 263Solutes 53

leakage 160, 223, 297Sorbitol 298Sorption 269Sorption properties 242Sorption sites 129, 167Spectroscopy 119 et seqSperm cells 186, 355Spin labels 120 et seqSpin probes 22, 120 et seqSpin trapping 126Spores 4, 7 et seq, 150, 186 et seq

dispersal 186fungal 115

Sporocarp 193Sporophytes 7 et seq, 253Stachyose 27, 167, 172, 300 et seq

synthase 369Starch extrusion 347Steroids 123Stigma 186Stomata 208, 217, 320Storage 155, 242, 297 et seqStress 264 et seq, 294

chilling 348drought 30, 298, 335duration 102, 103freezing 298imbibition 344 et seqintensity 102, 103mechanical 70, 346 et seqmultiple 321osmotic 229, 298, 333oxidative 30, 114, 116, 192, 296physico-chemical 70salt 25, 333, 335water 25, 154, 162, 167, 172, 226, 266 et

seqStress strain 69, 266, 347Stress tolerance 30Stroma 227

410 Subject Index


Succulents, desiccation tolerant 10Sucrose 298, 324 et seq, 368 et seq

alcohols 229distribution 131glass formation 26 et seq, 302 et seqand membranes 299in mosses 26 et seq, 328phosphate 322phosphate synthase 322in pollen 189protection 164, 167, 227, 328in seeds 26 et seqsynthase 322, 323

Sugars 20, 24, 26 et seq, 115, 118, 168 et seq,294, 335, 368 et seq

alcohols 229 hydrophilic 27hydroxyl groups 27interaction with protein 135, 301, 311and membranes 168, 276phosphates 322and phospholipids 281, 300 et seqprotective 151, 276, 277, 297

Sulphuric acid 225Superoxide 295Superoxide dismutase 174, 295, 334Syrups 269

Tannins 348Tardigrades 7, 207t-DNA 335Teliospores 196Telomeres, telomerase 357, 359Temperature

and desiccation tolerance 213, 224, 225,327

evaporation rate 62extremes 265and free energy difference 94and glasses 113and injury 344and longevity 241monolayer hydration 65and survival time 213transition 169, 195, 297, 300, 302 et seq,

346 et seqTEMPO 123TEMPONE 121 et seqTesta 252, 347 et seq

amphiphiles 349glycoproteins 348and imbibitional injury 348leakage 348lignin polymers 348phenolics 348pigmentation 348

water uptake 348waxy 348

Tetrazolium testfungi 104viability 104

Thiobarbituric acid 117, 118Thylakoids 23, 116, 227Tocopherol 167, 174, 280, 296Tonoplast 129, 132, 272Toxin 321Transcription 351

activator 163, 322, 335factors 165, 326, 334, 336regulators 322

Transduction see Signalling pathwaysTransgenic plants 112, 330 et seqTransgenic studies 25, 31, 298, 330 et seqTranslation 351Translation factor 322Transpiration 320Transposon tagging 31, 335Tree seeds 9Trehalase 168Trehalose 13, 26, 115, 133, 168, 195, 197, 298 et

seq, 310, 324 et seqstabilization by 333

Trehalose phosphate phosphatase 168, 331Trehalose phosphate synthetase 31Triacylglycerol 118Tropics 17, 252Tubers 228Tundra 17Turgor 49, 55, 106, 226, 267 et seq, 321, 371

pressure 55 et seq, 264 et seq

Ubiquitin 25, 167, 334, 349see also Polyubiquitin

UDP-glucose 322Ultrastructure 153Umbelliferose 169Urea 298Urediniospores 196Uridine 351UV radiation 119, 354 et seqUV-B tolerance 8

Vacuoles 21, 227and cell shrinkage 271

Van der Waals interactions 135, 273Van’t Hoff relationship 63 et seqVascular bundle 129, 323Vascular factors 153Vascular separation 171, 268Vascular system 227Vegetative tissues 207 et seq, 272, 320 et seq

Subject Index 411


Vesicles 168Vesicular trafficking 28Viability 161, 186 et seq, 241, 278, 303, 353Vicilin 166Viscosity 124, 280, 281, 301 et seq

cytoplasmic 78, 113, 159, 165 et seqVitrification see GlassVivipary 154, 158, 164, 241, 245, 253Volatiles 114, 173

Wateractivity 50 et seq, 65apoplastic 55 et seq, 68, 73, 106binding 25, 59 et seq, 65, 67bulk 73, 126 et seq, 130, 186, 190,298chemical potential 51 et seqclustering 65 et seqcompartments 130concentration 105conservation 4content 48 et seq, 68, 99, 105 et seq, 112 et

seq, 128 et seq, 151 et seq, 213, 266 etseq , 345

diffusion 94dissociation 63distribution 130dry weight basis 48 et seq, 105 et seqequilibrium water content 48exchange 73fractions 130freezable 73, 151, 160gradient 113, gravitational potential 51hydration levels 74immobilized 73intercellular 48, 57 et seqand life 4, 48 et seqloss 114, 208, 264 et seqmatric potential 53 et seq

matrix bound 160molecular interactions 76, 79monolayer 61, 65multimolecular clusters 62non-freezable 73, 151, 160osmotic potential 52 et seq, 68, 264osmotically inactive 73partial molar volume 51potential 49 et seq, 97, 157, 208 et seq, 241

et seq, 267 et seq, 344, 368 et seqrate of loss 28status 48 et seq, 79storage 20stress 25, 154, 162, 226, 266 et seqstrongly bound 128symplastic 55 et seq, 68, 73, 106transfer routes 131vapour 18vapour pressure 51 et seq, 94 et seqwet weight basis 48 et seq, 105 et seq

Water channel proteins 166, 323Water relations 55 et seqWater replacement hypothesis 12, 276, 280, 299

et seqWoody plants 250

Xanthophyll 18, 265Xeromorphs, desiccation tolerant 10, 224, 226X-ray diffraction 75Xylem cavitation 12, 20Xylem potential 53Xylem refilling 227

Zeaxanthin 18, 227Zeeman splitting 120, 127Zygospores 196Zygote 156

412 Subject Index


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Desiccation and survival in plants, drying without dying