Desiccation and Survival in Plants - Black (2002)

8/9/2019 Desiccation and Survival in Plants - Black (2002)

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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 College University of London

UK

and

H.W. Pritchard

Royal Botanic Gardens, Kew Wakehurst Place

UK

CABI Publishing


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CABI Publishing is a division of CAB International

CABI Publishing CABI PublishingCAB International 10 E 40th StreetWallingford Suite 3203Oxon OX10 8DE New York, NY 10016UK USA

Tel: +44 (0)1491 832111 Tel: +1 212 481 7018Fax: +44 (0)1491 833508 Fax: +1 212 686 7993Email: [email protected] Email: [email protected] site: www.cabi-publishing.org

© 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 2002

581.4--dc21

2001043835

ISBN 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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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 293 Julia Buitink, Folkert A. Hoekstra and Olivier Leprince

11 Molecular Genetics of Desiccation and Tolerant Systems 319 Jonathan 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

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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, 150

Stamford Street, London SE1 6NN, UK. [email protected] Boubriak, The Oxford Research Unit, Open University, Foxcombe Hall, Boars Hill

OX1 5HR, UK. [email protected] Buitink, UMR Physiologie Moléculaire des Semences, Institut National

d’Horticulture, 16 Bd Lavoisier, F49045 Angers, France. [email protected] B. Dickie, Seed Conservation Department, Royal Botanic Gardens Kew, Wakehurst

Place, Ardingly, West Sussex RH17 6TN, UK. [email protected] M. Farrant, Department of Molecular and Cellular Biology, University of Cape Town,

7700, South Africa. [email protected] E. Finch-Savage, Horticulture Research International, Wellesbourne, Warwick CV35

9EF, 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] Walters, USDA-ARS National Seed Storage Laboratory, 1111 South Mason

Street, Fort Collins, CO 80521, USA. [email protected] Wesley-Smith, School of Life and Environmental Sciences, University of Natal,

Durban 4041, South Africa. [email protected]

viii Contributors


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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 of dehydration 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 certain

topics 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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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 30

1.8. References 31

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


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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 about100 MPa (Gaff, 1997). This kills over 99%of flowering plants.

Terrestrial plants appear to haveevolved two solutions to the problem of maintaining an aqueous self in a witheringworld. The majority solution, at least at thepresent evolutionary time, is never to dryout – to maintain a chronic disequilibrium between 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 of these 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 of desiccation tolerance, partly in the hopes of some 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 of plants 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 of the apparently excellent ability to toleratedesiccation raises a second, cautionaryquestion about desiccation tolerance: How does 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 brief summary 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

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‘moderately dry’ means 50–70% relativehumidity at 20–30°C . This definition isworkable because there seems to be a widegap between the maximum tolerance of sensitive 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

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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 of desiccation tolerance must involve at leasttwo key elements (Section 1.6). First, theremust be an orderly shutdown of metabo-lism during desiccation. Different metabolic 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 of minimizing 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 involve both 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 of water. 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 be better 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. Droughtmay 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,

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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 of water 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 of desiccation 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 of drying 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 of desiccation, and for measuring rates andfinal levels of recovery (Chapter 3). Rate of drying 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 of membranes, 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 molecular biology 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 the question 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



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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 of Selaginella were also more heat-tolerant.The fungus Schizophyllum commune pro-duced hyphae after 34 years in a vacuum of less 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 relative biomass of desiccation-tolerant plants in all but 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 of plant 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 of desiccation 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 will be 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



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individuals; and there is a continuum of degree 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 may be 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 of drought and disperse the offspring of aplant more widely in space and time,although orthodoxy is not a prerequisite fordormancy (Chapter 5). Two advantages of being 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 to believe 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 may be more desiccation-tolerant whenyounger. Younger leaves are more tolerantthan older ones in Chamaegigas intrepidus (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.



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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 et al ., 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 of capacity 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 have been 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 of bryophytes probably tolerate at least brief desiccation of low intensity (Chapter 7);the proportion of desiccation-tolerantspecies appears to differ between orders of mosses 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 of angiosperms 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; Drazicet 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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Fig.1.1.

Sou

thernAfricaisacentreofdiversity

fordesiccation-tolerantangiosperm

s,including(a)Craterostigmawilm

sii,(b)Xerophytaviscosa,(c)Xerop

hyta

retinervis,an

d(d)Myrothamnusflabellifolius.Eachisshowninitsdesiccated(left)andhydrated(right)state.(Photosb

yJ.M.Farrant.)

( a

)

( b )

( c

)

( d )


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organisms depends on the selection andsynthesis of sufficient concentrations of molecular 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 still bare (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)


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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 of resources. 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)


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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)


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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) with

prominent 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)


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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 andgeographical 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 and bryophytes 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 et al ., 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-



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fore in the rhythms of desiccation andgrowth they experience in nature (Tuba et al ., 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 rates between 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 et al ., 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 of mosses 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 of activity 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 carbon balance. 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 of anthocyanin 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).



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Little is known about the interaction between 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 if the moss has dried slowly, but may take24 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 of N, P or K in two mosses compared touptake during continuous hydration, andBadacsonyi et al . (2000) saw no difference between 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 have been 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) could be 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-off between 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 of selection 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



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prolong hydration but inhibits photosyn-thesis, since much of the water is typi-cally held externally or in upper layers of the 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 of desiccation-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 et al ., 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 of surviving 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 generally believed that the mechanisms of desicca-tion tolerance in plants were mechanical



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(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 et al ., 1999).

Bewley (1979) further defined threecritical features of desiccation tolerance based 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 of extensive 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 of mechanisms 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 desiccationThe 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;



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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 preparation because 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 et al ., 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 tissuesare part of a developing system.Nevertheless, observations tend to sug-gest that desiccation of tolerant plantsgenerates an ordered ‘collapse’ of thecellular 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 of

dehydration 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 have been demonstrated in drying and rehydrat-ing desiccation-tolerant cells (Crowe et al .,1989; Hoekstra et al ., 1992), it has been

generally 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 membranes are in a liquid crystalline state

before liquid water surrounds the rehydrat-ing cells. Since leakage does occur duringthe rehydration of these tissues (Hoekstraet al ., 1992; Tetteroo et al ., 1996), it has been concluded that leakage must occurthrough an intact lipid bilayer, as suggested by Senaratna and McKersie (1983b).

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



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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 of amphiphilic 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 to be 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 damage by free radicals generated during desicca-tion and rehydration. If so, imbibitionalleakage may be a necessary trade-off forprotection. Much work will be required before 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 occupied by 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 of two 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.



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1.6.2. Protection

Much of what we know of the cellular pro-tection mechanisms involved in desiccationtolerance in plants comes from studies of orthodox 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 of the 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 metabolic 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 also become 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% of the 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 of desiccation 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 of the LEA proteins, e.g. dehydrins (see below), 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-



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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 motif near 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 of LEA 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 of seed maturation just prior to desiccation.In addition, ABA-deficient (aba) and ABA-insensitive (abi3) double-mutants of Arabidopsis 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 of LEA 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



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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 of their 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 et al ., 1996) prior to desiccation. Alamillo et al . (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 et al ., 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 of desiccation 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 Craterostigma plantagineum, 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



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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 a biological glass, of the cytoplasm of drycells (Leopold et al ., 1994; Chapter 10).This mechanism has been the subject of intense research over the last 15 years.

Vertucci and Leopold (1986) suggestedthat desiccation tolerance in seeds had to be 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 et al ., 1994; Leprince and Walters-Vertucci,1995), pollen (Buitink et al ., 1996) and inleaf tissues of C . plantagineum (Wolkers et al ., 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 also been 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 relationship between 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 correlation between 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.



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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,

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Desiccation and Survival in Plants - Black (2002)