You are on page 1of 422

Desiccation prels 19/3/02 1:42 pm Page i

Desiccation and Survival in Plants

Drying Without Dying

Desiccation prels 19/3/02 1:42 pm Page ii
Desiccation prels 19/3/02 1:42 pm Page iii

Desiccation and Survival in Plants

Drying Without Dying

Edited by

M. Black

King’s College
University of London


H.W. Pritchard

Royal Botanic Gardens, Kew

Wakehurst Place

CABI Publishing
Desiccation prels 4/4/02 2:16 pm Page iv

CABI Publishing is a division of CAB International

CABI Publishing CABI Publishing
CAB International 10 E 40th Street
Wallingford Suite 3203
Oxon OX10 8DE New York, NY 10016
Tel: +44 (0)1491 832111 Tel: +1 212 481 7018
Fax: +44 (0)1491 833508 Fax: +1 212 686 7993
Email: Email:
Web site:

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

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

Library of Congress Cataloging-in-Publication Data

Desiccation 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

ISBN 0 85199 534 9

Typeset in Melior by Columns Design Ltd, Reading

Printed and bound in the UK by Biddles Ltd, Guildford and King’s Lynn
Desiccation prels 19/3/02 1:42 pm Page v


Contributors vii
Preface ix
1 Drying Without Dying 3
Peter Alpert and Melvin J. Oliver
2 Methods for the Study of Water Relations Under Desiccation Stress 47
Wendell Q. Sun
3 Experimental Aspects of Drying and Recovery 93
Norman 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 111
Olivier Leprince and Elena A. Golovina
5 Desiccation Sensitivity in Orthodox and Recalcitrant Seeds in Relation to
Development 149
Allison R. Kermode and Bill E. Finch-Savage
6 Pollen and Spores: Desiccation Tolerance in Pollen and the Spores of Lower
Plants and Fungi 185
Folkert A. Hoekstra
7 Vegetative Tissues: Bryophytes, Vascular Resurrection Plants and Vegetative
Propagules 207
Michael C.F. Proctor and Valerie C. Pence
8 Systematic and Evolutionary Aspects of Desiccation Tolerance in Seeds 239
John B. Dickie and Hugh W. Pritchard

Desiccation prels 19/3/02 1:42 pm Page vi

vi Contents


9 Desiccation Stress and Damage 263
Christina 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 343
Daphne J. Osborne, Ivan Boubriak and Olivier Leprince
13 Damage and Tolerance in Retrospect and Prospect 367
Michael Black, Ralph L. Obendorf and Hugh W. Pritchard
Glossary 373
Taxonomic Index 383
Subject Index 401
Desiccation prels 19/3/02 1:42 pm Page vii


Peter Alpert, Biology Department, University of Massachusetts, Amherst, Massachusetts

01003-5810, USA.
Dorothea Bartels, Institute of Botany, University of Bonn, Kirschallee 1, D-53115 Bonn,
Patricia Berjak, School of Life and Environmental Sciences, University of Natal, Durban
4041, South Africa.
Michael Black, Division of Life Sciences, King’s College, Franklin Wilkins Building, 150
Stamford Street, London SE1 6NN, UK.
Ivan Boubriak, The Oxford Research Unit, Open University, Foxcombe Hall, Boars Hill
OX1 5HR, UK.
Julia Buitink, UMR Physiologie Moléculaire des Semences, Institut National
d’Horticulture, 16 Bd Lavoisier, F49045 Angers, France.
John B. Dickie, Seed Conservation Department, Royal Botanic Gardens Kew, Wakehurst
Place, Ardingly, West Sussex RH17 6TN, UK.
Jill M. Farrant, Department of Molecular and Cellular Biology, University of Cape Town,
7700, South Africa.
Bill E. Finch-Savage, Horticulture Research International, Wellesbourne, Warwick CV35
9EF, UK.
Elena A. Golovina, Laboratory of Plant Physiology, Department of Plant Sciences,
University of Wageningen, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands
and Timiryazev Institute of Plant Physiology, Botanicheskaya 35, Moscow 127276,
Folkert A. Hoekstra, Laboratory of Plant Physiology, Department of Plant Sciences,
University of Wageningen, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands.
Allison R. Kermode, Department of Biological Sciences, Simon Fraser University,
Burnaby, BC, V5A 1S6, Canada.
Olivier Leprince, UMR Physiologie Moléculaire des Semences, Institut National
d’Horticulture, 16 Bd Lavoisier, F49045 Angers, France.
Ralph L. Obendorf, Seed Biology, Department of Crop and Soil Sciences, Cornell
University, Ithaca, New York, USA.
Melvin J. Oliver, USDA-ARS Plant Stress and Germplasm Development Unit, 3810 4th
Street, Lubbock, Texas 79415, USA.

Desiccation prels 19/3/02 1:42 pm Page viii

viii Contributors

Daphne J. Osborne, The Oxford Research Unit, Open University, Foxcombe Hall, Boars
Hill OX1 5HR, UK.
Norman W. Pammenter, School of Life and Environmental Sciences, University of Natal,
Durban 4041, South Africa.
Valerie C. Pence, CREW, Cincinnati Zoo and Botanical Garden, 3400 Vine Street,
Cincinnati, OH 45220, USA.
Jonathan R. Phillips, Max-Planck-Institute for Plant Breeding Research, Carl-von-Linné-
Weg 10, D-550829 Köln, Germany.
Hugh W. Pritchard, Seed Conservation Department, Royal Botanic Gardens Kew,
Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK.
Michael C.F. Proctor, School of Biological Sciences, University of Exeter, Washington
Singer Laboratories, Perry Road, Exeter EX4 4QG, UK.
Wendell Q. Sun, Department of Biological Sciences, National University of Singapore,
Kent Ridge Crescent, Singapore 119260.
Clare Vander Willigen, Department of Botany, University of Capetown, Private Bag,
Rondebosch 7701, South Africa.
Christina Walters, USDA-ARS National Seed Storage Laboratory, 1111 South Mason
Street, Fort Collins, CO 80521, USA.
James Wesley-Smith, School of Life and Environmental Sciences, University of Natal,
Durban 4041, South Africa.
Desiccation prels 19/3/02 1:42 pm Page ix


Plant survival of desiccation as sporophytic and gametophytic tissues was last

reviewed in detail in two books published in 1980. The first, by J. Levitt, on
Responses of Plants to Environmental Stresses, Volume II, Water, Radiation, Salt
and Other Stresses is a classic. The topic of plant water stress consumes about
200 pages and is set mainly at the introductory level but is still of sufficient
detail to stimulate post-graduate researchers. Interestingly, there is no mention
in Levitt’s book of desiccation sensitivity in seeds. However, this latter topic was
specifically covered in another book by H.F. Chin and E.H. Roberts (Recalcitrant
Seeds). At that time recalcitrant seed behaviour was something of a novelty and
the book deals mainly with descriptions of germination, a listing of species with
such 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 dealing
with the biology and biophysics of dehydration and in contributions to general
works on seeds but a comprehensive treatment of desiccation and plant survival
is not yet available.
Since 1980 there has been a revolution in plant science as new methods of
cell and molecular biology and biophysics have been applied to environmental
stress, particularly in relation to desiccation tolerance. The basic level of under-
standing of how plant cells cope with extreme water stress has increased
tremendously and considerable effort has been made in the last 10 years to
develop diagnostic markers for desiccation tolerance. At the physiological level,
studies have often focused on seed material and on the responses of resurrection
plants. At a more mechanistic level, model membrane systems have been used
extensively, and exploration of the molecular genetics of desiccation tolerance
has begun on developmental mutants, especially of seeds of crop species.
These progressive but fundamental changes in approach to investigating the
basis of survival of plant tissues under desiccation since the 1980s have meant
that our perceptions of this subject have altered significantly. It seems particular
appropriate now to take stock of these recent developments, to assess critically
the importance of the experimental systems available for investigation and to

Desiccation prels 19/3/02 1:42 pm Page x

x Preface

consider possible foci for future research work. This book sets out to address
these 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 damage
and tolerance; and (iv) a brief retrospect and prospect. It will not attempt to
address 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 in
Environmental 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 and
editors have attempted, as far as is possible, to avoid repetition of detail.
Extensive cross-referencing has been used, to aid the reader in identifying
where, within the special viewpoints of the treatments, similar subjects are
This comprehensive presentation on desiccation and survival in plants
will be of value to all researchers in the field, both beginners and the more
experienced, and to those with interests in basic and applied plant sciences –
physiology, ecology, conservation biology, agriculture and horticulture.

M. Black
H.W. Pritchard
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 1

Part I

01 Desiccation -Chap 1 18/3/02 1:53 pm Page 2
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 3

1 Drying Without Dying

Peter Alpert1 and Melvin J. Oliver2

1BiologyDepartment, 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 4
1.2. Defining and Measuring Desiccation Tolerance 4
1.2.1. Operational and conceptual definitions 4
1.2.2. Measuring tolerance 6
1.3. A Brief History of Research on Desiccation Tolerance 6
1.3.1. Early work (1702–1860) on the question of whether life can
stand still 6
1.3.2. The next step: establishing records 7
1.4. The Occurrence of Desiccation Tolerance in Plants: Rarity and Ubiquity 8
1.4.1. Seeds, pollen and spores 8
1.4.2. Vegetative tissues 9
1.5. The Ecology of Desiccation Tolerance in Plants: a Diversity of Cycles in
Marginal Habitats 13
1.5.1. Habitats 17
1.5.2. Cycles 17
1.5.3. Hypotheses 19
1.6. Mechanisms of Desiccation Tolerance 20
1.6.1. Damage 21 Damage during desiccation 21 Damage during rehydration 22 Poikilochlorophylly 23
1.6.2. Protection 24 Proteins 24 Sugars 26
1.6.3. Repair 28
1.7. 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
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 4

4 P. Alpert and M.J. Oliver

1.1. Introduction Though some desiccation-tolerant plants

can survive droughts more intense and pro-
Water is a universal requirement for life as longed than any that occur almost any-
we know it. Water is the most abundant where on earth, tolerant plants are in the
compound in all active cells, it is essential minority. Desiccation-sensitive plants dom-
for metabolism and all organisms must take inate the world’s vegetation. The rarity of
in water to survive. Living things therefore the apparently excellent ability to tolerate
face a major problem whenever they desiccation raises a second, cautionary
emerge above ground on land: the air is question about desiccation tolerance: How
almost always drier than they are and takes does surviving desiccation affect plant sur-
water from them. This is a life and death vival? These two questions, one largely
problem for most organisms in most habi- genetic and biochemical and the other
tats, because the air is at least sometimes mainly physiological and ecological, frame
deadly dry. For example, when the relative the topic of desiccation and plant survival.
humidity is about 50% and the tempera- The purpose of this introductory chap-
ture 28°C, a plant cell that dries to equilib- ter is to summarize some of the current
rium will drop to a water potential of about answers to these questions and lead into
100 MPa (Gaff, 1997). This kills over 99% the more detailed reviews of questions and
of flowering plants. answers about desiccation and plant sur-
Terrestrial plants appear to have vival in the chapters that follow. We begin
evolved two solutions to the problem of with some terms and techniques that pro-
maintaining an aqueous self in a withering vide concepts and methods for research on
world. The majority solution, at least at the desiccation tolerance in plants, and a brief
present evolutionary time, is never to dry summary of the surprisingly lively history
out – to maintain a chronic disequilibrium of research on desiccation tolerance. We
between wet cells and dry air. Some of the then give an overview of the range and
most universal features of plant form, such ecology of desiccation tolerance in plants,
as waxy coatings on shoots, and pores that subjects that bear on how surviving desic-
can open and close on leaves, seem largely cation affects plant survival. Last, we dis-
designed to conserve water. cuss mechanisms of desiccation tolerance
The minority solution is to dry up but in plants, the keys to understanding how
not die – to desiccate during drought and plants survive desiccation, and consider
rehydrate and resume growth when the potential for breeding crops that can
drought ends. About 300 species of flower- dry without dying. We will sometimes
ing plants, or perhaps 0.1% of those named, abbreviate desiccation tolerance to ‘toler-
are known to tolerate desiccation ance’, and we will call plants that cannot
(Porembski and Barthlott, 2000). Some of tolerate desiccation ‘desiccation-sensitive’
these species can lose all of the free water or ‘sensitive’. We will consider desiccation
in their cells or remain dry for up to 5 years tolerance in plants and in some organisms
and still recover (Gaff, 1977). These prodi- that are not in the plant kingdom, mainly
gious abilities raise the first and fundamen- cyanobacteria, algae and fungi.
tal question about desiccation tolerance:
How do plants survive desiccation? Most
recent research on desiccation tolerance has 1.2. Defining and Measuring
focused on discovering the mechanisms of Desiccation Tolerance
desiccation tolerance, partly in the hopes of
some day engineering tolerance in econom- 1.2.1. Operational and conceptual definitions
ically important species and banishing the
spectre of famine from drought. Desiccation tolerance can be operationally
However, the ability to survive desicca- defined as the ability to dry to equilibrium
tion may not always increase the ability of with moderately dry air and then resume
plants to survive in natural systems. normal function when rehydrated, where
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 5

Drying Without Dying 5

‘moderately dry’ means 50–70% relative 2000). Some tolerant species are more dam-
humidity at 20–30°C . This definition is aged by being held at intermediate water
workable because there seems to be a wide contents than at full hydration or complete
gap between the maximum tolerance of desiccation (Gaff, 1997), and one advantage
sensitive plants and the minimum toler- of rapid desiccation may be to minimize
ance of tolerant ones (Gaff, 1997). Almost time spent at intermediate levels of hydra-
all species known to recover from complete tion (Kappen and Valladares, 1999; Proctor,
drying at 80% relative humidity also 2000; Chapters 3 and 5). Second, cells must
recover from drying at 50% (but see preserve enough cellular organization and
Bochicchio et al., 1998). functional enzymes so that metabolism can
The reason why a gap exists between the resume after rewetting. Preserving a skele-
ranges of tolerance of drying in desiccation- tal machinery for metabolism must involve
sensitive and desiccation-tolerant plants both protection and repair (Section 1.6).
may be that desiccation tolerance depends Enzymes and membranes must be pro-
on the ability to reversibly cease metab- tected from loss of configuration and orga-
olism as cells dry out. There may not be nization, and the damage that accumulates
very many marginally desiccation-tolerant from degradative non-metabolic reactions
plants because, once metabolism has while plants are inactive must be repaired.
stopped, it cannot be stopped further. Clegg Differences in effectiveness of protec-
(1973) argued on biochemical grounds that tion may explain much of why desiccation-
metabolism, defined as ‘systematically con- tolerant plants do differ in the intensity
trolled pathways of enzymatic reactions’ (minimum water content or water poten-
(Clegg, 2001), cannot take place at a cell tial) of desiccation that they can stand. For
water content of less than 0.1 g H2O g1 instance, tolerant angiosperms tend to sur-
dry mass because not enough water vive equilibration with lower relative
remains to hydrate intracellular proteins. humidities than do tolerant pteridophytes
Organisms this dry do show chemical in South Africa (Gaff, 1977). Species with a
activity. For instance, dried pollen can greater degree of protection of molecular
incorporate water vapour into organic com- configuration and cellular organization
pounds (Wilson et al., 1979). However, may survive with smaller fractions of
chemical reactions, even some characteris- water. Differences in effectiveness of repair
tic of living things such as oxygen uptake, may help explain why species also differ in
do not necessarily require metabolism: iron the duration of desiccation (length of time
rusts (Clegg, 1986). We propose that desic- in the dried state) that they can stand (e.g.
cation tolerance can be conceptually Sagot and Rochefort, 1996). Those with
defined as reversible cessation of metab- more effective repair mechanisms may be
olism in response to water loss. better able to undo non-metabolic degrada-
This suggests that the mechanisms of tion suffered while dry.
desiccation tolerance must involve at least There has been some confusion about
two key elements (Section 1.6). First, there the difference between ‘desiccation toler-
must be an orderly shutdown of metabo- ance’ and ‘drought tolerance’. We would
lism during desiccation. Different meta- like to propose that desiccation tolerance is
bolic pathways must slow at compatible one form of drought tolerance. Drought
rates to avoid fatal accumulations of inter- may be defined as any level of water avail-
mediates and generation of free radicals. ability that is low enough to reduce plant
Oxidation is a major hazard of desiccation performance. ‘Drought tolerance’ is most
(e.g. Smirnoff, 1993), and the advantages of often used to refer to tolerance of water
minimizing photo-oxidation may explain availabilities that are suboptimal but not
why some desiccation-tolerant plants cease low enough to cause complete drying to
photosynthesis at relatively high water equilibrium with the air, i.e. desiccation.
contents during drying (e.g. Sherwin and Mechanisms of drought tolerance include
Farrant, 1998; Tuba et al., 1998; Farrant, ways of maintaining cell water content,
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 6

6 P. Alpert and M.J. Oliver

such as osmotic regulation and stomatal undergo cycles of drying and wetting.
closure, whereas desiccation tolerance con- Chapter 4 reviews the rapidly expanding
sists of ways of surviving the nearly com- range of non-invasive techniques available
plete loss of water. Some papers on to study the diffusion of water, the configu-
intertidal algae maintain the confusion by rations and interactions of macromole-
using ‘desiccation’ to refer to any amount of cules, metabolism, thermal events
water loss (e.g. Leuschner et al., 1998; Bjork associated with membrane phase transi-
et al., 1999). They are probably wrong, tions, ultrastructure, oxidative stress, fer-
since the Oxford English Dictionary (1989) mentation and the physical properties of
defines ‘to desiccate’ as ‘to make quite dry; membranes, cytoplasm and protein com-
to deprive thoroughly of moisture’. plexes during desiccation and rehydration.
Chapter 7 summarizes some of the techni-
cal developments in infrared gas analysis
1.2.2. Measuring tolerance and fluoroscopy that have improved our
capacity to quantify responses to desicca-
Techniques for quantifying the degree of tion on the physiological level.
desiccation tolerance in different species
are reviewed in Chapters 2–4 and 7.
Chapter 2 discusses the advantages and 1.3. A Brief History of Research on
limitations of different measures of water Desiccation Tolerance
content and techniques for distinguishing
water properties in plant cells. Chapter 3 The 300-year history of the science of desic-
notes how the survival and recovery rates cation tolerance began with a lengthy
of seeds and vegetative tissues vary with period of discovery and doubt. In the
rate of drying, light conditions during dry- course of discovering desiccation tolerance,
ing, storage conditions and length of time scientists confronted the nature of life. The
in the dehydrated state. In general, highly next step was to enumerate the organisms
desiccation-tolerant bryophytes can sur- that tolerate desiccation and test the limits
vive rapid drying but tolerant angiosperms of their tolerance. In the 1960s, researchers
cannot; this seems to be related to differ- started to investigate the physiological ecol-
ences in their mechanisms of tolerance ogy of desiccation tolerance in plants, espe-
(Section 1.6). A few species appear insensi- cially the cycles of wetting and drying and
tive to rate of drying, but most probably their effects on carbon uptake in bryophytes
have an optimal rate or optimal range of and lichens. Since the 1980s, emphasis has
drying rates. For instance, desiccation in shifted to the biochemistry and molecular
less than 6 hours or over more than 7 days biology of desiccation tolerance. We now
kills the otherwise highly tolerant pterido- know more about how plants survive desic-
phyte Selaginella lepidophylla (Eickmeier, cation than about how tolerating desicca-
1983). Rates and final levels of recovery tion affects plant survival.
can decrease with increasing intensity or
duration of desiccation (e.g. Gaff, 1977;
Alpert and Oechel, 1987; Davey, 1997). 1.3.1. Early work (1702–1860) and the
Quantifying desiccation tolerance therefore question of whether life can stand still
also requires techniques for imposing
known rates, intensities and durations of It took scientists one and a half centuries to
desiccation, and for measuring rates and establish that desiccation tolerance exists
final levels of recovery (Chapter 3). Rate of (Keilin, 1959). At the end of an often ran-
drying is particularly hard to standardize corous debate, the nature of life had been
across species. called into question: Can life stop, be con-
Investigating the mechanisms of desic- tained in a static array of molecules and
cation requires techniques for measuring restart? Anthony von Leeuwenhoek was
processes and states in cells as they apparently the first to glimpse desiccation
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 7

Drying Without Dying 7

tolerance, soon after he invented the micro- can be cooled to within 0.5°C of absolute
scope. In 1702, he wrote to a friend (see zero, absolute zero being the temperature at
Schierbeek, 1959): which all molecular motion is believed to
stop, and then revive when rewarmed and
The following day the sky was very hot and
rehydrated (Becquerel, 1951). The discov-
dry and, about nine in the morning, I took
some of the sediment which has been in the
ery of desiccation tolerance has shown us
leaden gutter … and poured on it a small that living things can come to exist in three
quantity of rain-water taken out of my stone states: alive, dead and still (Clegg, 2001).
cistern … so that if there were still any living
animalcules in it they might issue forth;
though I confess I never thought that there 1.3.2. The next step: establishing records
could be any living creatures in a substance
so dried as this was. The scientific battle over whether living
I was, however, mistaken; for scarce an things could dry without dying was fought
hour had elapsed, when I saw at least a
over animals. Starting in the 1960s, toler-
hundred of the animalcules before described.
ance was identified in the larvae of at least
These animals were rotifers. By the mid- one insect and of some other arthropods
1800s, others had seen desiccation toler- (Hinton, 1968; Crowe et al., 1992) but has
ance in two more phyla of animals, never been found in any life stage of any
nematodes and tardigrades (Keilin, 1959; vertebrate or in the adults of any animals
Alpert, 2000). However, others still flatly except microscopic rotifers, nematodes and
denied it was possible to survive drying tardigrades. In contrast, tolerance was
out. A French biologist, Felix Pouchet found to be widespread in plants. Tolerant
(1859), wrote that: ‘Dry and completely bryophytes were reported by 1886, fern
mummified animals cannot be resuscitated gametophytes by 1914, fern sporophytes by
by hydration. Rational beliefs, observation, 1931 and angiosperms by 1921 (tables and
and experiment unite to demonstrate it.’ citations in Kappen and Valladares, 1999;
The Société de Biologie in Paris convened Alpert, 2000; Chapter 7).
a special commission and conducted its The intensity and duration of desicca-
own tests on rotifers. Its report effectively tion that plants and plant-like organisms
settled the matter: ‘[organisms] reaching could survive were shown to be remark-
the most complete degree of desiccation able. Like tardigrades (Doyère, 1842), cer-
that can be realized … may yet retain the tain lichens, bryophytes, pteridophytes and
ability to revive in water’ (Broca, 1860). angiosperms survived equilibration with
However, the commission was silent on air of nearly 0% relative humidity, in
the deeper question of whether this means closed volumes over concentrated H2SO4
that life can stop and restart. As phrased by or P2O5 (e.g. Lange, 1953; Hosokawa and
Pouchet’s main scientific opponent Kubota, 1957; Gaff, 1977). The liverwort
(Doyère, 1842), ‘Has there been a mere Riccia macrocarpa produced new apical
slowing down of the vital phenomena, … cells after 23 years of air-dryness (Breuil-
or truly an absolute destruction that one Sée, 1993); the moss Grimmia laevigata
could compare to death itself?’ grew when rehydrated after 10 years in a
The modern consensus on this question herbarium (Keever, 1957); lichens survived
seems to be that some organisms can slow 10 years of being desiccated and frozen
their metabolism at least to the point at (Larson, 1988); and leaves of ferns and
which it cannot be detected against the flowering plants took up neutral red dye or
background of physical chemical reactions excluded Evans blue dye after 5 years of
and then resume normal metabolism air-dryness (Gaff, 1977). Some desiccation-
(Keilin, 1959; Hinton, 1968; Clegg, 2001). tolerant plants clearly survive longer and
The most convincing evidence may be that more intense drought than ever occurs
some desiccated tardigrades, rotifers, where they grow, raising the question of
seeds, spores, algae, lichens and mosses what has selected for such tolerance.
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 8

8 P. Alpert and M.J. Oliver

Dessicated plants were also shown to Porembski and co-workers (e.g. Porembski
tolerate other extreme stresses. Various taxa and Barthlott, 2000) are probably the clos-
survived extreme cold (Becquerel, 1951; est approaches to surveys for whole plants.
Pence, 2000), and some mosses survived The overall pattern one sees is taxonomic
heating to 100°C (Glime and Carr, 1974; and geographic breadth contrasted with
Norr, 1974). Eickmeier (1986) found that ecological narrowness.
more desiccation-tolerant populations of
Selaginella were also more heat-tolerant.
The fungus Schizophyllum commune pro- 1.4. The Occurrence of Desiccation
duced hyphae after 34 years in a vacuum of Tolerance in Plants: Rarity and Ubiquity
less than 0.01 mm Hg (Bisby, 1945), show-
ing long-term tolerance of both desiccation Part of the puzzle of desiccation tolerance in
and lack of oxygen. Takács et al. (1999) cor- plants is that it is both very uncommon and
related desiccation and UV-B tolerance in a nearly universal (Alpert, 2000). The relative
set of bryophyte species. biomass of desiccation-tolerant plants in all
The correlation between tolerance of des- but the most arid or frigid habitats is very
iccation and tolerance of cold, heat and low, and fewer than one in a thousand
anoxia has suggested that there may be species of flowering plants is known to toler-
some basic properties or mechanisms that ate desiccation. At the same time, desicca-
confer ‘broad-spectrum’ tolerance. Since tion-tolerant species are found on all
freezing often dehydrates cells, cold and continents, in all major plant groups except
desiccation stress have an obvious func- gymnosperms, and among species of all
tional link. Another parallel between desic- growth forms except trees; and the great
cation and cold tolerance is that both can be majority of flowering plants and also gym-
‘softened’ by periods of low stress and nosperms have desiccation-tolerant seeds or
‘hardened’ by ones of moderate stress. pollen or both. Desiccation tolerance appears
Plants may lose some of their desiccation to be a universal evolutionary potential of
tolerance after prolonged periods of full plant cells that has been little selected for
hydration (e.g. Gaff, 1977; Schonbeck and except in resting stages of the life cycle and
Bewley, 1981; Kappen and Valladares, in organisms that have not evolved effective
1999). Desiccation tolerance can vary sea- ways of avoiding desiccation.
sonally (Dilks and Proctor, 1976; Gaff, 1980) Detailed reviews of the occurrence of
and increase in winter (Kappen, 1964). desiccation tolerance in seeds, pollen and
However, the correlation between tolerance other spores, and vegetative tissues are
of desiccation and other stresses is not given in Chapters 5, 6, 7 and 8. Other recent
absolute. Wood and Gaff (1989) saw no cor- reviews of the occurrence of tolerance in
relation between desiccation and salinity adult plants and non-plant non-animals
tolerance in species of the grass Sporobolus. include those of Kappen and Valladares
As records of desiccation tolerance (1999), Alpert (2000), and Porembski and
accumulated, pictures emerged of the taxo- Barthlott (2000). In this section, some of the
nomic and geographic ranges of desicca- main points in these reviews will be dis-
tion tolerance in plants. These pictures cussed, a few of the examples they give will
remain somewhat haphazard because there be mentioned and some additional exam-
have been few systematic surveys for desic- ples and points will be presented.
cation tolerance within taxa or habitats.
Relatively extensive lists exist for seeds
(Chapter 8). A survey of all the soil algae at 1.4.1. Seeds, pollen and spores
one site was published by Evans (1959).
The lists of tolerant vascular plants from As in adult plants, the desiccation toler-
different regions published by Gaff and co- ance of seeds can vary greatly between
workers (e.g. Gaff, 1977, 1986; Gaff and species within genera, between individuals
Latz, 1978) and from rock outcrops by within species and between tissues within
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 9

Drying Without Dying 9

individuals; and there is a continuum of 1.4.2. Vegetative tissues

degree of tolerance across species
(Chapters 5 and 8). However, whereas des- Desiccation tolerance appears common
iccation tolerance is rare in adult flowering though not universal in bryophytes (e.g.
plants, it is so much the rule in their seeds Richardson, 1981; Proctor, 1990), common
that tolerant seeds are traditionally known in lichens (Kappen and Valladares, 1999),
as ‘orthodox’ and desiccation-sensitive uncommon in pteridophytes and rare in
seeds as ‘recalcitrant’. Desiccation sensitiv- angiosperms (Chapter 7). No gymnosperms
ity may be a derived character in seeds, are known to tolerate desiccation (Gaff,
evolved through neoteny, and is probably 1980; Chapter 7), even though gym-
associated with large seeds and trees nosperms may have desiccation-tolerant
(Chapter 8). Another difference between seeds or pollen (Chapters 5 and 6).
desiccation tolerance in adult plants and Desiccation tolerance occurs in non-lich-
seeds is that tolerance and desiccation are enized fungi, cyanobacteria and algae (Ried,
environmentally induced in adults but may 1960; Mazur, 1968; Bertsch, 1970;
be developmentally programmed in seeds. Schonbeck and Norton, 1978; Potts, 1994,
Seeds become tolerant as part of develop- 1999; Dodds et al., 1995) but little is known
ment and dry because the parent withholds about its extent. It must be very common in
or withdraws water from them. Once they free-living algae and bacteria that grow on
germinate, the seedlings of desiccation-sen- the surface of plants or soil, where they are
sitive species with desiccation-tolerant very probably subject to desiccation.
seeds lose their tolerance within hours. Different vegetative parts of a plant
The obvious ecological advantage of ortho- may have different degrees of tolerance.
doxy is that seeds can survive periods of There seem to be two main patterns. First,
drought and disperse the offspring of a in some species only the perennating
plant more widely in space and time, structures survive desiccation, such as
although orthodoxy is not a prerequisite for corms in Limosella grandiflora (Gaff and
dormancy (Chapter 5). Two advantages of Giess, 1986) or special dry-season organs
being recalcitrant are that seeds need never in the small shrub Satureja gilliesii
stop growing and may germinate more (Montenegro et al., 1979). As in plants
rapidly – as in whole plants, there may be that are desiccation-sensitive but have
a trade-off between desiccation tolerance desiccation-tolerant seeds, tolerance in
and productivity in seeds. these species is confined to relatively
Desiccation tolerance is probably also inactive plant parts. Second, leaves may
the rule rather than the exception in pollen be more desiccation-tolerant when
and spores, and tolerance and desiccation younger. Younger leaves are more tolerant
are developmentally programmed in spores than older ones in Chamaegigas intre-
as in seeds (Chapter 6). However, there are pidus (Gaff and Giess, 1986) and some
at least three differences between tolerance species of Borya (Gaff, 1989). In the leaves
in seeds and in spores. Tolerant pollen has of some grasses, only the basal meristem-
no dormancy, it survives no more than a atic zone tolerates drying (Gaff and
few months of dry storage at room tempera- Sutaryono, 1991). This suggests that some
ture, and spores of some pteridophytes can tissues may lose tolerance as they differ-
survive cycles of drying and wetting. entiate or age; the processes involved
Desiccation-sensitive pollen is relatively could conceivably parallel those that
common in species of Poaceae, cause loss of tolerance after germination
Cucurbitaceae and Araceae (Chapter 6), of seeds. In all these examples of differen-
and may be associated with hot, humid tial tolerance in leaves, there are con-
habitats. The prevalence of desiccation tol- geners whose leaves remain tolerant as
erance in seeds and spores is one reason to they mature, offering inviting systems for
believe that the genetic potential to tolerate comparative studies of the ecology and
desiccation exists in all plants. mechanisms of desiccation tolerance.
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 10

10 P. Alpert and M.J. Oliver

No one appears to have assessed the rel- and Valladares, 1999; Porembski and
ative prevalence of desiccation tolerance in Barthlott, 2000). Desiccation-tolerant mono-
different taxa of bacteria, cyanobacteria, cotyledons outnumber tolerant dicotyle-
fungi and algae. Acinetobacter radioresis- dons. The monocotyledonous family
tans survives 150 days at 31% relative Velloziaceae may have over 200 tolerant
humidity, which helps make it a persistent species (Kubitzki, 1998). At least 39 species
source of infection in hospitals (Jawad et of Poaceae tolerate desiccation (Gaff, 1997).
al., 1998). At least 400 species of algae and One very small family of angiosperms, the
cyanobacteria tolerate desiccation (e.g. Myrothamnaceae, is entirely desiccation-
Davis, 1972; Potts, 1994, 1999; Trainor and tolerant (Porembski and Barthlott, 2000). At
Gladych, 1995). Evans (1959) found that the other extreme, some species, such as
many but not all of the freshwater algae in Borya nitida (Liliaceae), contain both toler-
pond mud survived desiccation in the ant and sensitive individuals (Gaff, 1981).
field; at least two species survived 69 days Phylogenetic analysis suggests that desicca-
of desiccation in the laboratory without tion tolerance in active phases of the life
forming resting stages. Two interesting phe- cycle has evolved at least eight separate
nomena that have been reported from some times in vascular plants (Oliver et al., 2000).
green algae but apparently not from other Desiccation-tolerant angiosperms are
groups are dependence of tolerance on also widely but unevenly geographically
nutrient availability (McLean, 1967, cited distributed. They occur on all continents
in Chandler and Bartels, 1999) and loss of except Antarctica, but very few species are
capacity to reproduce after desiccation known from Europe or North America. The
(Hsu and Hsu, 1998). We know of few European species are all in two genera from
reports of desiccation tolerance in non- one family (Ramondia and Haberlea in the
lichenized fungi (Bisby, 1945; Gesneriaceae) (Muller et al., 1997; Drazic
Zimmermann and Butin, 1973), but there is et al., 1999). The North American species
an extensive literature on tolerance in include three grasses (Iturriaga et al., 2000).
lichens, at least 50 species of which have The greatest concentrations of known desic-
been shown to tolerate desiccation cation-tolerant angiosperms are in southern
(Kappen and Valledares, 1999). Africa, western Australia and eastern South
Desiccation tolerance is broadly but America (Figs 1.1 and 1.2; Gaff, 1977, 1987;
unevenly distributed among taxa in plants. Gaff and Latz, 1978; Porembski and
Most of the 25,000–30,000 species of Barthlott, 2000). Different taxa predominate
bryophytes probably tolerate at least brief in each of these three areas.
desiccation of low intensity (Chapter 7); Desiccation-tolerant plants have a wide
the proportion of desiccation-tolerant range of morphological and physiological
species appears to differ between orders of characteristics (Porembski and Barthlott,
mosses and to be higher in mosses than in 2000). There are desiccation-tolerant annuals
liverworts. There are also desiccation-toler- and perennials, graminoids and forbs, and
ant hornworts (Oliver et al., 2000). herbs, shrubs and arborescent rosette plants.
Porembski and Barthlott (2000) estimated Tolerant species may be caespitose, stolonif-
that there are 275–325 desiccation-tolerant erous or rhizomatous. Some species are xero-
species of vascular plants. At least nine fam- morphic, such as B. nitida (Gaff and
ilies of pteridophytes and seven families of Churchill, 1976); others are not, such as Boea
angiosperms contain desiccation-tolerant hygroscopica (Gaff, 1981). A few desiccation-
sporophytes (Chapter 7). Some fern gameto- tolerant species, like C. intrepidus, have mor-
phytes also tolerate desiccation (e.g. Pence, phological features typical of aquatic plants
2000). Groups of ferns and allies that seem (Gaff and Giess, 1986), and at least one
to be relatively rich in desiccation-tolerant species is succulent (Barthlott and
species include the family Pteridaceae and Porembski, 1996). Desiccation-tolerant
the genera Cheilanthes and Selaginella angiosperms can have crassulacean acid
(Gaff, 1977; Gaff and Latz, 1978; Kappen metabolism (Barthlott and Porembski, 1996;

01 Desiccation -Chap 1
1:53 pm

(c) (d)
Page 11

Drying Without Dying

Fig. 1.1. Southern Africa is a centre of diversity for desiccation-tolerant angiosperms, including (a) Craterostigma wilmsii, (b) Xerophyta viscosa, (c) Xerophyta

retinervis, and (d) Myrothamnus flabellifolius. Each is shown in its desiccated (left) and hydrated (right) state. (Photos by J. M. Farrant.)
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 12

12 P. Alpert and M.J. Oliver


(b) (c)

(d) (e)

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 of
the 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, shown
desiccated), a dominant, mat-forming species on inselbergs in West Africa. (Photos by S. Porembski.)

Markovska et al., 1997) and probably C4 pho- The wide distribution of desiccation tol-
tosynthesis (Lazarides, 1992). However, no erance in plants has suggested to some
plants more than 3 m tall and hence no trees authors that the basic mechanism of toler-
are known to tolerate desiccation, possibly ance must be simple (Chandler and Bartels,
because they cannot re-establish upward 1999). According to the ‘water replacement
movement of water once the xylem cavitates hypothesis’ of Crowe et al. (1998a), the
during desiccation (e.g. Sherwin et al., 1998). evolution of desiccation tolerance in all
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 13

Drying Without Dying 13

organisms depends on the selection and habitats where desiccation-sensitive plants

synthesis of sufficient concentrations of do not live (Fig. 1.3). In habitats where
molecular substitutes for water (Clegg, water availability and temperature are
2001). Under certain circumstances, tre- moderate and sensitive plants are abun-
halose may even induce desiccation toler- dant, desiccation-tolerant vascular plants
ance in human cells (Guo et al., 2000). grow mostly on outcrops of bare rock
However, tolerance in plants also involves (Porembski and Barthlott, 2000). In the
other mechanisms (Section 1.6), and the driest and coldest habitats, especially
ecology of desiccation-tolerant plants sug- where dew and fog are major water sources,
gests that the evolution of tolerance in desiccation-tolerant bryophytes, lichens,
plants is constrained by its consequences algae or cyanobacteria may form the only
for growth and competition. vegetation (e.g. Thompson and Iltis, 1968;
Friedmann and Galun, 1974; Davey, 1997).
Despite the ability of some of these species
1.5. The Ecology of Desiccation to tolerate a drought that is longer and
Tolerance in Plants: a Diversity of Cycles more intense than occurs in these habitats,
in Marginal Habitats the most xeric microsites are often still
bare (e.g. Alpert, 1985). On rocks and soil
Desiccation-tolerant plants grow mainly in in the desert, small differences in exposure
the interstices and on the margins of the to the sun may determine whether a patch
world’s vegetation, in microhabitats and of soil or stone is colonized or not.

(a) (b)

(c) (d)

Fig. 1.3. (a) Exposed surfaces of granitic boulders in the western foothills of the Cuyamaca Mountains in
southern 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), shown
hydrated. Crevices support desiccation-tolerant pteridophytes such as Pentagramma triangularis (gold-back
fern), shown (c) desiccated and (d) hydrated. (Photos by P. Alpert.)
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 14

14 P. Alpert and M.J. Oliver

(a) (b)


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 is
shown in its desiccated and its rehydrated state. (Photos by M.C.F. Proctor.)
These patterns appear tied to the differ- dry out again in hours. Desiccation-tolerant
ent sources of water that different desicca- vascular plants are only known to rewet
tion-tolerant plants can use to rehydrate, from rain; they recover in hours to days
the rates at which they rewet and dry out, and dry out in days to weeks. The cumula-
and their ability to recover after desicca- tive effect of repeated cycles of desiccation
tion and achieve a cumulative net gain of on net photosynthesis and growth may
resources. Lichens and bryophytes may explain why desiccation-tolerant plants fail
rewet from dew, recover in minutes and to survive in the most exposed microsites.
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 15

Drying Without Dying 15




Fig. 1.5. The most intensively studied desiccation-tolerant bryophyte is (a) Tortula ruralis, shown in the fully
hydrated state (top), after slow drying (lower right), and after rehydration for 2 min (lower left). T. ruralis is
common in dry habitats in North America, as shown on rocks at Mesa Verde National Park, USA (b). One of the
most studied desiccation-tolerant angiosperms is the grass Sporobolus stapfianus (c), shown after drying in a pot
for 14 days (left) and after subsequent immersion in water for 24 h (right). (Photos by M.J. Oliver and B. Mishler.)
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 16

16 P. Alpert and M.J. Oliver

(a) (b)


Fig. 1.6. Effects of desiccation and rehydration on ultrastructure in leaves of the moss Tortula ruralis. The
transmission electron micrographs (after Bewley and Pacey, 1978) show papillose cells in (a) the fully
hydrated 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 had
been rehydrated for 5 min (note that the chloroplasts are swollen but the nucleus (N) is not). The
freeze–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 the
mitochondrion (M) outside the chloroplast.)
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 17

Drying Without Dying 17

Trade-offs between tolerance and growth pensation point for photosynthesis

and competition with sensitive plants may (Schipperges and Rydin, 1998). In warm
explain why desiccation-tolerant plants, and cold deserts, tolerant algae and lichens
though they can rise again from ‘apparent grow inside or on the underside of translu-
death’ (Doyère, 1842), have not dominated cent rocks (Friedmann and Galun, 1974;
the earth. Kappen, 1993; Nienow and Friedmann,
1993). Species of Nostoc, Anacystis and
other cyanobacteria form desiccation-
1.5.1. Habitats tolerant crusts on bare walls and rocks
from the tropics to the boreal zone (Potts,
In contrast to the wide taxonomic and 1994; Lüttge, 1997). Algae, lichens and
geographical ranges and the broad mor- bryophytes join cyanobacteria to form
phological diversity of desiccation-tolerant crusts on desert soils, which are important
vascular plants, their ecological range is in nitrogen cycling (e.g. Nash and Moser,
narrowly confined to chronically or sea- 1982; Lange et al., 1994, 1997). Since
sonally dry habitats or microhabitats lichens and bryophytes dry so rapidly, they
where desiccation-sensitive plants are may be active mostly when conditions are
sparse or absent. Porembski and Barthlott effectively mesic and function as ‘shade
(2000) estimated that 90% of desiccation- plants’, even in exposed, xeric habitats
tolerant vascular plants are associated (Green and Lange, 1994; Proctor, 2000).
with rock outcrops, mainly in tropical to Degree of desiccation tolerance seems to
lower temperate latitudes. Some species explain some of the relative ability of toler-
grow on exposed rock surfaces, while oth- ant species to occupy xeric microsites or
ers are associated with crevices (Nobel, habitats (e.g. Hernandez-Garcia et al., 1999;
1978; Gildner and Larson, 1992). Franks and Bergstrom, 2000). For instance,
Ephemeral pools on rock outcrops in ability to tolerate desiccation (Mitchell et
Africa harbour a set of aquatic, desicca- al., 1999), to maintain photosynthesis dur-
tion-tolerant vascular plants (e.g. Volk, ing desiccation (Robinson et al., 2000) and
1984; Gaff and Giess, 1986). Tolerant to recover photosynthesis after repeated
angiosperms and pteridophytes also grow cycles of desiccation (Davey, 1997) are
in semiarid or desert grasslands, especially associated with occurrence of bryophytes
(Eickmeier, 1983; Gaff, 1987; Kappen and in relatively dry sites in Antarctica. The
Valladares, 1999) though not invariably ability to tolerate prolonged desiccation
(Gaff and Sutaryono, 1991), on shallow and to recover quickly upon rehydration
soils. There are exceptions to this narrow- appeared necessary but not sufficient to
ness of ecological range. A few tolerant allow mosses to colonize highly insolated
vascular species, such as Boeah hygro- surfaces on boulders in chaparral in
scopica (Gaff, 1981) and Pentagramma California (Alpert, 1985; Alpert and
triangularis (P. Alpert, personal obser- Oechel, 1987). A species of Selaginella
vation), occur in forest understoreys. from dry habitats recovered net photosyn-
Desiccation-tolerant bryophytes, lichens thesis faster than one from moister habitats
and algae occupy a much wider ecological (Eickmeier, 1980). Shirazi et al. (1996)
range than do tolerant vascular plants, reported differences in desiccation toler-
including both less and more arid sites ance between populations of lichens from
(Fig. 1.4). For example, tolerant bryophytes different habitats.
and lichens are common on rocks, trunks
and soil in moderately moist forests. They
may be common in tundra, although the 1.5.2. Cycles
Sphagnum species characteristic of tundra
do not necessarily recover net photosyn- Desiccation-tolerant plants vary greatly in
thesis after losing more than about 10% the rates at which they dry out, rehydrate
of the water content they hold at the com- and recover upon rehydration and there-
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 18

18 P. Alpert and M.J. Oliver

fore in the rhythms of desiccation and Xerophyta scabrida began to respire within
growth they experience in nature (Tuba et 20 min after rehydration, reached full rates
al., 1998; Kappen and Valladares, 1999; of respiration within 6 h, began to synthe-
Chapter 7). In general, bryophytes and size chlorophyll after 12 h and did not
lichens dry out in hours in the sun, complete synthesis until 36 h (Tuba et al.,
whereas ferns and angiosperms take a day 1994). Poikilochlorophylly appears to be a
or more. Minimum times to net photosyn- programmed rather than a pathological
thesis after rehydration range from minutes response to desiccation. For instance, it is a
in lichens and mosses rewetted with liquid necessary component of tolerance in the
water, to hours in lichens and mosses rehy- leaves of some species: when these leaves
drated with water vapour and in some vas- are detached before drying, they stay green
cular plants rewetted with liquid, to days as they dry but they die (Gaff, 1981).
in other vascular plants (Fig. 1.5; Lange, Natural cycles of wetting and drying
1969; Lange and Kilian, 1985; Gaff and have been followed for a number of
Giess, 1986; Reynolds and Bewley, 1993a; mosses and lichens (e.g. Kappen et al.,
Scott and Oliver, 1994; Scheidegger et al., 1979; Lange et al., 1994; Sancho et al.,
1997; Tuba et al., 1998). 1997) but very few desiccation-tolerant
Desiccated lichens can resume net pho- vascular plants (Nobel, 1978; Gaff and
tosynthesis by taking up water vapour Giess, 1986). During a year in the Negev
(Hahn et al., 1993; Schroeter et al., 1994), Desert, thalli of the lichen Ramalina maci-
but only if the phycobiont is a green alga formis underwent a cycle of wetting and
rather than a cyanobacterium (Kappen and drying almost daily, mostly from dew
Valladares, 1999). A few mosses can (Kappen et al., 1979). Some bryophytes in
recover at least very slow rates of net pho- semiarid grasslands can likewise experi-
tosynthesis by taking up water vapour after ence diurnal desiccation cycles driven by
desiccation (Lange, 1969; Rundel and dew during dry seasons (Csintalan et al.,
Lange, 1980). Both bryophytes and lichens 2000). At the other extreme, some
can rehydrate with dew (Lange et al., 1994; angiosperms may undergo a single period
Csintalan et al., 2000). Despite the lack of a of desiccation per year, with a cycle of
cuticle, differences in thallus, leaf and activity almost like that of an annual plant
shoot morphology and packing produce (e.g. Gratani et al., 1998).
several-fold differences in drying rates Three factors that determine how cycles
between different species of bryophytes of desiccation translate into growth are
and lichens (e.g. Gimingham and Smith, light damage, nutrient relations and carbon
1971; Proctor, 1982; Scott, 1982; balance. Photodamage can occur as plants
Valladares, 1994); differences in morpho- dry or while they are desiccated, due at
logical control of water loss may help least in part to light absorption without
explain differences in ability to colonize energy transfer to photosynthesis (e.g. Seel
xeric microhabitats. et al., 1992; Gauslaa and Solhaug, 1996).
Desiccation-tolerant angiosperms are Desiccation-tolerant plants show a variety
known to rehydrate in nature only after of mechanisms likely to reduce photodam-
rain. Woody angiosperms may take longer age, including leaf curling, accumulation of
to desiccate and rehydrate than herbaceous anthocyanin and carotenoids, and xantho-
ones (Sherwin and Farrant, 1996; Farrant et phyll metabolism (e.g. Muslin and
al., 1999). The slowest to recover from des- Homann, 1992; Eickmeier et al., 1993;
iccation are the poikilochlorophyllous des- Lebkeucher and Eickmeier, 1993;
iccation-tolerant plants, monocots that Calatayud et al., 1997; Deltoro et al., 1998;
dismantle their photosynthetic machinery Beckett et al., 2000; Farrant, 2000).
when they dry and reassemble it again Antarctic mosses, which could be subject
when they rehydrate (Sherwin and Farrant, to photodamage during freezing, show
1996; Tuba et al., 1998). In one desiccation reversible photoinhibition and zeaxanthin
study, the poikilochlorophyllous species activity (Lovelock et al., 1995).
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 19

Drying Without Dying 19

Little is known about the interaction These factors are most important in des-
between desiccation tolerance and nutrient iccation-tolerant plants that tend to have
relations. Uptake and metabolism of min- short periods of hydration or frequent
eral nutrients must be interrupted from cycles of desiccation, such as lichens and
some point during drying to some point mosses in arid habitats, and are probably
during rehydration and recovery. In one reason why these species grow so
mosses, leakage of solutes during rehydra- slowly (Stark, 1997; Kappen and
tion could also reduce net nutrient uptake. Valladares, 1999; Badacsonyi et al., 2000).
Increased frequency of desiccation cycles Since brief periods of hydration can result
decreases potassium content but not cumu- in net carbon loss (Lange et al., 1994;
lative phosphorus uptake in Tortula ruralis Csintalan et al., 2000), it is possible that
(Badacsonyi et al., 2000). Activity of nitrate some desiccation-tolerant plants may have
reductase decreases rapidly during desicca- been selected for traits that help prevent
tion in T. ruralis (Mahan et al., 1998; rewetting by small amounts of water. Water
Badacsonyi et al., 2000); activity can repellence in epilithic lichens (Bertsch,
recover in less than 8 h after rehydration if 1966) and hair points on some epilithic
the moss has dried slowly, but may take mosses (P. Alpert, unpublished data) could
24 h after rapid drying (Mahan et al., be examples.
1998), and may decrease during the first
hour of rehydration (Marschall, 1998).
However, Bates (1997) found that weekly, 1.5.3. Hypotheses
24 h desiccation did not decrease uptake of
N, P or K in two mosses compared to The rarity of desiccation-tolerant vascular
uptake during continuous hydration, and plants in habitats where other vascular
Badacsonyi et al. (2000) saw no difference plants are abundant suggests that surviving
between the effect of low water potential desiccation may have negative as well as
on nitrate reductase activity in desiccation- positive effects on survival overall. One
tolerant and sensitive mosses. hypothesis is that there is a trade-off
Cycles of desiccation tend to reduce net between tolerance and growth, and that tol-
carbon gain by favouring respiration over erant plants are out-competed by sensitive
photosynthesis and by decreasing the ones where the latter can survive, because
amount of time that plants are active. the latter grow faster and larger. This
Desiccation increases the ratio of respira- should cause selection against tolerance in
tion to photosynthesis because: (i) photo- habitats where plants can acquire and con-
synthesis ceases before respiration during serve enough water to avoid desiccation.
drying and resumes after respiration during An alternative possibility is that tolerance
rehydration; (ii) respiration in some species is merely lost in plants that are not
increases above normal levels during recov- exposed to desiccation, due to lack of
ery from desiccation; and (iii) plants tend to selection pressure to maintain tolerance.
stay hydrated at night when they cannot Does desiccation tolerance entail a
photosynthesize but do respire, and to des- reduction in growth rate or maximum
iccate most rapidly when light levels are size? There are a number of reasons to
high (e.g. Alpert, 1979; Proctor, 1982; Lange suppose this, but little direct evidence.
et al., 1994; Tuba et al., 1998). Tuba et al. Kappen and Valladares (1999) proposed
(1999) examined the hypothesis that an that some morphological features that pro-
increase in atmospheric CO2 might improve mote tolerance also conflict with produc-
carbon balance during cycles of desicca- tivity. In angiosperms, hairs or scales that
tion. Elevated CO2 does prolong photosyn- reduce water loss and can thus prolong
thesis during drying in X. scabrida, but the periods of net photosynthesis also inhibit
authors concluded that this aspect of global rehydration (Kappen and Valladares,
change was unlikely to favour desiccation- 1999). In lichens and bryophytes, having
tolerant over sensitive species. high maximum water content tends to
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 20

20 P. Alpert and M.J. Oliver

prolong hydration but inhibits photosyn- tats? This hypothesis has been partially
thesis, since much of the water is typi- tested in bryophytes and lichens (e.g.
cally held externally or in upper layers of Alpert, 1990; Pintado et al., 1997; Williams
the lichen thallus and so slows gas diffu- and Flanagan, 1998; Kappen and
sion (Green and Lange, 1994; Valladares, Valladares, 1999). For example, during a
1994; Lange et al., 1996; Tuba et al., morning after nocturnal rain or dew,
1996a; but see Sojo et al., 1997). bryophytes and lichens growing on sloped
Populations of Ramalina capitata in dry, surfaces in north temperate latitudes tend
bright sites tend to have greater capacity to dry more rapidly if they are on surfaces
to store water and slower gas diffusion that face south or east than if they are on
than populations in more shaded sites surfaces that face north or west (Kappen et
(Pintado et al., 1997), suggesting that al., 1980; Alpert and Oechel, 1985). Those
selection favours water storage more when on north- and west-facing surfaces are
light is less limiting. Proctor (2000) pro- more likely to recoup the respiratory losses
posed that bryophytes have been selected incurred during the night before desicca-
for rapid desiccation to minimize time tion arrests photosynthesis in the morning.
spent at intermediate water contents, This probably at least partly explains why
which most dispose plants to damage. mosses are ‘more common on the north side
Rapid desiccation would also reduce time of the tree’. There appear to be no studies
available for photosynthesis and growth. on the effect of microsite on carbon balance
Cellular mechanisms of tolerance such as in desiccation-tolerant vascular plants.
sugar and protein synthesis (Section 1.6) Oliver et al. (2000) hypothesized that
seem likely to impose metabolic costs and desiccation tolerance was once the major-
thus reduce growth. If cavitation during ity solution to the problem of living in dry
desiccation precludes desiccation-tolerant air. They suggested that tolerance is a prim-
plants from exceeding 3 m in height itive characteristic in green plants that
(Sherwin and Farrant, 1998), then they allowed them to colonize the land. Once
will be overtopped wherever trees can plants evolved vascular tissues and effi-
grow. Some comparative studies on cient internal water transport, they lost
mosses (Bates, 1997; Arscott et al., 2000) their tolerance of desiccation except in
and anecdotal reports on grasses (Gaff, parts that had to be cut off from water
1989) have found that more productive transport – their spores, seeds and pollen.
species are less desiccation-tolerant. The Tolerance in adult plants then re-evolved
long-standing hypothesis (Grime, 1979) several times in different lineages.
that stress tolerance conflicts with pro- Porembski and Barthlott (2000) proposed
ductivity is intuitively appealing but that this re-evolution occurred as
mechanically elusive. Further compara- angiosperms colonized the bare rock out-
tive studies on desiccation-tolerant plants crops where their desiccation-tolerant
could help reveal mechanisms that dictate species are now most diverse. If this sce-
trade-offs between tolerance and growth. nario is correct, then desiccation tolerance
The absence of desiccation-tolerant in plants has evolved not just as a way of
plants in some highly xeric habitats where surviving in marginal habitats, but as a way
no other plants occur suggests that surviv- of colonizing frontiers, first from water on
ing desiccation may not assure survival, to land and then from soil on to stone.
even where competition is not a factor. One
hypothesis to explain why tolerant plants
are not more abundant in barren habitats is 1.6. Mechanisms of Desiccation
that the plants cannot maintain a cumula- Tolerance
tive positive carbon balance under certain
regimes of water availability (Ried, 1960). Until the mid-1970s, it was generally
Does carbon balance limit the survival of believed that the mechanisms of desicca-
desiccation-tolerant plants in xeric habi- tion tolerance in plants were mechanical
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 21

Drying Without Dying 21

(see reviews by Bewley, 1979; Oliver and 1.6.1. Damage

Bewley, 1984). Structural features such as
flexible cell walls, small vacuoles and Before discussing what is known of the
lack of plasmodesmata were suggested as cellular protection and repair mecha-
key elements in tolerance (Gaff, 1980; nisms of desiccation tolerance, it is worth
Bewley and Krochko, 1982; Oliver and reviewing the effects of desiccation and
Bewley, 1984). In a landmark paper, rehydration on cellular integrity in desic-
Bewley (1979) articulated the alternative cation-tolerant plants. The critical ques-
view that desiccation tolerance is primar- tion, when deciding what type of
ily protoplasmic in nature. This theory mechanisms desiccation-tolerant plants
argues that certain plants and plant tis- employ, is when might damage occur? Is
sues achieve desiccation tolerance as a it during the drying process or upon rehy-
result of the inherent properties of their dration? For instance, if damage does not
cellular contents (protoplasm). Most evi- actually occur during desiccation, then
dence now supports this view, though there is good reason to believe that pro-
structural features are clearly important tective mechanisms are in place. If dam-
in desiccation tolerance in some cases age occurs upon rehydration, and the cell
(Sherwin and Farrant, 1996; Farrant et subsequently recovers, repair mecha-
al., 1999). nisms are probably operative. In addition,
Bewley (1979) further defined three the amount of damage and the rate at
critical features of desiccation tolerance which cells return to a normal status
based on the observation that many desic- measure the effectiveness of protective
cation-tolerant plants exhibit cellular and repair processes and the overall level
changes, some of which can be described of desiccation tolerance.
as extensive damage, during and follow-
ing desiccation. The plant or tissue must: Damage during desiccation
(i) limit damage to a repairable level; (ii)
maintain its physiological integrity in the The timing of damage is still controversial,
dried state (perhaps for extended periods but a consensus is building that little dam-
of time); and (iii) mobilize mechanisms age occurs during drying in desiccation-
upon rehydration that repair damage suf- tolerant tissues. Much of the work in this
fered during desiccation and rehydration. area has focused on the plasma membrane.
These criteria laid the experimental foun- All desiccation-tolerant tissues leak
dation for the field from the 1980s solutes during rehydration (Simon, 1978;
onwards and continue to influence the Bewley, 1979; Bewley and Krochko, 1982),
way we think about how plants survive indicating that the cell membrane has been
desiccation. In particular, it is now compromised. Early electron microscopy
widely accepted that the cells of desicca- of seeds (Webster and Leopold, 1977;
tion-tolerant plants employ mechanisms Morrison-Baird et al., 1979) and bryophyte
that protect them from the rigours of tissues (reviewed by Oliver and Bewley,
extensive water loss and also mecha- 1984) suggested that membranes in dried
nisms, at least in the case of vegetative plant cells were completely disorganized.
cells, that repair damage suffered during With the advent of more sophisticated
desiccation or rehydration (Bewley and technologies, these observations were
Oliver, 1992). This introductory overview determined to be artefacts of sample
of the mechanisms of desiccation toler- preparation and chemical fixation (Bewley,
ance will therefore concentrate on cellu- 1979; Thompson, 1979; Bewley and
lar features (the so-called ‘inherent Krochko, 1982; Oliver and Bewley, 1984).
properties’ of desiccation-tolerant cells) The use of non-aqueous fixatives elimi-
that have been suggested to play a major nated some of these artefacts but the heavy
role in protection and repair. Details are use of chemical treatments still made
covered in subsequent chapters. interpretation difficult (Thompson, 1979;
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 22

22 P. Alpert and M.J. Oliver

Öpik 1980, 1985; Tiwari et al., 1990; Damage during rehydration
Smith, 1991). Freeze–fracture electron
microscopy, however, has yielded the most As noted above, all plant tissues leak
reliable data. Dried tissues are eminently solutes when rehydrated following a dry-
suited for freeze–fracture preparation ing event. In desiccation-tolerant tissues,
because their low water content virtually however, this is a transient event (Simon,
eliminates the formation of ice crystals, 1978; Bewley, 1979; Bewley and Krochko,
which make high-quality replicas difficult 1982). Several hypotheses have been
to obtain. Freeze–fracture studies clearly offered to explain imbibitional (or rehydra-
demonstrated that the membranes of seeds tive) leakage (Simon, 1974; Senaratna and
(Thompson and Platt-Aloia, 1982; Bliss et McKersie, 1983a,b; Crowe et al., 1989,
al., 1984) and pollen (Platt-Aloia et al., 1992; Hoekstra et al., 1992). The prevailing
1986) could retain normal bilayer organi- hypothesis is that imbibitional leakage is
zation and dispersal patterns of intramem- the result of lipid-phase transitions occur-
branous particles at water contents as low ring in the plasma membrane as a result of
as 0.08 g H2O g1 dry mass. The plasma dehydration and rehydration (Crowe et al.,
and organelle membranes of vegetative 1992). During drying, membranes pass
cells of the desiccation-tolerant pterido- from the liquid crystalline to the gel phase,
phyte Selaginella lepidophylla and the and they return to the liquid crystalline
moss Tortula ruralis also retain normal phase during rehydration. In artificial
organization and dispersal patterns in the membranes, this transition can lead to a
dried state (Platt et al., 1994; Fig. 1.6). transient leakage event (Hammoudah et al.,
The effects of desiccation on cellular 1981), and, since phase transitions have
components that cannot be observed by been demonstrated in drying and rehydrat-
freeze–fracture microscopy are more diffi- ing desiccation-tolerant cells (Crowe et al.,
cult to evaluate, largely due to the likeli- 1989; Hoekstra et al., 1992), it has been
hood of partial rehydration and the generally accepted that phase transition is
production of artefacts during chemical the basis of imbibitional leakage in most
fixation. In seeds, the uncertainty is com- desiccation-tolerant tissues. In seeds, how-
pounded by the fact that the tissues ever, it is thought that membrane-phase
are part of a developing system. changes do not occur because of the pres-
Nevertheless, observations tend to sug- ence of a seed coat, which impedes the
gest that desiccation of tolerant plants passage of water to the dried cells.
generates an ordered ‘collapse’ of the Hoekstra et al. (1999) suggested that the
cellular milieu that results in little ultra- slow rate of penetration of water may set
structural damage (Oliver and Bewley, up a ‘pre-hydration’ state where the mem-
1984; Gaff, 1989; Goldsworthy and branes are in a liquid crystalline state
Drennan, 1991; Sherwin and Farrant, before liquid water surrounds the rehydrat-
1996; Farrant et al., 1999). If desiccation- ing cells. Since leakage does occur during
tolerant plants successfully avoid damage the rehydration of these tissues (Hoekstra
during the dehydration process, as it et al., 1992; Tetteroo et al., 1996), it has
appears they do, is there any consequence been concluded that leakage must occur
at all of desiccation in these plants? The through an intact lipid bilayer, as suggested
answer appears to be yes. All desiccation- by Senaratna and McKersie (1983b).
tolerant plants and plant tissues show Recently, a new hypothesis has emerged
signs of cellular damage when the dried from some exciting new studies on dehy-
tissue is rehydrated. It is, however, debat- drating and rehydrating pollen (Hoekstra et
able whether or not the damage occurs al., 1997, 1999; Golovina et al., 1998;
during the drying process (but is not Buitink et al., 2000; Chapter 10). This body of
observable at an ultrastructural level) or work using amphiphilic spin probes demon-
as the result of the inrush of water into strates that during dehydration endogenous
the cells during rehydration. amphiphilic substances partition from the
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 23

Drying Without Dying 23

aqueous cytoplasm into pollen membranes. Within minutes after rehydration, the
Using data obtained from liposome-based ex- chloroplasts of the green gametophytic tis-
periments, Golovina et al. (1998) suggested sues of desiccation-tolerant bryophytes
that it is the presence of these amphiphiles appear swollen and globular. Their outer
in the membrane that causes imbibitional membranes are folded and separated from
leakage and that as the pollen rehydrates the the thylakoids, which are no longer com-
amphiphilic substances move out of the pacted (Oliver and Bewley, 1984). The
membranes and leakage stops. This hypo- extent of thylakoid disruption increases
thesis could explain how transient leakage with the rate of prior desiccation. The
can occur through an intact membrane. In a chloroplasts of desiccation-tolerant
more recent study, Buitink et al. (2000) angiosperms tend to be more resistant to
demonstrated that the movement of disruption than those of bryophytes,
amphiphilic compounds into membranes although vesicularization within the
also occurs in imbibing radicles of peas and chloroplast internal membranes is common
cucumbers. This study, using electron para- (Gaff and Hallam, 1974; Gaff et al., 1976;
magnetic resonance (EPR) spectroscopy and Sherwin and Farrant, 1996). In all desicca-
inserted nitroxide spin probes, demon- tion-tolerant plants, mitochondria swell
strated a difference in partitioning behav- and exhibit disruption of the cristae
iour between desiccation-tolerant and (reviewed by Bewley and Krochko, 1982).
sensitive tissues. Spin probes partitioned Swelling and disruption of mitochondria
into the membranes at higher water content are not affected by rate of desiccation. In
in desiccation-sensitive tissues than in toler- all cases, organelles regain normal struc-
ant tissues. These authors suggest, from in ture within 24 h.
vitro portioning experiments, that it is the
microviscosity of the cytoplasm that con- Poikilochlorophylly
trols portioning of amphiphilic compounds
into the plasma membrane. What remains to At least eight genera of desiccation-tolerant
be determined is the role of the native monocots are ‘poikilochlorophyllous’, i.e.
amphiphilic compounds in membrane dam- they reversibly lose their chlorophyll and
age and, if they are important in desiccation dismantle their chloroplasts during desic-
tolerance, the role they play in the long-term cation (Gaff, 1989; Tuba et al., 1998). The
stability of membranes in the dried state. thylakoid system within desiccated chloro-
Golovina et al. (1998) speculated that plasts is completely replaced by small
amphiphiles may have antioxidant proper- groups of plastoglobuli and by osmophilic,
ties that protect membranes from damage stretched lipid material, which appears to
by free radicals generated during desicca- occupy the positions previously occupied
tion and rehydration. If so, imbibitional by the thylakoids (Hallam and Luff, 1980;
leakage may be a necessary trade-off for Tuba et al., 1993a,b; Sherwin and Farrant,
protection. Much work will be required 1996). After 10–12 h rehydration, when
before the importance of native full turgor and maximum leaf water con-
amphiphilic compounds in desiccation tol- tent are reached, synthesis of chlorophylls
erance can be determined, but amphiphiles and carotenoids and the reassembly of thy-
are an intriguing new development in our lakoids begin. Early in reassembly, sets of
understanding of desiccation tolerance. two primary thylakoids stack to form
Rehydration-induced damage other than grana. Within 72 h the chloroplasts appear
leakage is difficult to distinguish from nor- normal and full photosynthetic capacity is
mal development in seeds and pollen but restored (Tuba et al., 1993b, 1994). From
is clearly evident in the tissues of most these studies and later physiological in-
desiccation-tolerant vegetative tissues, vestigations (Tuba et al., 1997), it appears
especially in organelles (reviewed by Bewley that these changes can be classified as
and Krochko, 1982; Oliver and Bewley, genetically programmed responses to des-
1984; Gaff, 1989; Oliver and Wood, 1997). iccation rather than damage.
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 24

24 P. Alpert and M.J. Oliver

1.6.2. Protection reach a maximum about three days before

the seed begins to desiccate (Galau and
Much of what we know of the cellular pro- Hughes, 1987; Galau et al., 1987). The
tection mechanisms involved in desiccation other class contains 12 transcripts, which
tolerance in plants comes from studies of appear late in maturation and achieve max-
orthodox seeds (Bewley and Black, 1994; imum expression just before and during
Chapter 5) and, to a slightly lesser extent, desiccation. LEA proteins make up 30% of
pollen (Crowe et al., 1992; Hoekstra et al., the non-storage protein and 2% of the total
1992). The ability of seeds to withstand soluble protein in the mature cotton
desiccation is acquired during their devel- embryos and are uniformly localized
opment. This acquisition is usually sub- throughout the cytoplasm (Roberts et al.,
stantially earlier than the culmination of 1993). LEA proteins and the acquisition of
the drying event itself, which is the termi- desiccation tolerance during seed matura-
nal event in orthodox seed maturation. tion have been linked in other dicots (e.g.
Seeds of some species can withstand pre- soybean: Blackman et al., 1995) and in
mature desiccation well before the mid- monocots (e.g. maize: Mao et al., 1995;
point of their development (Bewley and Wolkers et al., 1998).
Black, 1994; Chapter 5). Among the meta- A set of LEA proteins arises in develop-
bolic changes that take place just prior to or ing barley and maize embryos at the time
during drying is the synthesis of proteins that tolerance of desiccation is acquired. A
and sugars, which have long been postu- small subset of these proteins is induced
lated to form the basis of a series of overlap- when barley embryos at the intolerant stage
ping protective mechanisms that limit are cultured in abscisic acid (ABA) (Bartels
damage to cellular constituents (Bewley, et al., 1988; Bochicchio et al., 1991), and a
1979; Leprince et al., 1993; Oliver and causal relationship between ABA and lea
Bewley, 1997). These two components have gene expression has been suggested.
since been widely implicated as being criti- Evidence for, and against, this relationship
cal for desiccation tolerance in all plant exists in the literature. In cotton embryos,
cells including vegetative cells (Ingram and high expression of the first class of lea
Bartels, 1996; Oliver and Bewley, 1997; genes occurs as ABA content increases.
Scott, 2000). Over the years it has also High expression of the second set of lea
become clear that the synthesis of antioxi- genes, however, occurs at the start of, and
dants and enzymes involved in oxidative during, maturation drying, when the
metabolism also play a critical role in cellu- endogenous ABA content is low. There are
lar protection and desiccation tolerance explanations for this lack of correlation,
(Chapter 10). However, this aspect of pro- e.g. there is an early-regulated, ABA-con-
tection will not be addressed here. trolled mechanism, which operates only
later when drying commences. On the
other hand, an ABA-independent pathway Proteins
may be involved in the synthesis of the
Only one subset of proteins that accumu- second group of LEA proteins.
late at the time of the acquisition of desic- LEA proteins have been identified in the
cation tolerance has been extensively vegetative tissues of all desiccation-tolerant
investigated, the late embryogenesis abun- plants studied so far (Ingram and Bartels,
dant (LEA) proteins, first described in cot- 1996; Oliver and Bewley, 1997; Blomstedt
ton (Galau and Hughes, 1987; Galau et al., et al., 1998) and proteins related to some of
1987, 1991; Chapter 5). The genes that the LEA proteins, e.g. dehydrins (see
encode LEA proteins in developing cotton- below), have been associated with the
seeds are comprised of two distinct classes response of non-tolerant plants to water
whose regulation is coordinated. One class stress (Skriver and Mundy, 1990; Bray,
contains six different lea transcripts, which 1997). In nearly all instances, the induction
appear relatively early in development and of LEA protein synthesis in vegetative tis-
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 25

Drying Without Dying 25

sues can be elicited by exogenous ABA LEA protein synthesis is also highly
application (Ingram and Bartels, 1996; induced in the vegetative tissues of desic-
Campalans et al., 1999). cation-tolerant angiosperms during drying
LEA proteins fall into five groups by (Bartels et al., 1993; Blomstedt et al., 1998;
virtue of sequence similarities (Dure et al., Bartels, 1999). Callus derived from vegeta-
1989; Ingram and Bartels, 1996; Cuming, tive tissue of the desiccation-tolerant plant
1999). All are highly hydrophilic and all are Craterostigma plantagineum is not inher-
very stable, as evidenced by their resistance ently tolerant but can be made so by the
to the denaturing effects of boiling (with the application of ABA (Bartels et al., 1990).
exception of Group 5 LEA proteins). Group The application of ABA to this tissue
1 LEA proteins are characterized by a 20- results in the synthesis of novel proteins,
amino acid motif and are represented by the some of which are LEA proteins including
wheat Em protein, the first LEA protein the Group 2 LEA proteins, the dehydrins
identified (Cuming and Lane, 1979). Group (Bartels et al., 1993). The desiccation-toler-
2 LEA proteins are characterized by a 15- ant moss T. ruralis utilizes a more primi-
amino acid motif, the K-segment, a stretch tive mechanism of desiccation tolerance
of serine residues and a conserved motif (Oliver et al., 2000), which involves a con-
near the N-terminus of the protein (Close, stitutive cellular protection strategy, and in
1997). This group of proteins is also called this plant, unlike others, dehydrins are not
the dehydrins and these are the most wide- induced by dehydration or by ABA but are
spread and most studied of the LEA pro- constitutively expressed (Bewley et al.,
teins. Group 3 LEA proteins share a 1993). Dessication-sensitive species
characteristic 11-amino acid repeat motif exposed to sub-lethal dehydration stress
(Dure et al., 1989), which is predicted to also respond by synthesizing LEA proteins
form an amphipathic -helix. These amphi- and LEA-like proteins, in particular dehy-
pathic helices are postulated to form intra- drins (Close, 1997). These examples and
and intermolecular interactions that may many more all point to the importance of
have important consequences for their func- LEA proteins in dehydration responses and
tion (Baker et al., 1988; Dure, 1993a). The desiccation tolerance.
least studied of the LEA proteins are those in The most convincing pieces of evidence
Groups 4 and 5, which are somewhat to suggest that LEA proteins have an
atypical (Dure, 1993b; Galau et al., 1993). important role in cellular protection come
Group 5 LEA proteins are more hydrophobic from transgenic studies using a barley
than other LEA proteins and are not resistant Group 3 lea gene, HVA1. This gene, when
to high temperature. Most of the LEA protein expressed in a constitutive fashion in
groups have been identified in many differ- transgenic rice, increased its tolerance to
ent plants. All groups are thought to play a water and salt stress (Xu et al., 1996).
role in desiccation tolerance, and the evi- HVA1 overexpression in wheat, driven by a
dence for this viewpoint is growing. maize ubiquitin promoter, resulted in
The evidence for the involvement of transgenic lines that performed in a supe-
LEA proteins in desiccation tolerance is rior fashion under soil-water deficits
circumstantial but compelling. LEA protein (Sivamani et al., 2000).
synthesis in seeds, as mentioned above, is There are a variety of suggested mecha-
associated with both the acquisition of des- nisms by which LEA proteins might pro-
iccation tolerance and the final stage of tect cellular components. Many LEA
seed maturation just prior to desiccation. proteins have extensive regions of random
In addition, ABA-deficient (aba) and ABA- coiling, which has been postulated to pro-
insensitive (abi3) double-mutants of mote the binding of water, helping to main-
Arabidopsis seeds do not dry on the parent tain a minimum water requirement (Ingram
plant, do not tolerate desiccation and lack and Bartels, 1996). For instance, the Em
several LEA proteins (Koorneef et al., 1989; protein of wheat is considerably more
Meurs et al., 1992). hydrated than most common proteins, and
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 26

26 P. Alpert and M.J. Oliver

over 70% of the Em protein is configured in accumulation during desiccation.

as random coils (McCubbin et al., 1985). Constitutive expression of HSPs is unusual
Baker et al. (1988) suggested that the ran- in vegetative tissues and resembles the
dom coil nature of some LEA proteins may expression pattern of these proteins in
allow them to conform to the shape of cel- seeds. In addition, exogenous ABA
lular constituents and thus, by virtue of induced both the expression of HSPs and
their hydroxyl groups, help to maintain the acquisition of desiccation tolerance in
their solvation state when water is C. plantagineum callus tissues (Alamillo et
removed. These authors also suggested that al., 1995). Finally, a LEA-like HSP, HSP-12,
the Group 2 LEA proteins (dehydrins), by from yeast was shown to be capable of pro-
virtue of their amphipathic helical repeats, tecting liposomal membranes from the
provide surfaces when bundled together damaging effects of desiccation in a way
that would sequester ions. This may be similar to that seen with the sugar tre-
crucial as the increasing ionic strength dur- halose (Sales et al., 2000). Thus it appears
ing drying could cause irreversible damage that small HSPs may also play a role in cel-
to cellular proteins and structural compo- lular protection during desiccation: per-
nents. Recently, Velten and Oliver (2001) haps this capability is related to their
described an LEA-like protein from T. chaperonin-like activities, which may help
ruralis that contains 15 15-amino-acid maintain protein structure under denatur-
repeats predicted to form amphipathic ing conditions. Other proteins whose tran-
helices. This protein appears to be synthe- scripts accumulate during the dehydration
sized during the rehydration event and phases of vegetative desiccation-tolerant
may serve to trap valuable ions that would angiosperms have been identified but little
otherwise be lost. Studies using individual has been done to confirm their roles in des-
LEA proteins in in vitro assays also add to iccation tolerance (Kuang et al., 1995;
the possible mechanisms by which these Ingram and Bartels, 1996; Blomstedt et al.,
proteins exert protection of cellular compo- 1998; Bockel et al., 1998; Neale et al.,
nents. Wolkers (1998) suggested from data 2000). See Chapters 5 and 11 for further
obtained from the study of a pollen Group discussion of all these proteins.
3 LEA protein and its effect on sucrose
glass formation that LEA proteins may act Sugars
as anchors in a structural network that sta-
bilizes cytoplasmic components during The accumulation of soluble sugars is also
drying and in the dried state. strongly correlated to the acquisition of
At this point it seems likely that each desiccation tolerance in plants and other
individual group of LEA proteins may have organisms (for reviews see Crowe et al.,
different, complementary effects. Most des- 1992; Leprince et al., 1993; Vertucci and
iccation-tolerant tissues contain a represen- Farrant, 1995; Chapters 5 and 10). Soluble
tative of most, if not all, of the different sugars, especially sucrose, accumulate in
groups of LEA proteins, and it is also likely seeds (Leprince et al., 1993), pollen
that all are needed to achieve the highest (Hoekstra et al., 1992) and in desiccation-
degree of desiccation tolerance. tolerant vegetative tissues (Bewley and
There is mounting evidence that another Krochko, 1982; Ingram and Bartels, 1996;
class of proteins, the small heat-shock pro- Oliver and Bewley, 1997). In Craterostigma
teins (HSPs), may play a role in cellular plantagineum, 2-octulose stored in the
protection during desiccation. Small HSPs hydrated leaves is converted to sucrose
accumulate in maturing seeds of many during drying to such an extent that in the
plant species (Vierling, 1991; Wehmeyer et dried state it comprises about 40% of the
al., 1996) prior to desiccation. Alamillo et dry weight (Bianchi et al., 1991).
al. (1995) reported that small HSPs are Sucrose is the only free sugar available
expressed constitutively in the vegetative for cellular protection in desiccation-toler-
tissues of C. plantagineum and increased ant mosses, including Tortula ruraliformis
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 27

Drying Without Dying 27

and T. ruralis (Bewley et al., 1978; Cytoplasmic glass formation has also
Smirnoff, 1992). The amount of this sugar been postulated to maintain the structural
in gametophytic cells of T. ruralis is and functional integrity of macromolecules
approximately 10% of dry mass, which is (Sun and Leopold, 1997; Crowe et al.,
sufficient to offer membrane protection 1998b), which has been well demonstrated
during drying, at least in vitro (Strauss and with in vitro models (Roos, 1995).
Hauser, 1986). Moreover, neither drying Intracellular glasses, by virtue of their high
nor rehydration in the dark or light results viscosity, drastically reduce molecular
in a change in sucrose concentration, sug- movement and impede diffusion of reac-
gesting that it is important for cells to tive compounds in the cell. It is by this
maintain sufficient amounts of this sugar property that glasses are thought to prolong
(Bewley et al., 1978). The lack of an the longevity of desiccated tissues by slow-
increase in soluble sugars during drying ing down degradative processes during
appears to be a common feature of desicca- storage. Buitink et al. (1998) recently
tion-tolerant mosses (Smirnoff, 1992). demonstrated a strong relationship
It is thought that sugars protect the cells between molecular mobility and storage
during desiccation by two mechanisms. longevity in both pollen and pea seeds.
First, the hydroxyl groups of sugars may Thus, although glass formation may not be
substitute for water to maintain important in the initial acquisition of des-
hydrophilic interactions in membranes and iccation tolerance, it may be crucial for sur-
proteins during dehydration (Crowe et al., vival of the dried state (as suggested by
1992). This has so far only been demon- Buitink, 2000; Chapter 10).
strated in vivo, using liposomes and iso- Other carbohydrates besides sucrose
lated proteins (Crowe et al., 1992). accumulate in desiccation-tolerant tissues,
Secondly, sugars are a major contributing the principal ones being the oligosaccha-
factor to vitrification, the formation of a rides stachyose and raffinose (Horbowicz
biological glass, of the cytoplasm of dry and Obendorf, 1994), and have been postu-
cells (Leopold et al., 1994; Chapter 10). lated to play a part in desiccation toler-
This mechanism has been the subject of ance. The presence of these compounds
intense research over the last 15 years. has also been correlated with seed
Vertucci and Leopold (1986) suggested longevity (Hoekstra et al., 1994;
that desiccation tolerance in seeds had to Horbowicz and Obendorf, 1994), which
be associated with some feature or solute has linked them to a possible role in the
combination that would avoid crystalliza- stabilization of intracellular glasses
tion of the cytoplasm as dehydration pro- (Leopold et al., 1994; Bernal-Lugo and
gressed. Burke (1986) proposed that high Leopold, 1995; Sun, 1997). However,
concentrations of sugars lead to vitrification Buitink et al. (2000) demonstrated that the
of the cytoplasm during desiccation and reduction in oligosaccharides in primed
thus prevent crystallization. Glass forma- seeds did not alter Tg (the glass-to-liquid
tion has since been demonstrated in seeds transition temperature) or viscosity and
(Williams and Leopold, 1989; Leopold et thus they contended that oligosaccharides
al., 1994; Leprince and Walters-Vertucci, do not affect the stability of intracellular
1995), pollen (Buitink et al., 1996) and in glasses. These results support the earlier
leaf tissues of C. plantagineum (Wolkers et studies of Black et al. (1999), which had
al., 1998). Walters (1998) went as far as to shown a lack of a temporal correlation
say that glass formation is an intrinsic prop- between the induction of desiccation toler-
erty of any complex system that can survive ance by a mild dehydration treatment and
desiccation. However, glass formation may the appearance of raffinose in wheat
not be sufficient to confer desiccation toler- embryos. These studies cast doubt on the
ance since desiccation-sensitive tissues are role of oligosaccharides in the acquisition
capable of forming cytoplasmic glasses of tolerance and the maintenance of viabil-
(Sun et al., 1994; Buitink et al., 1996). ity in the dried state.
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 28

28 P. Alpert and M.J. Oliver

1.6.3. Repair In the desiccation-tolerant fern

Polypodium virginianum rehydrating tis-
The repair processes associated with des- sues do accumulate novel transcripts but
iccation tolerance have been difficult to their identities have not been investigated
detail and characterize. In seeds, repair (Reynolds and Bewley, 1993b). Recent
mechanisms are difficult to separate from work with the desiccation-tolerant grass
events that are associated with germina- Sporobolus stapfianus has identified two
tion and early seedling growth, but evi- transcripts that accumulate during the
dence for repair does exist. In vegetative early phases of dehydration but also accu-
angiosperms, the major emphasis appears mulate during rehydration. The first of
to be on effective cellular protection and these is a transcript coding for a plant
much of the research in angiosperms has Rab2, a small GTP-binding protein that in
focused on this component. The most other systems is an important protein in
promising models for investigating a direct the targeting of membrane vesicles in
role for cellular repair in desiccation toler- vesicular trafficking pathways and a path-
ance appear to be the highly desiccation- way directly involved in membrane con-
tolerant bryophytes. struction (O’Mahony and Oliver, 1999a).
In seeds, most of the evidence for cellu- The second transcript encoded a polyubi-
lar repair derives from investigations into quitin, a protein involved in protein
the causal relationship between cellular turnover (O’Mahony and Oliver, 1999b). In
damage and loss of viability during storage C. plantagineum very few transcripts were
(Bewley and Black, 1994). Consequently, identified as being specific for the rehydra-
one has to keep in mind that the repair tion process. Those that were appeared to
processes that have been identified may be involved in the metabolism of sugars
not play a major role in desiccation toler- that is required to re-establish the pools of
ance per se but rather in the ability to sur- octulose required for the generation of
vive long term in the dried state. There are sucrose during dehydration (Bernacchia et
two reports, however, that indicate the al., 1996).
repair of cellular components, proteins and Cellular repair, as a component of desic-
DNA, which may directly affect desicca- cation tolerance mechanisms, is more easily
tion tolerance as well as storage longevity. defined in desiccation-tolerant bryophytes.
Mudgett et al. (1997) demonstrated that Desiccation-tolerant bryophytes are thought
proteins containing abnormal L-isoaspartyl to employ a mechanism for desiccation tol-
residues could be repaired in aged barley erance that represents the most primitive
seeds by the activity of the enzyme L-isoas- form expressed in land plants (Oliver et al.,
partyl methyltransferase. These authors 2000). Unlike the acquisition of desiccation
argue that this type of repair is particularly tolerance in seeds, which may be develop-
important during dehydration where pro- mentally programmed, and in desiccation-
tein turnover rates are slow. Boubriak et al. tolerant angiosperms, which is
(1997) demonstrated that one of the earliest environmentally induced by drying, desic-
activities seen in imbibing cereal grains is cation tolerance in most bryophytes
the repair of damage to genomic DNA appears to be constitutive (Oliver and
incurred whilst the seeds were dry and in Bewley, 1997). The difference in mecha-
storage. If the repair processes were nisms of tolerance in these systems is
blocked during imbibition then DNA reflected in their biology. Desiccation-toler-
degradation became severe. If universal, ant angiosperms have morphological and
DNA repair would certainly qualify as a physiological adaptations in place that can
key process in the mechanism of desicca- retard the loss of water. The mechanism of
tion tolerance in seeds (Chapter 12). desiccation tolerance that has evolved in
The identification of repair processes in these plants takes advantage of these adap-
vegetative tissues of desiccation-tolerant tations by being inducible. As the rate of
tracheophytes has received little attention. water loss is relatively slow, there is time
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 29

Drying Without Dying 29

to establish the protective measures bolism when rehydrated. During the first 2
required, and the plant can thus survive a h following rehydration of dried T. ruralis,
drying event. If water loss is too rapid, the synthesis of 25 proteins (termed
these plants succumb to the damaging hydrins) is terminated or substantially
effect of water loss and die (Gaff, 1989; decreased, and the synthesis of 74 proteins
Bewley and Oliver, 1992; Oliver and (termed rehydrins) is initiated or substan-
Bewley, 1997). Bryophytes, on the other tially increased (Oliver, 1991). Controls
hand, have little in the way of adaptations over changes in synthesis of these two
to retain water within the plant and, as a groups of proteins are not mechanistically
result, the internal water content of these linked. It takes a certain amount of prior
plants rapidly equilibrates to the water water loss to fully activate the synthesis of
potential of the environment (Proctor et al., rehydrins upon rehydration. Perhaps this
1998). A consequence of this is that many is a strategy that has evolved to link the
bryophytes experience drying rates that are amount of energy expended in repair to
extreme and therefore have insufficient the amount of damage potentiated by dif-
time to induce and set in place protective fering degrees of drying.
measures. Thus, it appears that bryophytes In T. ruralis, there also appears the
have evolved a constitutive mechanism for capability of preparing the cell for a rapid
desiccation tolerance, one that has protec- recovery if drying rates are sufficiently
tive measures that are always in place. This slow (4–6 h). Using cDNA clones corre-
conclusion is supported by the observa- sponding to T. ruralis transcripts that are
tions that both sucrose and LEA proteins preferentially translated during rehydra-
are maintained at constant levels in desic- tion (Scott and Oliver, 1994), it was deter-
cation-tolerant bryophytes during drying mined that several ‘recovery’ transcripts
and rehydration (see above). The constitu- accumulate during slow drying (Oliver
tive protection mechanism appears to be and Wood, 1997; Wood and Oliver, 1999).
particularly effective in preventing damage Recent studies clearly demonstrate that
to the photosynthetic apparatus, as evi- these transcripts are sequestered in the
denced by the very rapid recovery of pho- dried gametophytes in messenger ribo-
tosystem II activity (Tuba et al., 1996b; nucleoprotein (mRNP) particles (Wood
Csintalan et al., 1999; Proctor and and Oliver, 1999). Of 18 rehydrin cDNAs
Smirnoff, 2001). isolated (Scott and Oliver, 1994) and
How does this relate to cellular repair sequenced (Oliver et al., 1997; Wood et
and the uniqueness of bryophytes for al., 1999) in T. ruralis, only three exhibit
studying this aspect of desiccation toler- significant sequence homology to known
ance? It appears that the level of protec- genes in the Genbank databases. Tr155 has
tion that bryophytes are capable of a strong sequence similarity to an alkyl
maintaining is not sufficient to completely hydroperoxidase linked to seed dormancy
prevent damage, especially to membranes, in barley (Aalen et al., 1994) and
during rehydration. To achieve desicca- Arabidopsis embryos (Haslekas et al.,
tion tolerance, bryophytes thus rely heav- 1998), and in rehydrated but dormant
ily on repair mechanisms induced during Bromus secalinas seeds (Goldmark et al.,
the initial phases of hydration following 1992). Tr213 exhibits a high degree of sim-
rewetting (Oliver and Bewley, 1997; ilarity to polyubiquitins from several
Oliver et al., 1998). plant sources, suggesting that protein
Most work on repair in mosses has cen- turnover may be an important part of
tred on the proteins whose synthesis is repair in mosses as well as in
induced immediately upon rehydration of angiosperms. Tr288 encodes an LEA-like
desiccated gametophytic tissue. Early protein (see above), which suggests that
work (see Bewley, 1979, for review) estab- LEA proteins may have a protective role
lished the ability of T. ruralis and other during rehydration and a role in cellular
mosses to rapidly recover synthetic meta- repair (Velten and Oliver, 2001).
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 30

30 P. Alpert and M.J. Oliver

1.7. Future Prospects and Agricultural tolerance and thus the genetic information
Significance necessary for expanding their drought tol-
erance may not be exploitable or indeed
Our present agricultural system is almost present. In contrast, more may be gained by
totally dependent upon the ability of ortho- understanding how stress-tolerant plants or
dox seeds to tolerate desiccation. The iden- plant structures accomplish tolerance, and
tification and functional analysis of genes from such sources genes that contribute
involved in the developmentally pro- directly to tolerance can be identified. As
grammed desiccation tolerance stage of pointed out by Bartels and Nelson (1994),
seeds is a vital step in understanding this the limiting factor for the improvement of
complex trait. The knowledge gained from abiotic stress tolerance in crops is ‘the
such studies will impact on many diverse availability of structural genes and regula-
areas of agricultural concerns such as tory elements which positively contribute
germplasm preservation, seed production to stress tolerance improvement’. If our
and seedling establishment. Apart from efforts to utilize our understanding of the
genetic considerations, the ongoing studies underlying mechanisms of desiccation tol-
on the role of biological glasses in desicca- erance are to bear fruit, we must discover
tion tolerance and the determination of and identify the genes that are central to
which proteins and sugars are important in this trait.
their stability will affect our ability to pre- Cushman and Bohnert (2000) describe a
serve viable germplasm for longer periods strategy for cataloguing genes central to
of time, preserving genetic diversity for particular traits in their discussion on
future breeding needs. This goal will also genomic approaches to plant stress. The
be benefited by our continued progress in initial phase of gene discovery is the large-
understanding of how desiccation-tolerant scale sequencing of randomly selected
tissues deal with oxidative stress. cDNA clones, termed Expressed Sequence
Over 35% of the world’s land surface is Tags (ESTs), which are synthesized from
considered to be arid or semiarid, experi- mRNA pools representing a specific devel-
encing precipitation that is inadequate for opmental stage or response state. EST col-
most agricultural uses. Ramanathan (1988) lections that will enhance gene discovery
has argued, based on predictions of global in desiccation tolerance have been started,
environmental changes, that developing the most extensive of which is that for
crops that are more tolerant to water developing seeds of Arabidopsis thaliana
deficits while maintaining productivity (Girke et al., 2000). Smaller EST collec-
will become a critical requirement in the tions have been made for C. plantagineum
early part of this century. Understanding leaves that had been dried for an hour or
how plant cells tolerate water loss is a vital fully dried (Bockel et al., 1998) and S. stap-
prerequisite for developing strategies that fianus leaves during dehydration
can influence agricultural and horticultural (Blomstedt et al., 1998; Neale et al., 2000).
crop productivity and survival under these Wood et al. (1999) have established a lim-
conditions of decreasing water availability. ited sample of ESTs (152) from a cDNA
Much has been accomplished in the dis- library developed from the mRNP fraction
covery and characterization of those genes of slowly-dried T. ruralis gametophytes. In
that are expressed during the response of the EST collections from vegetative desic-
crop and model plants to water deficit or cation-tolerant plant tissues, many of the
salt stress. From this work, our knowledge sequenced clones were of unknown iden-
of stress tolerance has improved tity (71% for Tortula) and/or not previ-
immensely and some success has been ously associated with water stress.
achieved, but these traits are very complex Cushman and Bohnert (2000) suggested
and breeding progress has been slow. This that this may indicate, as suggested above,
approach is also restricted in that most that these plants may possess ‘unique gene
crops have a limited capacity for drought complements or regulatory processes that
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 31

Drying Without Dying 31

contribute to desiccation stress’ and, by The next step in the search for insight
inference, novel genes that may prove use- into gene function and regulatory controls
ful in breeding for drought stress tolerance. in desiccation tolerance will come from the
The cataloguing of gene products that are use of expression profiling with cDNA
expressed during the acquisition and estab- microarrays. Such work is under way but
lishment of desiccation tolerance is only the little has been reported as yet. The first
first step. The ultimate goal is to determine information is likely to come from the
which genes are central to desiccation toler- analysis of Arabidopsis EST microarrays in
ance and what functions they perform. The extensions of the studies reported by Girke
major approach used to gain a functional et al. (2000), which may pinpoint tran-
understanding of individual gene products scripts to the exact time of the acquisition
has been the overexpression of genes in of desiccation tolerance in developing
transgenic plants. We have already men- seeds. Direct approaches to elucidate func-
tioned the studies of Xu et al. (1996) and tionality of individual genes or gene fami-
Sivamani et al. (2000) concerning the over- lies will be slower to develop. The vast
expression of HVA1, a Group 3 lea gene, and array of genetic tools, such as transgenic
their success in improving drought toler- capability, mutant generation and screen-
ance in rice and wheat. Two other groups ing tools, T-DNA and transposon-tagged
have attempted to modify sugar metabolism knockouts, and map-based cloning tech-
to improve tolerance by engineering tre- nologies, will make Arabidopsis and seed
halose 6-phosphate synthetase genes from desiccation tolerance the initial foci of
non-plant sources, from yeast (Holmström et functional studies. Nevertheless, tools are
al., 1996) and from bacteria (Pilon-Smits et becoming available for vegetative desicca-
al., 1998) into tobacco. The aim was to pro- tion-tolerant model plants and these will
mote trehalose accumulation in leaf cells, play an important role in evaluating target
and both groups were successful and genes. An example of this has been the
achieved greater tolerance to water deficits exciting use of activation tagging, by trans-
in tobacco. The exciting conclusion from genic random insertion of a highly active
these studies, and those with the Group 3 foreign promoter to ‘activate’ native genes,
LEA protein, is that the engineering of a sin- by Furini et al. (1997) to isolate a gene
gle gene can achieve results that affect such (cDT-1) involved in regulation of the
a complex trait as drought tolerance. It will response of C. plantagineum callus to des-
be interesting to see how these results will iccation. Advances in the fields of molecu-
translate into an advancement in our under- lar biology, genetics, genomics and
standing of how these gene products func- biophysics have put us on the threshold of
tion to achieve greater tolerance and if these a new era in our quest to understand one of
plants will have an impact on drought- the most complex and important traits in
tolerance breeding efforts. plant biology: desiccation tolerance.

1.8. References

Aalen, R.B., Opsahl-Ferstad, H.G., Linnestad, C. and Olsen, O.A. (1994) Transcripts encoding an
oleosin and a dormancy-related protein are present in both the aleurone layer and in the embryo
of developing barley (Hordeum vulgare L.). The Plant Journal 5, 385–396.
Alamillo, J., Roncarati, R., Heino, P., Velasco, R., Nelson, D., Elster, R., Brenacchia, G., Furini, A.,
Schwall, G., Salamini, F. and Bartels, D. (1995) Molecular analysis of desiccation tolerance in
barley embryos and in the resurrection plant Craterostigma plantagineum. Agronomie 2,
Alpert, P. (1979) Desiccation of desert mosses following a summer rainstorm. Bryologist 82, 65–71.
Alpert, P. (1985) Distribution quantified by microtopography in an assemblage of saxicolous mosses.
Vegetatio 64, 131–139.
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 32

32 P. Alpert and M.J. Oliver

Alpert, P. (1990) Microtopography as habitat structure for mosses on rocks. In: McCoy, E., Bell, S.A.
and Mushinsky, H. (eds) Habitat Structure: the Physical Arrangement of Objects in Space.
Chapman & Hall, London, pp. 120–140.
Alpert, P. (2000) The discovery, scope, and puzzle of desiccation tolerance in plants. Plant Ecology
151, 5–17.
Alpert, P. and Oechel, W.C. (1985) Carbon balance limits the distribution of Grimmia laevigata, a des-
iccation-tolerant plant. Ecology 66, 660–669.
Alpert, P. and Oechel, W.C. (1987) Comparative patterns of net photosynthesis in an assemblage of
mosses with contrasting microdistributions. American Journal of Botany 74, 1787–1796.
Arscott, D.B., Bowden, W.B. and Finlay, J.C. (2000) Effects of desiccation and temperature/irradiance
on the metabolism of 2 arctic stream bryophyte taxa. Journal of the North American
Benthological Society 19, 263–273.
Badacsonyi, A., Bates, J.W. and Tuba, Z. (2000) Effect of desiccation on phosphorus and potassium
acquisition by a desiccation-tolerant moss and lichen. Annals of Botany 86, 621–627.
Baker, J., Steele, C. and Dure, L., III (1988) Sequence and characterization of 6 LEA proteins and their
genes from cotton. Planta 175, 485–492.
Bartels, D. (1999) Late embryogenesis abundant (LEA) proteins: expression and regulation in the resur-
rection 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. and Nelson, D. (1994) Approaches to improve stress tolerance using molecular genetics.
Plant, Cell and Environment 17, 659–667.
Bartels, D., Singh, M. and Salamini, F. (1988) Onset of desiccation-tolerance during development of
the barley embryo. Planta 175, 485–492.
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 plant
Craterostigma plantagineum. Planta 181, 27–34.
Bartels, D., Alexander, R., Schneider, K., Elster, R., Velasco, R., Alamillo, J., Bianchi, G., Nelson, D.
and Salamini, F. (1993) Desiccation-related gene products analyzed in a resurrection plant and
in barley embryos. In: Close, T.J. and Bray, E.A. (eds) Plant Responses to Cellular Dehydration
during Environmental Stress. Current Topics in Plant Physiology Series Vol. 10, American
Society of Plant Physiologists, Rockville, USA, pp. 119–127.
Barthlott, W. and Porembski, S. (1996) Ecology and morphology of Blossfeldia liliputana (Cactaceae):
a poikilohydric and almost astomate succulent. Botanica Acta 109, 161–166.
Bates, J.W. (1997) Effects of intermittent desiccation on nutrient economy and growth of two ecologi-
cally contrasted mosses. Annals of Botany 79, 299–309.
Beckett, R.P., Csintalan, Z. and Tuba, Z. (2000) ABA treatment increases both the desiccation toler-
ance of photosynthesis and non-photochemical quenching in the moss Atrichum undulatum.
Plant Ecology 151, 65–71.
Becquerel, P. (1951) La suspension de la vie des spores des algues, lichens, et mousses aux confins
du zéro absolue et rôle de la synérèse réversible pour leur survie au dégel expliquant l’existence
de la flore polaire et des hautes altitudes. Comptes Rendues de l’Académie des Sciences de Paris
232, 22–25.
Bernacchia, G., Salamini, F. and Bartels, D. (1996) Molecular characterization of the rehydration
process in the resurrection plants Craterostigma plantagineum. Plant Physiology 111, 1043–1050.
Bernal-Lugo, I. and Leopold, A.C. (1995) Seed stability during storage: raffinose content and seed
glassy state. Seed Science Research 5, 75–80.
Bertsch, A. (1966) Uber den CO2-Gaswechsel einiger Flechten nach Wasserdampfaufnahme. Planta
70, 157–166.
Bertsch, A. (1970) CO2-Gaswechsel und Wasseraushalt der aerophilen Grünalge Apatococcus
lobotus. Planta 70, 46–72.
Bewley, J.D. (1979) Physiological aspects of desiccation-tolerance. Annual Review of Plant
Physiology 30, 195–238.
Bewley, J.D. and Black, M. (1994) Seeds. Physiology of Development and Germination, 2nd edn.
Plenum Press, New York.
Bewley, J.D. and Krochko, J.E. (1982) Desiccation-tolerance. In: Lange, O.L., Nobel, P.S., Osmond,
C.B. and Ziegler, H. (eds) Encyclopedia of Plant Physiology, Vol 12B, Physiological Ecology II.
Springer-Verlag, Berlin, pp. 325–378.
01 Desiccation -Chap 1 4/4/02 2:16 pm Page 33

Drying Without Dying 33

Bewley, J.D. and Oliver, M.J. (1992) Desiccation-tolerance in vegetative plant tissues and seeds: protein
synthesis in relation to desiccation and a potential role for protection and repair mechanisms. In:
Osmond, C.B. and Somero, G. (eds) Water and Life: a Comparative Analysis of Water Relationships
at the Organismic, Cellular and Molecular Levels. Springer-Verlag, Berlin, pp. 141–160.
Bewley, J.D. and Pacey, J. (1978) Desiccation-induced ultrastructural changes in drought-sensitive
and drought-tolerant plants. In: Crowe, J.H. and Clegg, J.S. (eds) Dry Biological Systems.
Academic Press, London, pp. 53–73.
Bewley, J.D., Halmer, P., Krochko, J.E. and Winner W.E. (1978) Metabolism of a drought-tolerant and
a 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.
Bewley, J.D., Reynolds, T.L. and Oliver, M.J. (1993) Evolving strategies in the adaptation to desicca-
tion. In: Close, T.J. and Bray, E.A. (eds) Plant Responses to Cellular Dehydration During
Environmental Stress. Current Topics in Plant Physiology Series, Vol. 10. American Society of
Plant Physiologists, Rockville, USA, pp. 193–201.
Bianchi, G., Gamba, A., Murelli, C., Salamini, F. and Bartels, D. (1991) Novel carbohydrate metabo-
lism in the resurrection plant Craterostigma plantagineum. The Plant Journal 1, 355–359.
Bisby, G.R. (1945) Longevity of Schizophyllum commune. Nature 155, 732–733.
Bjork, M., Uku, J., Weil, A. and Beer, S. (1999) Photosynthetic tolerances to desiccation of tropical
intertidal seagrasses. Marine Ecology–Progress Series 191, 121–126.
Black, M., Corbineau, F., Gee, H. and Côme, D. (1999) Water content, raffinose, and dehydrins in the
induction of desiccation tolerance in immature wheat embryos. Plant Physiology 120, 463–471.
Blackman, S.A., Obendorf, R.L. and Leopold, A.C. (1995) Maturation proteins and sugars in desicca-
tion tolerance of developing soybean seeds. Plant Physiology 100, 225–230.
Bliss, R.D., Platt-Aloia, K.A. and Thompson, W.W. (1984) Changes in plasmalemma organization in
cowpea radicle during imbibition in water and NaCl solutions. Plant Cell and Environment 7,
Blomstedt, C.K., Neale, A.D., Gianello, R.D., Hamill, J.D. and Gaff, D.F. (1998) Isolation and charac-
terization of cDNAs associated with the onset of desiccation tolerance in the resurrection grass,
Sporobolus stapfianus. Plant Growth Regulation 24, 219–228.
Bochicchio, A., Vazzana, C., Velasco, R., Singh, M. and Bartels, D. (1991) Exogenous ABA induces
desiccation tolerance and leads to the synthesis of specific gene transcripts in immature
embryos of maize. Maydica 36, 11–16.
Bochicchio, A., Vazzana, C., Puliga, S., Alberti, L., Singanelli, S. and Vernieri, P. (1998) Moisture con-
tent of the dried leaf is critical to desiccation tolerance in detached leaves of the resurrection
plant Boea hygroscopica. Plant Growth Regulation 24, 163–170.
Bockel, C., Salamini, F. and Bartels, D. (1998) Isolation and characterization of genes expressed dur-
ing early events of the dehydration process in the resurrection plant Craterostigma plan-
tagineum. Journal of Plant Physiology 152, 158–166.
Boubriak, L., Kargiolaki, H., Lyne, L. and Osborne, D.J. (1997) The requirement for DNA repair in
desiccation tolerance of germinating embryos. Seed Science Research 7, 97–105.
Bray, E.A. (1997) Plant responses to water deficit. Trends in Plant Science 2, 48–54.
Breuil-Sée, A. (1993) Recorded desiccation-survival times in bryophytes. Journal of Bryology 17,
Broca, P. (1860) Rapport sur la question soumise à la Société de Biologie au sujet de la reviviscence
des animaux desséchés. Mémoires de la Société de Biologie, Paris 2, 1–140.
Buitink, J. (2000) Biological glasses: nature’s way to preserve life. PhD thesis, Wageningen
Agricultural University, The Netherlands.
Buitink, J., Walters-Vertucci, C., Hoekstra, F.A. and Leprince, O. (1996) Calorimetric properties of
dehydrating pollen: analysis of a desiccation-tolerant and an intolerant species. Plant Physiology
111, 235–242.
Buitink, J., Claessens, M.M.A.E., Hemmings, M.A. and Hoekstra, F.A. (1998) Influence of water con-
tent and temperature on molecular mobility and intracellular glasses in seeds and pollen. Plant
Physiology 118, 531–541.
Buitink, J., Hemmings, M.A. and Hoekstra, F.A. (2000) Is there a role for oligosacharrides in seed
longevity? An assessment of intracellular glass stability. Plant Physiology 122, 1217–1224.
Burke, M.J. (1986) The glassy state and survival of anhydrous biological systems. In: Leopold, A.C.
(ed.) Membranes, Metabolism and Dry Organisms. Cornell University Press, Ithaca, New York,
pp. 358–363.
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 34

34 P. Alpert and M.J. Oliver

Calatayud, A., Deltoro, V.I., Barreno, E. and del Valle-Tascón, S. (1997) Changes in in vitro chloro-
phyll fluorescence quenching in lichen thalli as a function of water content and suggestion of
zeaxanthin-associated photoprotection. Physiologia Plantarum 101, 93–102.
Campalans, A., Messeguer, R., Goday, A. and Pages, M. (1999) Plant responses to drought, from ABA
signal transduction events to the action of the induced proteins. Plant Physiology and
Biochemistry 37, 327–340.
Chandler, J. and Bartels, D. (1999) Plant desiccation. In: Lerner, H.R. (ed.) Plant Responses to Animal
Stress – from Phytohormones to Genome Organization. Marcel Dekker, New York, pp. 575–590.
Clegg, J.S. (1973) Do dried cryptobiotes have a metabolism? In: Crowe, J.H. and Clegg, J.S. (eds)
Anhydrobiosis. Dowden, Hutchinson & Ross, Stroudsburg, Pennsylvania, pp. 141–146.
Clegg, J.S. (1986) The physical properties and metabolic status of Artemia cysts at low water con-
tents: the ‘water replacement hypothesis’. In: Leopold, A.C. (ed.) Membranes, Metabolism, and
Dry Organisms. Cornell University Press, Ithaca, New York, pp. 169–187.
Clegg, J.S. (2001) Cryptobiosis – a peculiar state of biological organization. Comparative
Biochemistry and Physiology 128, 613–624.
Close, T. (1997) A commonality in the response of plants to dehydration and low temperature.
Physiologia Plantarum 100, 291–296.
Crowe, J.H., Hoekstra, F.A. and Crowe, L.M. (1989) Membrane phase transitions are responsible for
imbibitional damage in dry pollen. Proceedings of the National Academy of Sciences USA 86,
Crowe, J.H., Hoekstra, F.A. and Crowe, L.M. (1992) Anhydrobiosis. Annual Review of Physiology 54,
Crowe, J.H., Clegg, J.S. and Crowe, L.M. (1998a) Anhydrobiosis: the water replacement hypothesis.
In: Ried, D.S. (ed.) The Properties of Water in Foods. Chapman & Hall, New York, pp. 440–455.
Crowe, J.H., Hoekstra, F.A. and Crowe, L.M. (1998b) The role of vitrification in anhydrobiosis.
Annual Reviews of Physiology 60, 73–103.
Csintalan, Z., Proctor, M.C.F. and Tuba, Z. (1999) Chlorophyll fluorescence during drying and rehy-
dration in the mosses Rhytidiadelphus loreus (Hedw.) Warnst., Anomodon viticulosus (Hedw.)
Hook. & Tayl. and Grimmia pulvinata (Hedw.) Sm. Annals of Botany 84, 235–244.
Csintalan, Z., Takács, Z., Proctor, M.C.F., Nagy, Z. and Tuba, Z. (2000) Early morning photosynthesis
of the moss Tortula ruralis following summer dew fall in a Hungarian temperate dry grassland.
Plant Ecology 151, 51–54.
Cuming, A.C. (1999) LEA proteins in seed proteins. In: Shewry, P.R. and Casey, R. (eds) Seed
Proteins. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 753–780.
Cuming, A.C. and Lane, B.G. (1979) Protein synthesis in imbibing wheat embryos. European Journal
of Biochemistry 99, 217–224.
Cushman, J.C. and Bohnert, H.J. (2000) Genomic approaches to plant stress tolerance. Current
Opinions in Plant Biology 3, 117–124.
Davey, M.C. (1997) Effects of continuous and repeated dehydration on carbon fixation by bryophytes
from the maritime Antarctic. Oecologia 110, 25–31.
Davis, J.S. (1972) Survival records in the algae, and the survival role of certain algal pigments, fat and
mucilaginous substances. Biologist 54, 52–93.
Deltoro, V.I., Calatayud, A., Gimeno, C., Abadia, A. and Barreno, E. (1998) Changes in chlorophyll a
fluorescence, photosynthetic CO2 assimilation and xanthophyll cycle interconversions during
dehydration in desiccation-tolerant and intolerant bryophytes. Planta 207, 224–228.
Dilks, T.J.K. and Proctor, M.C.F. (1976) Seasonal variation in desiccation tolerance in some British
bryophytes. Journal of Bryology 9, 239–247.
Dodds, W.K., Gudder, D.A. and Mollenbauer, D. (1995) The ecology of Nostoc. Journal of Phycology
31, 2–18.
Doyère, P. (1842) Sur la faculté qui possèdent les tardigrades, les rotifers, les anguillules des toits, et
quelques autres animalcules, de revenir à la vie après avoir été complètement desséchées.
Annales de Science Naturelle Partie Zoologique 18, 5–35.
Drazic, G., Mihailovic, N. and Stevanovic, B. (1999) Chlorophyll metabolism in leaves of the higher
poikilohydric plants Ramonda serbica Panc. and R. nathaliae Panc. & Petrov. during dehydra-
tion and rehydration. Journal of Plant Physiology 154, 379–384.
Dure, L. III (1993a) A repeating 11-mer amino acid motif and plant desiccation. The Plant Journal 3,
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 35

Drying Without Dying 35

Dure, L. III (1993b) Structural motifs in Lea proteins. In: Close, T.J. and Bray, E.A. (eds) Plant Responses
to Cellular Dehydration During Environmental Stress. Current Topics in Plant Physiology Series
Vol. 10, American Society of Plant Physiologists, Rockville, Maryland, pp. 91–103.
Dure, L. III, Crouch, M., Harada, J., Ho, T.-H.D., Mundy, J., Quatrano, R., Thomas, T. and Sung, Z.R.
(1989) Common amino sequence domains among LEA proteins of higher plants. Plant Molecular
Biology 12, 475–486.
Eickmeier, W.G. (1980) Photosynthetic recovery of resurrection spike mosses from different hydra-
tion regimes. Oecologia 46, 380–385.
Eickmeier, W.G. (1983) Photosynthetic recovery of the resurrection plant Selaginella lepidophylla:
effects of prior desiccation rate and mechanisms of desiccation damage. Oecologia 58, 115–120.
Eickmeier, W.G. (1986) The correlation between high temperature and desiccation tolerance in a
poikilohydric desert plant. Canadian Journal of Botany 64, 611–617.
Eickmeier, W.G., Casper, C. and Osmond, C.B. (1993) Chlorophyll fluorescence in the resurrection
plant Selaginella lepidophylla (Hook & Grev.) Spring during high-light and desiccation stress,
and evidence for zeaxanthin-associated photoprotection. Planta 189, 30–38.
Evans, J.H. (1959) Survival of freshwater algae during dry periods. Part II. Drying experiments. Part
III. Stratification of algae in pond margin litter and mud. Journal of Ecology 47, 55–81.
Farrant, J.M. (2000) A comparison of mechanisms of desiccation tolerance among three angiosperm
resurrection plant species. Plant Ecology 151, 29–39.
Farrant, J.M., Cooper, K., Kruger, L.A. and Sherwin, H.W. (1999) The effect of drying rate on the sur-
vival of three desiccation-tolerant angiosperm species. Annals of Botany 84, 371–379.
Franks, A.J. and Bergstrom, D.M. (2000) Corticolous bryophytes in microphyll fern forests of south-east
Queensland: distribution on Antarctic beech (Nothofagus moorei). Australian Ecology 25, 386–393.
Friedmann, E.I. and Galun, M. (1974) Desert algae, lichens, and fungi. In: Brown, G.W. Jr (ed.) Desert
Biology. Academic Press, New York, pp. 165–212.
Furini, A., Koncz, C., Salamini, F. and Bartels, D. (1997) High level transcription of a member of a
repeated gene family confers dehydration tolerance to callus tissue of Craterostigma plan-
tagineum. EMBO Journal 16, 3599–3608.
Gaff, D.F. (1977) Desiccation tolerant vascular plants of southern Africa. Oecologia 31, 95–109.
Gaff, D.F. (1980) Protoplasmic tolerance of extreme stress. In: Turner, N.C. and Kramer, P.J. (eds)
Adaptation of Plants to Water and High Temperature Stress. John Wiley & Sons, New York,
pp. 207–230.
Gaff, D.F. (1981) The biology of resurrection plants. In: Pate, J.S. and McComb, A.J. (eds) The Biology
of Australian Plants. University of Western Australia Press, Nedlands, pp. 114–146.
Gaff, D.F. (1986) Desiccation tolerant ‘resurrection’ grasses from Kenya and West Africa. Oecologia
70, 118–120.
Gaff, D.F. (1987) Desiccation tolerant plants in South America. Oecologia 74, 133–136.
Gaff, D.F. (1989) Responses of desiccation tolerant ‘resurrection’ plants to water stress. In: Kreeb,
K.H., Richter, H. and Hinckley, T.M. (eds) Structural and Functional Responses to
Environmental Stresses. SPB Academic Publishing, The Hague, The Netherlands, pp. 255–268.
Gaff, D.F. (1997) Mechanisms of desiccation tolerance in resurrection vascular plants. In: Basra, A.S.
and Basra, R.K. (eds) Mechanisms of Environmental Stress Resistance in Plants. Harwood
Academic Publishers, London, pp. 43–58.
Gaff, D.F. and Churchill, D.M. (1976) Borya nitida Labill. – an Australian species in the Liliaceae
with desiccation-tolerant leaves. Australian Journal of Botany 24, 209–224.
Gaff, D.F. and Giess, W. (1986) Drought resistance in water plants in rock pools of southern Africa.
Dinteria 18, 17–37.
Gaff, D. and Hallam, N.D. (1974) Resurrecting desiccated plants. In: Bieleski, R.L., Ferguson, A.R.
and Cresswell, M.M. (eds) Mechanisms of Regulation of Plant Growth. Royal Society of New
Zealand Bulletin 12, Wellington, pp. 389–393.
Gaff, D.F. and Latz, P.K. (1978) The occurrence of resurrection plants in the Australian flora.
Australian Journal of Botany 26, 485–492.
Gaff, D.F. and Sutaryono, Y.A. (1991) Grasses with complete desiccation tolerance. In: Proceedings of
IVth International Rangeland Congress. French Grasslands Society, Montpellier, France,
pp. 266–267.
Gaff, D., Bartels, S.-Y. and O’Brien, T.P. (1976) The fine structure of dehydrated and reviving leaves of
Borya nitida Labill. – a desiccation-tolerant plant. Australian Journal of Botany 24, 225–236.
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 36

36 P. Alpert and M.J. Oliver

Galau, G.A. and Hughes, D.W. (1987) Coordinate accumulation of homologous transcripts of seven cot-
ton lea gene families during embryogenesis and germination. Developmental Biology 123, 213–221.
Galau, G.A., Bijaisoradat, N. and Hughes, D.W. (1987) Accumulation kinetics of cotton late embryo-
genesis-abundant mRNAs: coordinate regulation during embryogenesis and the role of abscisic
acid. Developmental Biology 123, 198–212.
Galau, G.A., Jakobsen, K.S. and Hughes, D.W. (1991) The controls of late dicot embryogenesis and
early germination. Physiologia Plantarum 81, 280–288.
Galau, G.A., Wang, H.Y.C. and Hughes, D.W. (1993) Cotton Lea5 and Lea14 encode atypical late
embryogenesis-abundant proteins. Plant Physiology 101, 695–696.
Gauslaa, Y. and Solhaug, K.A. (1996) Differences in the susceptibility to light stress between epi-
phytic lichens of ancient and young boreal forest stands. Functional Ecology 10, 344–354.
Gildner, B.S. and Larson, D.W. (1992) Seasonal changes in photosynthesis in the desiccation-tolerant
fern Polypodium virginianum. Oecologia 89, 383–389.
Gimingham, C.H. and Smith, R.I.L. (1971) Growth form and water relations of mosses in the mar-
itime Antarctic. British Antarctic Survey Bulletin 25, 1–21.
Girke, T., Todd, J., Ruuska, S., White, J., Benning, C. and Ohlrogge, J. (2000) Microarray analysis of
developing Arabidopsis seeds. Plant Physiology 124, 1570–1581.
Glime, J.M. and Carr, R.E. (1974) Temperature survival of Fontinalis novae-angliae. Bryologist 77, 17–22.
Goldmark, P.J., Curry, J., Morris, C.F. and Walker-Simmons, M.K. (1992) Cloning and expression of an
embryo-specific mRNA up-regulated in hydrated dormant seeds. Plant Molecular Biology 19,
Goldsworthy, D.A. and Drennan, P.M. (1991) Anhydrous fixation of desiccated leaves of
Myrothamnus flabellifolius Welw. Electron Microscopy Society of South Africa 21, 105–106.
Golovina, E.A., Hoekstra, F.A. and Hemmings, M.A. (1998) Drying increases intracellular partitioning
of amphiphilic substances into the lipid phase. Plant Physiology 114, 975–986.
Gratani, L., Crescente, M.F. and Rossi, G. (1998) Photosynthetic performance and water use efficiency
of the fern Cheilanthes persica. Photosynthetica 35, 507–516.
Green, T.G.A. and Lange, O.L. (1994) Photosynthesis in poikilohydric plants: a comparison of lichens
and bryophytes. In: Schulze, E.-D. and Caldwell, M.M. (eds) Ecophysiology of Photosynthesis.
Springer-Verlag, New York, pp. 320–341.
Grime, J.P. (1979) Plant Strategies and Vegetation Processes. John Wiley & Sons, Chichester, UK.
Guo, N., Puhlev, I., Brown, D.R., Mansbridge, J. and Levine, F. (2000) Trehalose expression confers
desiccation tolerance on human cells. Nature Biotechnology 18, 168–171.
Hahn, S.C., Tenhunen, J.D., Popp, P.W., Meyer, A. and Lange, O.L. (1993) Upland tundra in the
foothills of the Brooks Range, Alaska: diurnal carbon dioxide exchange patterns of characteristic
lichen species. Flora 188, 125–143.
Hallam, N.D. and Luff, S.E. (1980) Fine structural changes in the mesophyll tissue of the leaves of
Xerophyta villosa during desiccation. Botanical Gazette 141, 173–179.
Hammoudah, M.M., Nir, S., Bentz, J., Mayhew, E., Stewart, T.P., Hui, S.W. and Kurland, R.J. (1981)
Interactions of La3+ with phosphatidylserine vesicles binding, phase transition, leakage, 31P-
NMR and fusion. Biochimica et Biophysica Acta 645, 102–114.
Haslekas, C., Stacy, R.A.P., Nygaard, V., Culianez Macia, F.A. and Aalen, R.B. (1998) The expression
of a peroxiredoxin antioxidant gene, AtPer1, in Arabidopsis thaliana is seed specific and related
to dormancy. Plant Molecular Biology 36, 833–845.
Hernandez-Garcia, C.D., Gonzales-Mancebo, J.M. and Losada-Lim, A. (1999) Water relations of some
mosses growing in pine forests of Tenerife, Canary Islands. Lindbergia 24, 15–22.
Hinton, H.E. (1968) Reversible suspension of metabolism and the origin of life. Proceedings of the
Royal Society of London B 171, 43–57.
Hoekstra, F.A., Crowe, J.H., Crowe, L.M. and van Bilsen, D.G.J.L. (1992) Membrane behavior and
stress tolerance in pollen. In: Ottaviano, E., Mulcahy, D.L., Sari Gorla, M. and Bergamini
Mulcahy, G. (eds) Angiosperm Pollen and Ovules. Springer-Verlag, New York, pp. 177–186.
Hoekstra, F.A., Haigh, A.M., Tetteroo, F.A.A. and van Roekel, T. (1994) Changes in soluble sugars in
relation to desiccation tolerance in cauliflower seeds. Seed Science Research 4, 143–147.
Hoekstra, F.A., Wolkers, W.F., Buitink, J., Golovina, E.A., Crowe, J.H. and Crowe, L.M. (1997) Mem-
brane stabilization in the dry state. Comparative Biochemistry and Biophysics 117A, 335–341.
Hoekstra, F.A., Golvina, E.A., van Elst, A.C. and Hemminga, M.A. (1999) Imbibitional leakage from
anhydrobiotes revisited. Plant, Cell, and Environment 22, 1121–1131.
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 37

Drying Without Dying 37

Holmström, K.O., Mantyla, E., Welin, B., Mandal, A. and Palva, E.T. (1996) Drought tolerance in
tobacco. Science 379, 683–684.
Horbowicz, M. and Obendorf, R.L. (1994) Seed desiccation tolerance and storability: dependence on
flatulence-producing oligosaccharides and cyclitols – review and survey. Seed Science Research
4, 385–405.
Hosokawa, T. and Kubota, H. (1957) On the osmotic pressure and resistance to desiccation of epi-
phytic mosses from a beech forest, south-west Japan. Journal of Ecology 45, 579–591.
Hsu, S.-J. and Hsu, B.-D. (1998) Flow cytometry of Chlorella after dehydration stress. Plant Science
134, 163–169.
Ingram, J. and Bartels, D. (1996) The molecular basis of dehydration tolerance in plants. Annual
Review of Plant Physiology and Plant Molecular Biology 47, 377–403.
Iturriaga, G., Gaff, D.F. and Zentella, R. (2000) New desiccation-tolerant plants, including a grass, in
the central highlands of Mexico accumulate trehalose. Australian Journal of Botany 48,
Jawad, A., Snelling, A.M., Heritage, J. and Hawkey, P.M. (1998) Exceptional desiccation tolerance of
Acinetobacter radioresistens. Journal of Hospital Infection 39, 235–240.
Kappen, L. (1964) Untersuchungen über den Jahreslauf der Frost-, Hitze- und Austrocknungsresistenz
von Sporophyten einheimischer Polypodiaceen (Filicinae). Flora 156, 101–115.
Kappen, L. (1993) Lichens in the Antarctic region. In: Friedmann, E.I. (ed.) Antarctic Microbiology.
Wiley-Liss, New York, pp. 433–490.
Kappen, L. and Valladares, F. (1999) Opportunistic growth and desiccation tolerance: the ecological
success of poikilohydrous autotrophs. In: Pugnaire, F.I. and Valladares, F. (eds) Handbook of
Functional Plant Ecology. Marcel Dekker, New York, pp. 10–80.
Kappen, L., Lange, O.L., Schulze, E.-D., Evenari, M. and Buschbom, U. (1979) Ecophysiological
investigations on lichens of the Negev desert. 6. Annual course of the photosynthetic production
of Ramalina maciformis (Del.) Bory. Flora 168, 85–108.
Kappen, L., Lange, O.L., Schulze, E.-D., Buschbom, U. and Evenari, M. (1980) Ecophysiological
investigations on lichens of the Negev desert. 7. Influence of the habitat exposure on dew imbi-
bition and photosynthetic productivity. Flora 169, 216–229.
Keever, C. (1957) Establishment of Grimmia laevigata on bare granite. Ecology 38, 422–429.
Keilin, D. (1959) The problem of anabiosis or latent life: history and current concept. Proceedings of
the Royal Society of London B 150, 149–191.
Koorneef, M., Hanhart, C.J., Hilhorst, H.W.M. and Karssen, C.M. (1989) In vivo inhibition of seed
development and reserve protein accumulation in recombinants of abscisic acid biosynthesis
and responsiveness mutants in Arabidopsis thaliana. Plant Physiology 90, 463–469.
Kuang, J., Gaff, D.F., Gianello, R.D., Blomstedt, C.K., Neale, A.D. and Hamill, J.D. (1995) Changes in
in vivo protein complements in drying leaves of the desiccation-tolerant grass Sporobolus stapfi-
anus and the desiccation-sensitive grass Sporobolus pyramidalis. Australian Journal of Plant
Physiology 22, 1027–1034.
Kubitzki, K. (1998) Velloziaceae. In: Kubitzki, K. (ed.) The Families and Genera of Vascular Plants.
Springer-Verlag, Berlin, pp. 459–467.
Lange, O.L. (1953) Hitze- und Trockenresistenz der Flechten in Bezeihung zu ihrer Verbreitung. Flora
140, 39–97.
Lange, O.L. (1969) CO2-Gaswechsel von Moosen nach Wasserdampfaufnahme aus dem Luftraum.
Planta 89, 90–94.
Lange, O.L. and Kilian, E. (1985) Reaktiviertung der Photosynthese trockener Flechten durch
Wasserdampfaufnahme aus dem Luftraum: artspezifisch unterschiedliches Verhalten. Flora
192, 1–15.
Lange, O.L., Meyer, A., Zellner, H. and Heber, U. (1994) Photosynthesis and water relations of lichen
soil crusts: field measurements in the coastal fog zone of the Namib Desert. Functional Ecology
8, 253–264.
Lange, O.L., Green, T.G.A., Reichenberger, H. and Ziegler, H. (1996) Photosynthetic depression at
high water contents in lichens: concurrent use of gas exchange and fluorescence techniques with
cyanobacterial and green algal Peltigera species. Botanica Acta 109, 43–50.
Lange, O.L., Belnap, J., Reichenberger, H. and Meyer, A. (1997) Photosynthesis of green algal soil
crust lichens from arid lands in southern Utah, USA: role of water content on light and tempera-
ture responses of CO2 exchange. Flora 192, 1–15.
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 38

38 P. Alpert and M.J. Oliver

Larson, D.W. (1988) The impact of ten years at 20°C on gas exchange in five lichen species.
Oecologia 78, 87–92
Lazarides, M. (1992) Resurrection grasses (Poaceae) in Australia. In: Chapman, G.P. (ed.) Desertified
Grasslands: Their Biology and Management. Academic Press, London, pp. 213–234.
Lebkeucher, J.G. and Eickmeier, W.G. (1993) Physiological benefits of stem curling for resurrection
plants in the field. Ecology 74, 1073–1080.
Leopold, A.C., Sun, W.Q. and Bernal-Lugo, I. (1994) The glassy state in seeds: analysis and function.
Seed Science Research 4, 267–274.
Leprince, O. and Walters-Vertucci, C. (1995) A calorimetric study of glass transition behaviors in
axes of bean with relevance to storage stability. Plant Physiology 109, 1471–1481.
Leprince, O., Hendry, G.A.F. and McKersie, B.D. (1993) The mechanisms of desiccation-tolerance in
developing seeds. Seed Science Research 3, 231–246.
Leuschner, C., Landwehr, S. and Mehlig, U. (1998) Limitation of carbon assimilation of intertidal
Zostera noltii and Z. marina by desiccation at low tide. Aquatic Botany 62, 171–176.
Lovelock, C.E., Jackson, A.E., Melic, D.R. and Seppelt, R.D. (1995) Reversible photoinhibition in
Antarctic moss during freezing and thawing. Plant Physiology 109, 955–961.
Lüttge, U. (1997) Cyanobacterial Tintenstrich communities and their ecology. Naturwissenschaften
84, 526–534.
Mahan, J.R., Oliver, M.J. and Sherman, T.D. (1998) Nitrate reductase activity during desiccation and
rehydration of the desiccation-tolerant moss Tortula ruralis. Environmental and Experimental
Botany 39, 67–76.
Mao, Z.Y., Palva, R., Kriz, A.L. and Juvik, J.A. (1995) Dehydrin gene expression in normal and vivip-
arous embryos of Zea mays during seed development and germination. Plant Physiology and
Biochemistry 33, 649–653.
Markovska, Y.K., Tsonev, T. and Kimenov, G. (1997) Regulation of CAM and respiratory recycling by
water supply in higher poikilohydric plants – Haberlea rhodopensis Friv. and Ramonda serbica
Panc. at transition from biosis to anabiosis and vice versa. Botanica Acta 110, 18–24.
Marschall, M. (1998) Nitrate reductase activity during desiccation and rehydration of the desicca-
tion-tolerant moss Tortula ruralis and the leafy liverwort Porella platyphylla. Journal of
Bryology 20, 273–285.
Mazur, P. (1968) Survival of fungi after freezing and desiccation. In: Ainsworth, G.C. and Sussman,
A.L. (eds) The Fungi. Academic Press, London, pp. 325–394.
McCubbin, W.D., Kay, C.M. and Lane, B.G. (1985) Hydrodynamic and optical properties of the wheat
germ Em protein. Canadian Journal of Biochemistry and Cell Biology 63, 803–811.
Meurs, C., Basra, A.S., Karssen, C.M. and van Loon, L.C. (1992) Role of abscisic acid in the induction
of desiccation tolerance in developing seeds of Arabidopsis thaliana. Plant Physiology 98,
Mitchell, D.N., Davidson, A.W. and Cooke, J.A. (1999) The bryophytes of magnesian limestone sea
cliffs, County Durham: a multivariate analysis of community–environment relationships.
Transactions of the Natural History Society of Northumbria 99, 59.
Montenegro, G., Hoffmann, A.J., Aljaro, M.E. and Hoffman, A.E. (1979) Satureja gillesii: a poikilohydric
shrub from the Chilean Mediterranean vegetation. Canadian Journal of Botany 57, 1206–1213.
Morrison-Baird, L.A., Leopold, A.C., Bramlage, W.J. and Webster, B.D. (1979) Ultrastructural modifi-
cations associated with imbibition of the soybean radicle. Botanical Gazette 140, 316–320.
Mudgett, M.B., Lowensen, J.D. and Clarke, S. (1997) Protein repair L-isoaspartyl methyltransferase in
plants. Phylogenetic distribution and the accumulation of substrate proteins in aged barley
seeds. Plant Physiology 115, 1481–1489.
Muller, J., Sprender, N., Bortlik, K., Boller, T. and Wiemken, A. (1997) Desiccation increases sucrose
levels in Ramonda and Haberlea, two genera of resurrection plants in the Gesneriaceae.
Physiologia Plantarum 100, 153–158.
Muslin, E.H. and Homann, P.H. (1992) Light as a hazard for the desiccation-resistant ‘resurrection’
fern Polypodium polypodioides L. Plant, Cell and Environment 15, 81–89.
Nash, T.H. and Moser, T.J. (1982) Vegetational and physiological patterns of lichens in North
American deserts. Journal of the Hattori Botanical Club 53, 331–336.
Neale, A.D., Blomstedt, C.K., Bronson, P., Le, T.-N., Guthridge, K., Evans, J., Gaff, D.F. and Hamill,
J.D. (2000) The isolation of genes from the resurrection grass Sporobolus stapfianus which are
induced during severe drought stress. Plant, Cell and Environment 23, 265–277.
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 39

Drying Without Dying 39

Nienow, J.A. and Friedmann, E.I. (1993) Terrestrial lithophytic (rock) communities. In: Friedmann,
E.I. (ed.) Antarctic Microbiology. Wiley-Liss, New York, pp. 343–412.
Nobel, P.S. (1978) Microhabitat, water relations, and photosynthesis of a desert fern, Notholaena par-
ryi. Oecologia 31, 293–309.
Norr, M. (1974) Hitzresistenz bei Moosen. Flora 163, 388–397.
Oliver, M.J. (1991) Influence of protoplasmic water loss on the control of protein synthesis in the des-
iccation-tolerant moss Tortula ruralis: ramifications for a repair-based mechanism of desicca-
tion-tolerance. Plant Physiology 97, 1501–1511.
Oliver, M.J. and Bewley J.D. (1984) Desiccation and ultrastructure in bryophytes. Advances in
Bryology 2, 91–131.
Oliver, M.J. and Bewley, J.D. (1997) Desiccation-tolerance of plant tissues: a mechanistic overview.
Horticultural Reviews 18, 171–214.
Oliver, M.J. and Wood, A.J. (1997) Desiccation-tolerance in mosses. In: Koval, T.M. (ed.) Stress
Induced Processes in Higher Eukaryotic Cells. Plenum Press, New York, pp. 1–26.
Oliver, M.J., Wood, A.J. and O’Mahony, P. (1997) How some plants recover from vegetative desicca-
tion: A repair based strategy. Acta Physiologia Plantarum 19, 419–425.
Oliver, M.J., Wood, A.J. and O’Mahony, P. (1998) ‘To dryness and beyond’ – preparation for the dried
state and rehydration in vegetative desiccation-tolerant plants. Plant Growth Regulation 24,
Oliver, M.J., Tuba, Z. and Mishler, B.D. (2000) The evolution of vegetative desiccation tolerance in
land plants. Plant Ecology 151, 85–100.
O’Mahony, P. and Oliver, M.J. (1999a) Characterization of a desiccation responsive small GTP-bind-
ing protein (Rab2) from the desiccation tolerant grass Sporobolus stapfianus. Plant Molecular
Biology 39, 809–821.
O’Mahony, P. and Oliver, M.J. (1999b) The involvement of ubiquitin in vegetative desiccation-toler-
ance. Plant Molecular Biology 41, 657–667.
Öpik, H. (1980) The ultrastructure of coleoptile cells in dry rice (Oryza sativa L.) grains after anhy-
drous fixation with osmium tetroxide vapour. New Phytologist 85, 521–529.
Öpik, H. (1985) The fine structure of some dry seed tissues observed after completely anhydrous
chemical fixation. Annals of Botany 56, 453–466.
Oxford English Dictionary (1989) Oxford English Dictionary, 2nd edn. Oxford University Press,
Oxford, UK.
Pence, V.C. (2000) Cryopreservation of in vitro grown fern gametophytes. American Fern Journal 90,
Pilon-Smits, E.A.H., Terry, N., Sears Tobin, K.H., Zayed, A., Hwang, S., van Dun, K., Voogd, E.,
Verwoerd, T.C., Krutwagen, R.W.H.H. and Goddijn, O.J.M. (1998) Trehalose-producing trans-
genic tobacco plants show improved growth performance under drought stress. Journal of Plant
Physiology 152, 525–532.
Pintado, A., Valladares, F. and Sancho, L.G. (1997) Exploring phenotypic plasticity in the lichen
Ramalina capitata: morphology, water relations and chlorophyll content in north- and south-
facing populations. Annals of Botany 80, 345–353.
Platt, K.A., Oliver, M.J. and Thomson, W.W. (1994) Membranes and organelles of dehydrated
Selaginella and Tortula retain their normal configuration and structural integrity: freeze fracture
evidence. Protoplasma 178, 57–65.
Platt-Aloia, K.A., Lord, E.M., DeMason, D.A. and Thompson, W.W. (1986) Freeze-fracture observations
on membranes of dry and hydrated pollen from Collomia, Phoenix, and Zea. Planta 168, 291–298.
Porembski, S. and Barthlott, W. (2000) Granitic and gneissic outcrops (inselbergs) as center of diver-
sity for desiccation-tolerant vascular plants. Plant Ecology 151, 19–28.
Potts, M. (1994) Desiccation tolerance of prokaryotes. Microbiology Review 58, 755–805.
Potts, M. (1999) Mechanisms of desiccation tolerance in cyanobacteria. European Journal of
Phycology 57, 43–68.
Pouchet, F.A. (1859) Expériences sur la résistance vitale des animalcules pseudo-ressuscitants.
Comptes Rendues de l’Académie des Sciences de Paris 49, 886–888.
Proctor, M.C.F. (1982) Physiological ecology: water relations, light and temperature responses, carbon
balance. In: Smith, A.J.E. (ed.) Bryophyte Ecology. Chapman & Hall, London, pp. 333–381.
Proctor, M.C.F. (1990) The physiological basis of bryophyte production. Botanical Journal of the
Linnean Society 104, 61–77.
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 40

40 P. Alpert and M.J. Oliver

Proctor, M.C.F. (2000) The bryophyte paradox: tolerance of desiccation, evasion of drought. Plant
Ecology 151, 41–49.
Proctor, M.C.F. and Smirnoff, N. (2001) Rapid recovery of photosystems on rewetting desiccation-tol-
erant mosses: chlorophyll fluorescence and inhibitor experiments. Journal of Experimental
Botany 51, 1695–1704.
Proctor, M.C.F., Nagy, Z., Csintalan, Z. and Takács, Z. (1998) Water-content components in bryophytes:
analysis of pressure–volume relationships. Journal of Experimental Botany 49, 1845–1854.
Ramanathan, V. (1988) The greenhouse theory of climate change: a test by an inadvertent global
experiment. Science 240, 293–299.
Reynolds, T.L. and Bewley, J.D. (1993a) Characterization of protein synthetic changes in a desicca-
tion-tolerant fern, Polypodium virginianum. Comparison of the effects of drying, rehydration
and abscisic acid. Journal of Experimental Botany 44, 921–928.
Reynolds, T.L. and Bewley, J.D. (1993b) Abscisic acid enhances the ability of the desiccation tolerant
fern Polypodium virginianum to withstand drying. Journal of Experimental Botany 44,
Richardson, D.H.S. (1981) The Biology of Mosses. Blackwell Scientific, New York.
Ried, A. (1960) Stoffwechsel und Verbreitungsgrenzen von Flechten. II. Wasser- und
Assimilationshaushalt, Entquellungs- und Submersionsresistenz von Krustenflechten benach-
barter Standorte. Flora 149, 345–385.
Roberts, J.K., DeSimone, N.A., Lingle, W.L. and Dure, L. III (1993) Cellular concentrations and uni-
formity of cell-type accumulation of two LEA proteins in cotton embryos. Plant Cell 5, 769–780.
Robinson, S.A., Wasley, J., Popp, M. and Lovelock, C.E. (2000) Desiccation tolerance of three moss
species from continental Antarctica. Australian Journal of Plant Physiology 27, 379–388.
Roos, Y.H. (1995) Phase Transitions in Foods. Academic Press, London.
Rundel, P.W. and Lange, O.L. (1980) Water relations and photosynthetic response of a desert moss.
Flora 169, 329–335.
Sagot, C. and Rochefort, L. (1996) Sphagnum desiccation tolerance. Cryptogamie, Bryologie et
Lichenologie 17, 171–183.
Sales, K., Brandt, W., Rumbak, E. and Lindsey, G. (2000) The Lea-like protein HSP 12 in
Saccharomyces cerevisiae has a plasma membrane location and protects membranes against desic-
cation and ethanol-induced stress. Biochimica et Biophysica Acta Biomembranes 1463, 267–278.
Sancho, L.G., Schroeter, B. and Valladares, F. (1997) Photosynthetic performance of two closely
related Umbilicaria species in central Spain: temperature as a key factor. Lichenologist 29,
Scheidegger, C., Frey, B. and Schroeter, B. (1997) Cellular water uptake, translocation and PSII acti-
vation during rehydration of desiccated Lobaria pulmonaria and Nephroma bellum. Bibliotheca
Lichenologica 67, 105–117.
Schierbeek, A. (1959) Measuring the Invisible World. Abelard-Schuman, London.
Schipperges, B. and Rydin, H. (1998) Response of photosynthesis of Sphagnum species from contrast-
ing microhabitats to tissue water content and repeated desiccation. New Phytologist 140, 677–684
Schonbeck, M.W. and Bewley, J.D. (1981) Responses of the moss Tortula ruralis to desiccation treat-
ments. II. Variations in desiccation tolerance. Canadian Journal of Botany 59, 2707–2712.
Schonbeck, M. and Norton, T.A. (1978) Factors controlling the upper limits of fucoid algae on the
shore. Journal of Experimental Marine Biology and Ecology 31, 303–313.
Schroeter, B., Green, T.G.A., Kappen, L. and Seppelt, R.D. (1994) Carbon dioxide exchange at subzero
temperatures. Field measurements on Umbilicaria aprina in Antarctica. Cryptogamic Botany 4,
Scott, G.A.M. (1982) Desert bryophytes. In: Smith, A.J.E. (ed.) Bryophyte Ecology. Chapman & Hall,
London, pp. 105–212.
Scott, H.B. II and Oliver, M.J. (1994) Accumulation and polysomal recruitment of transcripts in
response to desiccation and rehydration of the moss Tortula ruralis. Journal of Experimental
Botany 45, 577–583.
Scott, P. (2000) Resurrection plants and the secrets of eternal leaf. Annals of Botany 85, 159–166.
Seel, W.E., Baker, N.R. and Lee, J.A. (1992) The combined effects of desiccation and irradiance on
mosses from xeric and hydric habitats. Journal of Experimental Botany 43, 1023–1030.
Senaratna, T. and McKersie, B.D. (1983a) Dehydration injury in germinating soybean (Glycine max L.
Merr) seeds. Plant Physiology 72, 620–624.
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 41

Drying Without Dying 41

Senaratna, T. and McKersie, B.D. (1983b) Characterization of solute efflux from dehydration injured
soybean (Glycine max L. Merr) seeds. Plant Physiology 72, 911–914.
Sherwin, H.W. and Farrant, J.M. (1996) Differences in rehydration of three desiccation-tolerant
angiosperm species. Annals of Botany 78, 703–710.
Sherwin, H.W. and Farrant, J.M. (1998) Protection mechanisms against excess light in the resur-
rection plants Craterostigma wilmsii and Xerophyta viscosa. Plant Growth Regulation 24,
Sherwin, H.W., Pammenter, N.W., February, E., Vander Willigen, C. and Farrant, J.M. (1998) Xylem
hydraulic characteristics, water relations and wood anatomy of the resurrection plant
Myrothamnus flabellifolius Welw. Annals of Botany 81, 567–575.
Shirazi, A.M., Muir, P.S. and McCune, B. (1996) Environmental factors influencing the distribution of
the lichens Lobaria oregana and L. pulmonaria. Bryologist 99, 12–18.
Simon, E.W. (1974) Phospholipids and plant membrane permeability. New Phytologist 73, 377–420.
Simon, E.W. (1978) Membranes in dry and imbibing seeds. In: Crowe, J.H. and Clegg, J.S. (eds) Dry
Biological Systems. Academic Press, New York, pp. 205–224.
Sivamani, E., Bahieldin, A., Wraith, J.M., Al-Niemi, T., Dyer, W.E., Ho, T.H.D. and Qu, R.D. (2000)
Improved biomass productivity and water use efficiency under water deficit conditions in trans-
genic wheat constitutively expressing the barley HVA1 gene. Plant Science 155, 1–9.
Skriver, K. and Mundy, J. (1990) Gene expression in response to abscisic acid and osmotic stress.
Plant Cell 2, 503–512.
Smirnoff, N. (1992) The carbohydrates of bryophytes in relation to desiccation-tolerance. Journal of
Bryology 17, 185–191.
Smirnoff, N. (1993) The role of active oxygen in the response of plants to water deficit and desicca-
tion. New Phytologist 125, 27–58
Smith, M.T. (1991) Studies on the anhydrous fixation of dry seeds of lettuce (Lactuca sativa L.). New
Phytologist 119, 575–584.
Sojo, F., Valladares, F. and Sancho, L.G. (1997) Structural and physiological plasticity of the lichen
Catillaria corymbosa in different microhabitats of the maritime Antarctic. Bryologist 100,
Stark, L.R. (1997) Phenology and reproductive biology of Syntrichia inermis (Bryopsida, Pottiaceae)
in the Mojave Desert. Bryologist 100, 13–27.
Strauss, G. and Hauser, H. (1986) Stabilization of small uni-lamellar phospholipid vesicles by
sucrose during freezing and dehydration. In: Leopold, A.C. (ed.) Membranes, Metabolism and
Dry Organisms. Cornell University Press, Ithaca, New York, pp. 318–326.
Sun, W.Q. (1997) Glassy state and seed storage stability: the WLF kinetics of seed viability loss at T-
T8 and the plasticization effect of water on storage stability. Annals of Botany 79, 291–297.
Sun, W.Q. and Leopold, A.C. (1997) Cytoplasmic vitrification and survival of anhydrobiotic organ-
isms. Comparative Biochemistry and Physiology 117A, 327–333.
Sun, W.Q., Irving, T.C. and Leopold, A.C. (1994) The role of sugar, vitrification and membrane phase
transition in seed desiccation tolerance. Physiologia Plantarum 90, 621–628.
Takács, Z., Csintalan, Z., Sass, L., Laitat, E., Vass, I. and Tuba, Z. (1999) UV-B tolerance of bryophyte
species with different degrees of desiccation tolerance. Journal of Photochemistry and
Photobiology B: Biology 48, 210–215.
Tetteroo, F.A.A., deBruijn, A.Y., Henselmans, R.N.M., Wolkers, W.F., van Aelst, A.C. and Hoekstra,
F.A. (1996) Characterization of membrane properties in desiccation-tolerant and -intolerant car-
rot somatic embryos. Plant Physiology 111, 403–412.
Thompson, J.W. and Iltis, H.H. (1968) A fog-induced lichen community in the coastal desert of
southern Peru. Bryologist 71, 31–34.
Thompson, W.W. (1979) Ultrastructure of dry seed tissue after a non-aqueous primary fixation. New
Phytologist 82, 207–212.
Thompson, W.W. and Platt-Aloia, K.A. (1982) Ultrastructure and membrane permeability in cowpea
seeds. Plant, Cell, and Environment 5, 367–373.
Tiwari, S.C., Polito, V.S. and Webster, B.D. (1990) In dry pear (Pyrus communis L.) pollen, mem-
branes assume a tightly packed multilamellate aspect that disperses rapidly upon hydration.
Protoplasma 153, 157–168.
Trainor, F.R. and Gladych, R. (1995) Survival of algae in a desiccated soil: a 35-year study. Phycologia
34, 191–192.
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 42

42 P. Alpert and M.J. Oliver

Tuba, Z., Lichtenthaler, H.K., Maroti, I. and Csintalan, Z. (1993a) Resynthesis of thylakoids and func-
tional chloroplasts in the desiccated leaves of the poikilochlorophyllous plant Xerophyta
scabrida upon rehydration. Journal of Plant Physiology 142, 742–748.
Tuba, Z., Lichtenthaler, H.K., Csintalan, Z. and Pócs, T. (1993b) Regreening of desiccated leaves of
the poikilochlorophyllous Xerophyta scabrida upon rehydration. Journal of Plant Physiology
142, 103–108.
Tuba, Z., Lichtenthaler, H.K., Csintalan, Z., Nagy, Z. and Szente, K. (1994) Reconstitution of chloro-
phylls and photosynthetic CO2 assimilation upon rehydration of the desiccated poikilochloro-
phyllous plant Xerophyta scabrida (Pax) Th. Dur. et Schinz. Planta 192, 414–420.
Tuba, Z., Csintalan, Z. and Proctor, M.C.F. (1996a) Photosynthetic responses of a moss, Tortula
ruralis, ssp. ruralis, and the lichens Cladonia convoluta and C. furcata to water deficit and short
periods of desiccation, and their ecophysiological significance: a baseline study at present-day
CO2 concentration. New Phytologist 133, 353–361.
Tuba, Z., Lichtenthalter, H.K., Csintalan, Z., Nagy, Z. and Szente, K. (1996b) Loss of chlorophylls,
cessation of photosynthetic CO2 assimilation and respiration in the poikilochlorophyllous plant
Xerophyta scabrida during desiccation. Physiologia Plantarum 96, 383–388.
Tuba, Z., Smirnoff, N., Csintalan, Z., Szente, K. and Nagy, Z. (1997) Respiration during slow desicca-
tion of the poikilochlorophyllous desiccation tolerant plant Xerophyta scabrida at present-day
CO2 concentration. Journal of Plant Physiology and Biochemistry 35, 381–386.
Tuba, Z., Proctor, M.C.F. and Csintalan, Z. (1998) Ecophysiological responses of homoiochlorophyl-
lous and poikilochlorophyllous desiccation tolerant plants: a comparison and ecological per-
spective. Plant Growth Regulation 24, 211–217.
Tuba, Z., Proctor, M.C.F. and Takács, Z. (1999) Desiccation-tolerant plants under elevated air CO2: a
review. Zeitschrift für Naturforschung C 54, 788–796.
Valladares, F. (1994) Texture and hygroscopic features of the upper surface of the thallus in the
lichen family Umbilicariaceae. Annals of Botany 73, 493–500.
Velten, J. and Oliver, M.J. (2001) Tr288: a rehydrin with a dehydrin twist. Plant Molecular Biology 45,
Vertucci, C.W. and Farrant, J.M. (1995) Acquisition and loss of desiccation-tolerance. In: Kigel, J. and
Galili, G. (eds) Seed Development and Germination. Marcel Dekker, New York, pp. 237–271.
Vertucci, C.W. and Leopold, A.C. (1986) Physiological activities associated with hydration levels in
seeds. In: Leopold, A.C. (ed.) Membranes, Metabolism and Dry Organisms. Cornell University
Press, Ithaca, New York, pp. 35–49.
Vierling, E. (1991) The roles of heat shock proteins in plants. Annual Review of Plant Physiology and
Plant Molecular Biology 42, 579–620.
Volk, O.H. (1984) Pflanzenvergesellschaftungen mit Riccia-Arten in Südwestafrika (Namibia).
Vegetatio 55, 57–64.
Walters, C. (1998) Understanding the mechanisms and kinetics of seed aging. Seed Science Research
8, 223–244.
Webster, B.D. and Leopold, A.C. (1977) The ultrastructure of dry and imbibed cotyledons of soybean.
American Journal of Botany 64, 1286–1293.
Wehmeyer, N., Hernandez, L.D., Finkelstein, R.R. and Vierling, E. (1996) Synthesis of small heat
shock proteins is part of the developmental program of late seed maturation. Plant Physiology
112, 747–757.
Williams, R.J. and Leopold, A.C. (1989) The glassy state in maize embryos. Plant Physiology 89,
Williams, T.G. and Flanagan, L.B. (1998) Measuring and modelling environmental influences on pho-
tosynthetic gas exchange in Sphagnum and Pleurozium. Plant, Cell and Environment 21,
Wilson, A.T., Vickers, M. and Mann, L.R.B. (1979) Metabolism in dry pollen – a novel technique for
studying anhydrobiosis. Naturwissenschaften 66, 53–54.
Wolkers, W.F. (1998) The role of macromolecular stability in desiccation tolerance. PhD thesis,
Wageningen Agricultural University, The Netherlands.
Wolkers, W.F., Oldenhof, H., Alberda, M. and Hoekstra, F.A. (1998) A fourier transform infrared
microspectroscopy study of sugar glasses: application to anhydrobiotic higher plant cells.
Biochimica et Biophysica Acta 1379, 83–96.
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 43

Drying Without Dying 43

Wood, A.J. and Oliver, M.J. (1999) Translational control in plant stress: the formation of messenger
ribonucleoprotein particles (mRNPs) in response to desiccation of Tortula ruralis gametophytes.
The Plant Journal 18, 359–370.
Wood, A.J., Duff, R.J. and Oliver, M.J. (1999) Expressed sequence tags (ESTs) from desiccated Tortula
ruralis identify a large number of novel plant genes. Plant and Cell Physiology 40, 361–368.
Wood, J.N. and Gaff, D.F. (1989) Salinity studies with drought resistant Sporobolus species.
Oecologia 78, 559–564.
Xu, D., Duan, X., Wang, B., Hong, B., Ho, T.-H.D. and Wu, R. (1996) Expression of a late embryogene-
sis abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt stress in
transgenic rice. Plant Physiology 110, 249–257.
Zimmermann, M.H. and Butin, H. (1973) Untersuchungen über die Hitze- und Trockenresistenz
holzbewohnender Piltze. Flora 162, 393–419.
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 44
02 Dessication - Chap 2 18/3/02 1:53 pm Page 45

Part II

02 Dessication - Chap 2 18/3/02 1:53 pm Page 46
02 Dessication - Chap 2 18/3/02 1:53 pm Page 47

2 Methods for the Study of Water Relations

Under Desiccation Stress

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

2.1. Introduction 48
2.2. Expression of Water Status 48
2.2.1. Mass-based measures for tissue hydration 48 Water content 48 Relative water content 49
2.2.2. Thermodynamic measures for tissue hydration 50 Water activity 50 Chemical potential of water and water potential 51
2.3. Measurement of Tissue Water Potential 53
2.3.1. Psychrometric and hydrometric methods 53
2.3.2. Osmometric or cryoscopic method 54
2.3.3. Isothermal equilibrium method 55
2.4. Water Relations – the Thermodynamic Approach 55
2.4.1. The Höfler diagram and pressure–volume curve 55 Change of cell turgor pressure during desiccation 55 Change of osmotic potential during desiccation 57 The volume of water in symplast, apoplast and
intercellular spaces 57 Volumetric elasticity of the cell wall 59
2.4.2. Analysis of water sorption isotherms 60 Theoretical models 60 Temperature dependency of water sorption 62 Monolayer hydration and water-clustering function 65 Occupancy of water-binding sites 66
2.5. Measurement of Drying Rate and Desiccation Stress 68
2.5.1. Driving force for water loss and expression of drying rate 68
2.5.2. Quantification of desiccation stress 68
2.6. Water Relations – the Kinetic and Functional Approach 70
2.6.1. General considerations 72

© CAB International 2002. Desiccation and Survival in Plants: Drying Without Dying
(eds M. Black and H.W. Pritchard) 47
02 Dessication - Chap 2 18/3/02 1:53 pm Page 48

48 W.Q. Sun Time scale 72 Structural complexity and dynamics of
molecular ordering 72 The model-dependent interpretation: the pitfalls 73
2.6.2. Biophysical techniques 74 Differential scanning calorimetry 74 Thermally stimulated current (TSC) method 75 Nuclear magnetic resonance (NMR) 76 Electron spin resonance 77
2.7. Concluding Remarks 78
2.8. References 79
Appendix 84

2.1. Introduction of water relations that are directly related to

desiccation tolerance of plant tissues will be
In hydrated plant cells, water is the main introduced. The strengths and limitations of
constituent. The organization of cellular various methods or techniques of measure-
structures (both supramolecular assemblies ment of water relations during desiccation
and micromolecular structures) and the over- will be discussed. An effort will be made to
all biochemistry (the thermodynamics of bio- give a basic understanding of terms and
logical processes and their rate parameters) of concepts concerning cellular water status
an organism depend on water. Water is a sol- and the expression of dehydration stress.
vent and a medium in which diffusion of
solutes and biochemical reactions take place
in plant cells. It is often a participant and/or 2.2. Expression of Water Status
a product of various biochemical reactions.
In low-moisture systems such as naturally The most important quantity that has to be
dried pollen grains and plant seeds, cellular measured in all studies of desiccation toler-
water also plays an important role as a plasti- ance is the degree of dehydration stress. So
cizer, influencing the translational or rota- far, there is no agreed parameter of dehydra-
tional motions of entire molecules, or tion stress measurement. The change in
segments of macromolecules and intramolec- water content of plant tissues and organs is
ular motions. Water is involved in virtually often used as an indicator of dehydration.
every dynamic process in a living cell. However, insufficient attention has been
The loss of water from plant cells is an paid to problems commonly associated with
important environmental stress. Changes in the use of water content as an indicator of
the aqueous environment influence the dehydration stress. For example, different
complex thermodynamics and kinetics of concepts and approaches are currently used
structural stability and all aspects of biologi- by research groups working on biological
cal functions. The accurate measurement of systems, ranging from bacteria and fungal
the status of cellular water is essential for spores to microscopic animals, pollen
the study of both desiccation stress in plants grains, large seeds and resurrection plants.
and the mechanisms of plant desiccation
tolerance. The method of quantification and
2.2.1. Mass-based measures for tissue
interpretation must be applicable not just to
the narrowly defined desiccation condi-
tions, but also to all other types of physio- Water content
logical stresses with a dehydration
component, such as freezing and salinity. In Water content on a wet-weight basis (WC, %
this chapter, several fundamental principles w.b.) is widely used in the literature of desic-
02 Dessication - Chap 2 18/3/02 1:53 pm Page 49

Methods for Studying Water Relations Under Stress 49

cation studies, and is adopted by the WC (g g1 dw) = WC (% w.b.)/[100 – WC

International Seed Testing Association (% w.b.)] (3)
(ISTA, 1993) for the expression of seed water
content. WC (% w.b.) is the percentage mass Relative water content
fraction of water of the total tissue mass:
Relative water content (RWC) is another
WC (% w.b.) = (fresh weight  dry
mass-based parameter. RWC is widely used
weight)/fresh weight  100 (1)
in the pressure–volume analysis of plant
WC (% w.b.) is not a linear expression of tissue water stress. RWC is a simple and
water content in tissues, because fresh useful measure of the extent to which a tis-
weight appears in both the numerator term sue is in water deficit. It is related to tissue
and the denominator term in Equation (1). water content at full turgor (WCF). During a
When WC (% w.b.) is used to monitor the dehydration experiment, RWC is calcu-
loss of water during desiccation, the lated by dividing water content at a given
decrease of WC (% w.b.) does not necessar- time by water content at full turgor, and
ily reflect the exact extent of dehydration expressed as a fraction value or as a per-
stress. The change of WC (% w.b.) during centage. If water content in the tissue is
drying is, in fact, related to the change of determined as WC (g g1 dw), the calcula-
the reciprocal of tissue fresh weight. For tion of RWC is straightforward, being
example, when the tissue of 80% WC (% WC/WCF. But, if water content is deter-
w.b.) is dried to 70% and 60% water con- mined as WC (% w.b.), RWC is calculated
tent, the tissue actually loses 41.7% and by the equation:
62.5% of its initial water quantity, respec-
RWC = [WC (100 – WCF)]/[WCF
tively, not just 12.5% and 25% reduction as
(100 – WC)]  100 (4)
implied by the values of WC (% w.b.). The
quantity of water lost during dehydration RWC is a linear expression of moisture
from 80% to 70% WC (% w.b.) is twice as condition. The change in RWC over time
much as water loss during dehydration serves as a good indicator for the rate of
from 70% to 60% WC (% w.b.). dehydration. Physiological responses of
Water content on a dry-weight basis mea- plant water deficit are highly correlated
sures the mass ratio between water and the with RWC (Sinclair and Ludlow, 1985). The
dry mass in tissues, and is often expressed use of RWC is particularly advantageous for
by g water per g dry weight (i.e. g g1 dw): comparative studies, in which initial water
content at full turgor or full hydration
WC (g g1 dw) = (fresh weight  dry
varies considerably among different
weight)/dry weight (2)
species, different tissues of the same
WC (g g1 dw) is a linear expression of species or the same tissue at different
water content, and the change of WC (g g1 developmental stages, such as seeds. In cer-
dw) during drying is proportional to the tain cases, it may be even preferred over
loss of water in a tissue. On the mass basis, water potential, because RWC also accounts
a tissue with a WC of 0.20 g g1 dw is for the effect of osmotic adjustment in
hydrated exactly twice as much as the tis- affecting plant water status. For example,
sue with a WC of 0.10 g g1 dw, and four- two plants with the same leaf water poten-
fold as much as the sample with a WC of tial can have different RWCs if they differ
0.05 g g1 dw. For this reason, some in their ability for osmotic adjustment.
researchers have argued that WC (g g1 dw) In many desiccation studies on higher
is a more sensible expression than WC (% plants, water content of a tissue at full tur-
w.b.) for the measurement of dehydration. gor was not specifically determined, and
At WC < 15%, the difference between WC instead water content after full hydration in
(% w.b.) and WC (g g1 dw) is fairly small. water was used. Typically, leaf samples (e.g.
WC (% w.b.) is converted to WC (g g1 dw) discs or sections) of higher plant species
using the following equation: are taken and weighed immediately. The
02 Dessication - Chap 2 18/3/02 1:53 pm Page 50

50 W.Q. Sun

samples are then hydrated in distilled based parameters for the expression of
water for 4–6 h, after which they are dehydration stress has been shown by a
weighed again and their water contents are number of studies. The critical onset water
determined by drying the samples in an potential of Quercus rubra (Pritchard, 1991)
oven. In higher plants, the amount of inter- and Quercus robur (Pritchard and Manger,
cellular water is small or non-existent 1998) is about –3 MPa, but the correspond-
(Oertli, 1989). However, it should be noted ing mass water contents vary substantially
that some plant tissues do contain intercel- due to different seed oil content. Sun and
lular water, which is held in spaces Gouk (1999) studied the water relation
between cells of a tissue at relatively high responses of three recalcitrant (desiccation-
water potential (near zero). Therefore, water sensitive) seeds (Aesculus hippocastanum,
content at full turgor has different physio- Andira inermis, Q. rubra) during controlled
logical meaning from water content of the dehydration. The critical water potentials
tissue at full hydration when the tissue con- for seeds are quite similar for all three
tains intercellular water. If intercellular species (7 to 8 MPa), but their corre-
water is present, water content of the tissue sponding critical water contents are 0.45,
at full turgor has to be estimated from the 1.10 and 0.35 g g1 dw for A. hippocas-
plot of water potential on water content (g tanum, A. inermis and Q. rubra, respec-
g1 dw). To determine the water content of tively. If the critical water content were
the tissue at full turgor, two linear regres- used to express the relative desiccation tol-
sion lines can be fitted, respectively, with erance, one would conclude incorrectly
data points where water potential remains that seeds of A. hippocastanum and Q.
almost unchanged during initial water loss rubra are much more desiccation-tolerant
and the next few points where water poten- than seeds of A. inermis. Therefore, a mass-
tial starts to fall. The intercept of these two based parameter of water loss may not be a
regression lines gives the water content at reliable indicator for the degree of desicca-
full turgor. Beckett (1997) reported that the tion stress in plant tissues.
amount of intercellular water varied greatly
among species of bryophytes. If intercellu-
lar water exists in a tissue, correction needs 2.2.2. Thermodynamic measures for tissue
to be made to the raw RWC readings, which hydration
are calculated according to the water con-
tent at full hydration. The method used to The response of plant tissues to desiccation
correct the raw RWC readings was is related to the thermodynamic and
described in detail by Beckett (1997). kinetic status of tissue water, rather than to
There are shortcomings in using mass- actual water content. The water status of
based parameters for the expression of plant tissues can be expressed in terms of
water content. Plant tissues are heteroge- energy status of water molecules, i.e. the
neous, complex biological systems, in partial molar Gibbs’ free energy or water
which carbohydrates, proteins and lipids potential. This thermodynamic approach is
and other components have different hydra- preferred to the mass-based expression of
tion properties. As a consequence, when tissue hydration, because thermodynamic
plant tissues of various species are equili- parameters (i.e. energy status) are related
brated under given conditions of tempera- directly to the numerous biophysical and
ture and relative humidity, equilibrium physiological events that contribute to des-
water content varies considerably among iccation stresses and desiccation tolerance
species. For example, seeds with large lipid of plant tissues.
reserves equilibrate to lower water contents
than starchy seeds, even though the chemi- Water activity
cal potential of water molecules is the same
for all tissues when equilibrium is Water activity (aw) is used to describe
achieved. The disadvantage of using mass- water status in the studies of desiccation
02 Dessication - Chap 2 18/3/02 1:53 pm Page 51

Methods for Studying Water Relations Under Stress 51

tolerance and storage survival for spores, In such cases, the measured vapour pres-
pollen, seeds and resurrection plants (Ellis sure of water may not be the equilibrium
et al., 1990, 1991; Berjak and Pammenter, vapour pressure, but the vapour pressure of
1994; Vertucci et al., 1994, 1995; Walters, a ‘stationary’ state that is time-dependent.
1998a). Water activity is measured as the However, studies in food sciences have
ratio of the vapour pressure of water in a suggested that water activities measured
system to the vapour pressure of pure are likely to be close to equilibrium and the
water at the same temperature. It is related differences should be within the uncer-
to the equilibrium relative humidity (RH) tainty associated with the experimental
of the air surrounding the system (i.e. RH = determination (Chirife and Buera, 1996).
aw  100). Water activity can be viewed as The usefulness of the water activity con-
the ‘effective’ water content, which is ther- cept in seed storage stability has been dis-
modynamically available to various physi- cussed by Walters (1998b).
ological processes in cells. For the survival
of organisms under water stress, the ‘effec- Chemical potential of water and
tive’ water is more important than the total
water potential
amount of water present in the tissue.
Water activity of fresh plant tissues may The quantity of free energy of a component
vary only between 0.980 and 0.996. Within (µj) in a system is measured by its chemical
this narrow range, it is not useful for the potential. The chemical potential of water
expression of dehydration stress or tissue (µw) in a system is defined by:
water status. However, for the studies of –
µw = µ*
w + RT ln aw + VwP + zwFE + mwgh (5)
severe water stress and extreme desicca-
tion, water activity has several advantages where µ*w is the chemical potential of pure
over water content, including its concep- water at ideal reference conditions. The
tual simplicity, measurability, easy experi- second term RT ln aw is for water activity. R
mental manipulation, and its applicability is the gas constant (8.314  103 kJ mol1
to both simple and complex systems. A K1), T is the absolute temperature (K, in
number of physiological processes that are kelvin), and aw is water activity (RH/100).

relevant to desiccation tolerance or damage Vw is the partial molal volume of water (i.e.
have been shown to occur at specific water the differential increase or decrease in vol-
activities, and some of those are presented ume when a differential amount of water is

in Fig. 2.1. added or removed). Vw is influenced by the
Water activity in plant tissues can be presence of solutes and is also tempera-
determined using the hygrometric method ture-dependent (1.805  105 m3 mol1 at
and the isothermal equilibrium sorption 20°C). P is the hydrostatic pressure on
method. The hygrometric instrument water in excess of atmospheric pressure

method directly measures the equilibrium (MPa, 1 MPa = 103 kJ m3). The term VwP
RH of plant tissues in a closed chamber. represents the effect of pressure on the
With the equilibrium sorption method, chemical potential of water and is
samples of plant tissues are equilibrated to expressed in energy per mole (kJ mol1).
a series of known water activities at a spec- zwFE is the electrochemical potential of
ified temperature. The relationship water, which equals zero because water is
between water content and water activity uncharged (zw = 0). The last term mwgh is
upon equilibrium (i.e. the sorption the gravitational term, representing the
isotherm curve) is then used to calculate work needed to move 1 mole of water to a
water activity of plant tissues at different given height. Practically, mwgh will remain
water contents. Water activity is defined at constant in most circumstances of desicca-
equilibrium. However, plant tissues at low tion studies.
and intermediate moisture levels may not The water potential is proportional to
be in a true state of equilibrium at all, but the chemical potential of water (µw  µ*w) in
in an amorphous metastable state instead. a system as described in Equation (5).
02 Dessication - Chap 2 18/3/02 1:53 pm Page 52

52 W.Q. Sun

Therefore, water potential is actually the osmotic potential and h is the gravita-
potential energy of water per unit mass. tional potential. The total water potential is
While water content tells how much water the sum of hydrostatic, osmotic and gravita-
is in a sample, water potential tells you tional components. The gravitational term
how available that water is. By convention, (h) depends on the position of water in a
water potential is defined as follows: gravitational field and is not relevant to
 = P + π + h (6)
most desiccation studies. Osmotic potential
depends on the concentration of dissolved
where p = P and is hydrostatic pressure substances in water. Osmotic potential is
on water as defined in Equation (5), π is related to water activity by the equation:

Coffea species

Mean of minimum value for bacterial growth

Cell respiration starts to cease
Lysozyme activity stops
at lower aw
colonies in situ

Minimum required for photosynthesis

Nucleic acids and proteins fully hydrated
Water vapour above saturated NaCl solution
Water potential of water vapour (MPa)

seeds typically survive at lower aw

saturated CaCl2 solution

Desiccation tolerance of embryos of

DNA disordered and damaged

Typical exposure of Nostoc

Water vapour above



20 40 60 80 100

Relative humidity (%)

Fig. 2.1. Water activities (relative humidities) that limit physiological activities and cell growth. Physical
parameters 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 calculated
according 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 it
gives a clear concept about the possible sequence of potential physiological and biochemical events and
their interactions as the tissue loses water.
02 Dessication - Chap 2 18/3/02 1:53 pm Page 53

Methods for Studying Water Relations Under Stress 53

Vw π = RT ln aw (7) face. This technique, however, is unsuitable
for many desiccation-tolerant plant tissues,
During dehydration, the water content in
e.g. lichens, bryophytes, spores, pollen grain
seed tissues is reduced, resulting in an
and seeds. This is because the pressure
increase in the concentration of solutes and
chamber method measures the xylem ten-
thus a decrease in water activity and
sion, which is broadly equal to the leaf
osmotic potential (i.e. π becomes more
water potential. Water potential of plant
negative). A similar situation occurs during
parts that do not have vascular systems can-
freezing. The formation of ice leads to the
not be measured with the pressure chamber
dehydration of the protoplast and the con-
method. However, water potential of plant
centration of solutes.
tissues can be measured by a number of
The interactions of water with biological
other techniques. These techniques use
surfaces and interfaces are of great impor-
either the relationship of the sample water
tance to desiccation tolerance of plant tis-
potential to the equilibrium vapour pressure
sues, especially at low moisture levels. The
immediately around the sample or the prin-
influence of such interactions on water
ciple of the freezing-point depression in the
potential in a tissue is commonly called
liquid solution.
‘matric’ potential. Rapid water uptake by
dry seeds during the early stage of germina-
tion is mainly attributed to large matric
2.3.1. Psychrometric and hydrometric
potentials. Another example is the reduced
rate of water loss as the tissue is dried to
lower water content. Matric potential
Both methods are widely used for the mea-
depends on the adsorptive forces that bind
surement of tissue water potential. The mea-
water to a matrix. The amount of matrix-
surement of water potential by a
bound water in recalcitrant Q. robur
psychrometer and a hydrometer is called
embryonic axes is as high as 0.25–0.30 g
the wet-bulb depression method and the
g1 dw (Pritchard and Manger, 1998).
dew-point depression method, respectively.
However, the forces of such water–matrix
A psychrometer measures water potential of
interactions are adequately represented by
samples (placed in closed chambers)
their contributions to hydrostatic pressure
through its ability to determine the RH of
(P) and osmotic potential (π). For exam-
the closed environment. The instrument
ple, the presence of aqueous interfaces in
uses high-sensitivity thermometers to mea-
cells lowers water activity through interfa-
sure temperature reduction resulting from
cial attractions and binding of water near
the heat of vaporization of water in a sample
their surfaces, which has already been
relative to pure water. It can measure water
included in the osmotic component in
potential of solid tissue materials and
Equation (7). Therefore, matric potential
droplets of solutions. The sample is first
does not represent additional new forces.
sealed in a small chamber containing a ther-
mocouple. After an equilibration period, a
cooling current is applied to the thermocou-
2.3. Measurement of Tissue Water ple in order to condense water on the ther-
Potential mocouple junction. The amount of
condensed water is proportional to the
A pressure chamber (pressure bomb) is com- water potential of the tissue. The water is
monly used to measure directly leaf water allowed to evaporate, causing a change in
potential of higher plants. The detached leaf the thermocouple output, and the output is
is sealed in a steel chamber with the cut calibrated for water potential, using stan-
petiole protruding out. Pressure that is dard salt solutions. On the other hand, a
applied to the chamber is taken as the hydrometer maintains the dew-point
xylem (leaf water) potential when the sap depression temperature during the measure-
meniscus appears at the petiole xylem sur- ment using a thermocouple. The dew-point
02 Dessication - Chap 2 4/4/02 2:18 pm Page 54

54 W.Q. Sun

depression temperature is the temperature osmolal aqueous solution, the osmotic

to which the air in the sealed chamber must potential at 0°C is ideally 2.271 MPa, and
be reduced so that the air becomes saturated the freezing-point depression is 1.86°C.
with water vapour. Psychrometric and (An osmole is the mass of a substance that
hydrometric methods can be used to mea- when dissolved in 1 kg water generates an
sure both water potential and osmotic osmotic pressure equivalent to that pro-
potential of plant tissues. To measure duced by 1 mole of an ideal solute dis-
osmotic potential, a sample has to undergo solved in 1 kg water. After dissolving, an
the freeze–thaw cycles to disrupt the cellu- ideal solute gives 6.023  1023 osmotically
lar structures before the measurement, active particles.) Theoretically, the osmotic
whereas the water potential is measured potential of an unknown sample can be
using undisrupted tissues. estimated from the depression of its freez-
A psychrometer is very sensitive to tem- ing point by the following relationship:
perature change because it measures very
small temperature differences. A change in −2.271 MPa
Ψπ = ∆T = −1.221∆T (8)
water potential of 1.0 MPa is reflected by a 1.86°C
change in wet-bulb temperature depression where T is the depression of the freezing
of only approximately 0.085°C. A hydro- point. The effect of osmotic potential on
meter is less affected by the changes in freezing-point depression also holds for
ambient temperature during the measure- non-ideal solutions such as plant saps.
ment compared with a psychrometer. However, freezing-point depression is non-
Psychrometric and hydrometric methods linear with concentration changes during
are suitable only for plant tissues of high dehydration. Water potential (MPa at 0°C)
water content. At low water content, the can be derived by the empirical equation
equilibrium may take several hours to (Crafts et al., 1949):
achieve. The nominal range of the Peltier
Ψ = 1.206T + 0.0021T 2 (9)
thermocouple measurement is limited from
0 to 6.0 MPa for these two methods. Yet With the osmometric method, a sample
many desiccation-tolerant plant tissues can is usually supercooled a few degrees below
survive far below 6.0 MPa. Even if one its freezing point to induce immediate
uses the Richards thermocouple, it extends crystallization. As the heat of fusion is
only to –25 MPa and the accuracy released, the sample temperature rises to
decreases to –0.1 MPa at –10 MPa its freezing point, and its equilibrium tem-
(Decagon Devices Inc., Pullman, perature is measured. Alternatively, the
Washington, USA), corresponding to an RH temperature at which ice crystals start
of ~ 84% at 25°C. melting can be measured and taken as the
equilibrium freezing temperature (i.e.
Ramsay’s method). The applicability of
2.3.2. Osmometric or cryoscopic method Equations (8) and (9) to the measurement
of water potential or osmotic potential in
A freezing-point osmometer measures the plant tissues was examined by Sun and
osmotic concentration of a biological liquid Gouk (1999), using seed tissues that were
using the principle of the freezing-point pre-equilibrated with saturated salt solu-
depression. The freezing-point depression tions (from 3 to 35 MPa). The freezing-
is one of the four colligative properties of a point depression was determined with a
solution. The freezing point is the unique differential scanning calorimeter, using the
temperature at which the ice phase and the onset temperature for the exotherm on
liquid phase can coexist in equilibrium at cooling. Calculated water potentials were
standard pressure. When a solute is dis- found to be very close to the pre-freezing
solved in the water, the freezing point of water potentials of seed tissues, with
the water is lowered in proportion to the Equation (9) fitting the data slightly better
osmotic potential of the solution. For a 1.0 than Equation (8).
02 Dessication - Chap 2 18/3/02 1:53 pm Page 55

Methods for Studying Water Relations Under Stress 55

2.3.3. Isothermal equilibration method cessfully in seed desiccation studies by a

number of workers (Pritchard, 1991; Poulsen
It is more difficult to measure directly and Eriksen, 1992; Vertucci et al., 1994; Sun
water potential of low-moisture systems. et al., 1997; Tompsett and Pritchard, 1998).
Perhaps the convenient, yet accurate and
reliable method is first to establish the
empirical relationship between water con- 2.4. Water Relations – the
tent and water potential for a particular Thermodynamic Approach
plant tissue. Samples of plant tissues are
equilibrated over different salt solutions 2.4.1. The Höfler diagram and the
that would maintain a series of constant pressure–volume curve
water vapour pressures (i.e. RH) in closed
containers. Upon equilibrium, the water Water relations parameters of plant tissues
contents of tissue samples are determined can be presented by the Höfler diagram and
gravimetrically, and their water potential at the pressure–volume curve (PV curve). The
equilibrium is then the same as the water Höfler diagram shows the relationship
potential of the air in the closed containers, between water potential and relative water
which in turn equals the osmotic potential content (Fig. 2.2a). The PV curve is a plot
of the salt solutions used. Therefore, water between the reciprocal of water potential
potential of tissue samples is calculated by (1/) and RWC or water loss (1 – RWC)
the equation: during desiccation (Fig. 2.2b). Both the
Höfler diagram and the PV curve are
 = RT %RH
– ln (10) widely used to characterize water relations
Vw 100
of plant tissues. To construct a Höfler dia-
where R is the gas constant, T is the gram or a PV curve, the changes in water

absolute temperature (kelvin), Vw is the par- potential and RWC are monitored as the
tial molal volume of water, and %RH is the tissue is dehydrating. Several important
percentage relative humidity inside the parameters can be obtained by analysing
containers. (Note that Equation (10) is the components of cell water potential,
essentially the same as Equation (7).) The including the osmotic potentials at full tur-
empirical relationship between water con- gor and at the partially dehydrated state,
tent and water potential can be described the apoplastic and symplastic water vol-
by exponential and polynomial (Poulsen ume in tissues, a plot of turgor pressure
and Eriksen, 1992) or other functions (Sun (i.e. hydrostatic water potential in Equation
and Gouk, 1999). The derived mathematical (6)) as a function of RWC, and the tissue
expression is then used to calculate water bulk modulus of elasticity. Without know-
potential of plant tissues at any water con- ing these biophysical metrics, it would be
tent within the limit of experimental range. impossible to identify different kinds of
This method is particularly useful in moni- cellular stresses associated with the loss of
toring the change of tissue water potential water in the tissue and to examine the sig-
during desiccation. Water potential of dehy- nificance of an array of biochemical and
drating tissues can be calculated immedi- physiological responses during desicca-
ately from the data of water loss. tion. Moreover, valid comparisons of the
Constant RH can be achieved using satu- response of cell function to water stress
rated or non-saturated salt solutions, poly- among different organisms cannot be made
ethylene glycol solutions and glycerol without such knowledge.
solutions. Physico-chemical data of various
salts and their solutions are presented in the Change of cell turgor pressure during
Appendix. This technique does not need spe-
cial instruments to measure water potential,
and can avoid the difficulty in measuring RH In fully turgid cells, turgor pressure is equal
accurately. This method has been used suc- to the osmotic potential (with opposite
02 Dessication - Chap 2 18/3/02 1:54 pm Page 56

56 W.Q. Sun

(a) 2

Water potential (MPa)

–2 External


0.0 0.2 0.4 0.6 0.8 1.0 1.2
Relative water content (RWC)


Reciprocal of water potential

–1/ (MPa–1)

0.7 1.0 1.3
Incipient RWC
(p = 0)


–0.2 0.0 0.2 0.4 0.6 0.8 1.0

1 – RWC

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 π
follows a rectangular hyperbola (RWC = a + b/π). Osmotic potential at RWC = ~0.8–1.0 is extrapolated
from the rectangular hyperbola relationship. Turgor pressure is the difference between the measured water
potential and the extrapolated osmotic potential. (b) The pressure–volume curve showing the relationships
between , 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 (the
dashed line) of the tissue when the tissue is still turgid. The difference between the measured water potential
and the extrapolated osmotic potential is turgor pressure (inset).
02 Dessication - Chap 2 18/3/02 1:54 pm Page 57

Methods for Studying Water Relations Under Stress 57

signs). During dehydration, the PV curve of cation of negative turgor pressure. It is con-
a plant tissue initially displays a concave ceivable that the development of negative
region, beyond which the curve is linear turgor pressure may reduce mechanical
(Fig. 2.2b). The loss of turgor is marked by damage on cellular structures by preventing
the point at which the relation of 1/ to collapse of the cells.
(1  RWC) deviates away from linearity.
Turgor pressure (p) is calculated as the Change of osmotic potential during
difference between the extrapolated linear
portion of the PV curve and the water
potential actually measured, and is often When cell turgor pressure falls to zero dur-
plotted as a function of RWC. The relation- ing desiccation, the water potential of the
ship between turgor pressure and RWC can cell is equal to the osmotic potential (see
be described sufficiently by a quadratic or Equation (6)). As desiccation continues,
cubic function. osmotic potential and cell water potential
Certain plant tissues might develop nega- are equal and inversely proportional to the
tive turgor pressure before the cells collapse volume of osmotically active water. The rela-
and p can become zero under severe water tionship between RWC and the reciprocal of
stress. When negative p develops, the PV osmotic potential is a straight line. The
curve would fall below the extrapolated lin- osmotic potential at full turgor is calculated
ear portion of the graph (Fig. 2.3a). If the from the extrapolation of the linear portion
cells are sufficiently strong, do not collapse of the PV curve to the RWC at full turgor (i.e.
and the plasma membrane remains firmly RWC = 1.0 in Fig. 2.2b). In the Höfler dia-
attached to the cell wall, the formation of an gram, the relationship between osmotic
intracellular gas bubble will increasingly potential and RWC is represented by a rec-
become possible (cavitation). The develop- tangular hyperbolic function to the data
ment of negative turgor pressure and intra- points corresponding to the linear part of the
cellular cavitation appear to play some roles PV curve (dashed part of the π in Fig. 2.2a).
in desiccation tolerance of certain cells. A
good example of a cell surviving large nega- The volume of water in symplast,
tive turgor pressure and cavitation is the
apoplast and intercellular spaces
ascospore of Sordaria (Milburn, 1970). The
volume of Sordaria ascospores changes very In hydrated plant tissues, water may exist
little, and the protoplast remains in contact in the symplast, in the apoplast (i.e. the
with the spore wall at all times. Under porous spaces in the cell wall) and in the
water stress (by air-drying or in osmotic intercellular spaces (large voids) as dis-
solution), these cells might generate nega- cussed before. Intercellular water, also
tive p as much as –4 MPa. Beyond this called ‘external’ cell water by some work-
negative turgor pressure, a small bubble ers, may account for up to 35% of total
appears inside the protoplasm suddenly, water in certain plant tissues, such as
which increases slowly in size and lichens, liverworts, mosses and fern fronds
approaches the walls quite closely without (Beckett, 1997; Proctor, 1999) and develop-
losing its spherical appearance. Honegger ing embryos of higher plants (W.Q. Sun,
(1995) and Scheidegger et al. (1995) also unpublished data). During desiccation,
showed that ascomycetous lichen myco- water potential and turgor potential do not
bionts form large intracellular gas bubbles fall with initial water loss at RWC > 1.0
when desiccated. More recently, the PV (Fig. 2.2a and b inset). The volume of water
analysis by Beckett (1997) suggested the that is lost before turgor pressure starts to
existence of negative turgor pressure in veg- fall is assumed to be intercellular water.
etative cells of several desiccation-tolerant The volume of symplastic water represents
(poikilohydric) plants (e.g. Dumortiera hir- the amount of osmotically active water in
suta and Myrothamnus flabellifolia). PV the tissue, and is obtained by subtracting
curves of most plants do not show any indi- the apoplastic water volume from the water
02 Dessication - Chap 2 18/3/02 1:54 pm Page 58

58 W.Q. Sun

Reciprocal of water potential
–1/ (MPa–1)

1 0.6 0.8 1.0



–0.2 0.0 0.2 0.4 0.6 0.8 1.0

1 – RWC

(b) 1000

Water potential (–MPa)




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) and
intracellular 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 will
collapse 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 high
RWC break point corresponds to the turgor-loss point, whereas the low RWC break point corresponds to the
volume of apoplastic water. Drawn with data from Quercus rubra seeds (Sun, 1999).

content at full turgor. Symplastic water Apoplastic or osmotically inactive water

generally declines over a range of water is present in very small pores and strong
potential from about 0.5 to 10 MPa, in water-binding sites of biological surfaces in
line with that of osmotic potential. plant tissues. This fraction of water is held
02 Dessication - Chap 2 18/3/02 1:54 pm Page 59

Methods for Studying Water Relations Under Stress 59

by matric and molecular forces, and lost tion of cellular membrane and molecular
only when plant tissues are desiccated to a assemblies. So far, workers have paid little
water potential less than 15 MPa attention to the location of water in plant
(Meidner and Sheriff, 1976). The loss of tissues. The difference in the relative vol-
apoplastic water in some species extends ume of external, symplastic, and apoplastic
to approximately 800 MPa. The amount water should be taken into account in the
of such matrix-bound water in plant tissues comparative studies on mechanisms of des-
can be as high as 0.1–0.2 RWC or up to iccation tolerance among cells, tissues or
0.25–0.35 g g1 dw. This fraction of water plants. A similar analysis of water relations
does not generally act as a solvent in cells, was found to be very useful in developing a
and is not readily freezable. From the mechanistic understanding of the role of
Höfler diagram, the apoplastic volume is dehydration in freezing tolerance in earth-
estimated from the fitted hyperbolic func- worms (Holmstrup and Zachariassen, 1996).
tion. From the PV curve, the volume of
apoplastic water is commonly estimated by
extrapolation of the linear relationship Volumetric elasticity of the cell wall
between RWC and the reciprocal of The cell wall may undergo elastic expan-
osmotic potential to the (1  RWC) axis sion or contraction. Elastic (mechanical)
after the loss of turgor pressure. However, properties of cell walls play an important
the simple extrapolation from the PV curve role in cell water relations. For example,
is not a reliable method of estimating the the negative turgor pressure that can
apoplastic volume, and in some cases gives develop in a cell largely depends on the
negative values (Proctor et al., 1998). mechanical properties of the cell wall. The
The apoplastic volume of water should elasticity of the cell wall is represented by
be derived with data from the isothermal the volumetric elasticity module , where 
sorption study at low water potentials depends on both p (turgor pressure) and
(water activity), rather than the extrapola- V (cell volume) and is defined as:
tion method, because the linear relation-
ship between RWC and the reciprocal of p
= V (11)
osmotic potential does not hold for the V
apoplastic volume of water (which is where V is volume change caused by a
osmotically inactive). Compared to the given pressure change p. Equation (11)
removal of osmotically active (symplastic) indicates that a high value of  implies a
water, the measured osmotic potential rigid cell wall, whereas a low value implies
(including the term of matric potential) a more elastic cell wall. The  value can be
declines much more rapidly when the calculated from the relationship between
apoplastic water is removed. Therefore, the p and RWC (Steudle et al., 1977;
volume of apoplastic water is marked by Stadelmann, 1984). The change of  as a
the point at low water content at which the function of RWC is given by the first deriv-
relationship of 1/ to (1 – RWC) again ative of the quadratic or cubic function of
deviates away from linearity (Fig. 2.3b). turgor pressure on RWC. The value of the
The volume of apoplastic water roughly p/RWC derivative curve at RWC = 1.0 is
corresponds to the primary hydration in usually taken as the bulk modulus of elas-
tissues (including both strong and weak ticity and used for purposes of comparison.
water-binding sites). A pressure probe technique can be used
One can expect that plant tissues would directly to determine the turgor pressure
respond differently to the loss of external, and the  for individual plant cells. This
symplastic and apoplastic water. The loss of technique is useful for continuous mea-
symplastic water can cause osmotic pertur- surement of cell turgor pressure, cell wall
bation of physiological and biochemical elasticity and hydraulic conductivity of the
processes, whereas the loss of apoplastic cell membrane in single cells (Hüsken et
water may disrupt the structure and func- al., 1978). The intracellular hydrostatic
02 Dessication - Chap 2 18/3/02 1:54 pm Page 60

60 W.Q. Sun

pressure is transmitted to the pressure rated salt solutions in closed desiccators

transducer via an oil-filled microcapillary until constant weights are achieved,
introduced into the cell, which transforms whereas an adsorption isotherm is devel-
into a proportional voltage. This technique oped by rehydrating dried tissues over satu-
permits volume changes and turgor pres- rated salt solutions. A desorption curve can
sure changes to be determined with an also be developed during drying of tissues
accuracy of 105–106 µl and 3–5  103 in any atmospheric condition by measur-
MPa, respectively. ing, at various points in time, the water
At present, very little information is content of the tissue and the equilibrium
available on cell wall properties of desicca- RH of its surrounding air in a closed con-
tion-tolerant plant tissues. Proctor (1999) tainer. Similarly, the dry tissue can be rehy-
found that two highly desiccation-tolerant drate with a given quantity of water to raise
liverworts have low values of bulk elastic the water content and equilibrium RH.
modulus. He thought that extensible cell Sophisticated instruments such as con-
walls might be a part of structural adapta- trolled atmosphere microbalance and
tion to rapid changes of cell volume in dynamic vapour sorption systems (Surface
their intermittently desiccated habitats. Measurement Systems, London, UK) use
Ultrastructural studies on dry mesophyll the latter methods. Desorption and adsorp-
cells of desiccation-tolerant Selaginella tion isotherms are used, respectively, to
lepidophylla by Thomson and Platt (1997) study the properties of dehydration and
showed highly folded cell walls and con- rehydration of plant tissues. Desorption and
tinuous apposition of plasmalemma to the adsorption curves are rarely the same: the
walls. Vicre et al. (1999) studied the cell desorption curve usually gives a higher
wall architecture of leaf tissues of water content than the adsorption isotherm.
Craterostigma wilmsii (a resurrection The difference in the equilibrium water
plant), and also observed extensive folding content between two curves is called hys-
of the cell wall during desiccation. The teresis. Hysteresis is evidence of the irre-
folding of the cell wall allows the plasma versibility of the sorption process, and
membrane to remain firmly attached to the therefore indicates the limited validity of
wall as the cell loses water. Biochemical the equilibrium thermodynamic approach
modifications of the cell wall were to investigate the dehydration–rehydration
observed during desiccation and rehydra- properties of plant tissues. Hysteresis might
tion, leading to the change in its tensile be an important issue when considering
strength that may prevent the total collapse critical water activities for desiccation
of the walls in the dry tissue and avoid stress during dehydration–rehydration
rapid expansion upon rehydration. The cycles and when investigating storage stabil-
change in cell wall elasticity during desic- ity after manipulation of moisture content of
cation can be determined easily by taking seeds and pollen.
the first derivative of the function of turgor
pressure on RWC. Theoretical models
Plant tissues show a sigmoid sorption
2.4.2. Analysis of water sorption isotherms isotherm (Fig. 2.4a). The inflection point of
the isotherm is believed to indicate either a
The water sorption isotherm is the depen- change of water-binding capacity and/or
dence of water content on water activity of the relative amount of ‘bound’ or ‘free’
the surrounding environment at a given water. Water sorption data are normally
temperature. There are two types of sorp- analysed using theoretical models, from
tion isotherms: desorption isotherm and which useful biophysical parameters of
adsorption isotherm (Fig. 2.4a). water relations are derived. Commonly
Conventionally, a desorption isotherm is used models include the Brunauer–
developed by drying fresh tissues over satu- Emmett–Teller (BET) model, the
02 Dessication - Chap 2 18/3/02 1:54 pm Page 61

Methods for Studying Water Relations Under Stress 61

Guggenheim–Anderson–de Boer (GAB) aw C − 1  1

model and the D’Arcy–Watt model. =  aw + (12)
M w (1 − aw )  M m  M mC
The BET model (Brunauer et al., 1938)
is derived from statistical and thermody- where aw is the water activity, Mw is equi-
namic considerations. The equation can be librium water content in the tissue, Mm is
written as: the BET monolayer (water content corre-


Water content



Water activity

Sorption enthalpy

Free energy


Water content

Fig. 2.4. The analysis of water sorption isotherms. (a) The typical shape of desorption curves and adsorption
curves of plant tissues. The difference between these two curves shows hysteresis, which indicates the
irreversibility of water sorption in the tissues during dehydration and rehydration. The sigmoid shape of
sorption 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) and
entropy (S) of hydration. Desorption curves can be used to calculate H and S of tissue hydration. See
text for detailed discussion.
02 Dessication - Chap 2 4/4/02 2:18 pm Page 62

62 W.Q. Sun

sponding to the monolayer hydration) and amount of water in those three regions can
C is temperature dependence for sorption be estimated. K is the number of strong
excess enthalpy (Brunauer et al., 1938, water-binding sites, multiplied by the mole-
1940). BET equation parameters, Mm and C, cular weight of water and divided by
can be calculated by plotting aw/[Mw (1  Avogadro’s number (6.023  1023); K is the
aw)] against aw. The y-axis intercept of the strength of the attraction of the strong water-
straight line is equal to 1/(MmC) and the binding sites for water; c is a measure (lin-
slope is equal to (C  1)/Mm. The BET is ear approximation) of the affinity and the
valid only for aw < 0.5, thus data points number of weak water-binding sites; k
within that range are used to estimate the relates to the number of multimolecular
monolayer value (Mm). The BET model is water sorption sites; and k relates to the
an effective method for estimating the activity of water (D’Arcy and Watt, 1970).
amount of water bound specifically to The number of water-binding sites in tissues
polar sites (monolayer), but cannot be used can be calculated from the derived equation
to give a complete estimation of specific coefficients. The number of strong, weak
hydration parameters. and multimolecular water-binding sites are
The GAB model is an extension of the KN/M, cN/(Mo), and kN/M, respectively,
BET model, taking into consideration the where N is Avogadro’s number, M is the
modified properties of the sorbing materi- molecular weight of water and o is the satu-
als in the multilayer region and the bulk rated vapour pressure of pure water.
liquid properties through the introduction The D’Arcy–Watt model has been used
of a third constant, K. The GAB equation is extensively for the analysis of desiccation-
written as: tolerant and desiccation-intolerant plant
tissues (Vertucci and Leopold, 1986, 1987a,
Mw = (13) b; Sun et al., 1997). Both the GAB and the
(1  Kaw)(1  Kaw + CKaw) D’Arcy–Watt models are valid over a wider
range of water activities for plant tissues.
where C and K are temperature-dependent The GAB model has some advantages over
coefficients. Constants, Mm, C and K are the D’Arcy–Watt model, which assumes the
estimated via the curve fitting of sorption three types of water-binding sites. The GAB
data. In the field of food sciences, the GAB model does not have such an assumption.
model is the most widely accepted due to For biological systems it is more reasonable
its accuracy and its validity over a wide to assume that the number of water-binding
range of water activities from 0.05 to 0.9 sites is changing continually along with the
(Rahman and Labuza, 1999). binding energies. Moreover, the GAB model
The D’Arcy–Watt model was developed can be more easily applied to other thermal
for the analysis of sorption isotherms of analyses (e.g. water-clustering function).
non-homogeneous materials (D’Arcy and
Watt, 1970). This model assumes that there
is a fixed number of water-binding sites Temperature dependency of water
with different discrete binding energies. The sorption
D’Arcy–Watt equation can be written as: Desiccation involves the transfer of liquid
K Kaw kkaw
water in plant tissues into the vapour
Mw = + caw + (14) phase. Temperature influences evaporation
1 + Kaw 1 − κaw
rate through the heat supply as well as
where K, K, c, k and k are equation coeffi- through its effect on the partial water
cients (adjustable parameters). The equation vapour pressure in air and the energy sta-
has three terms, which represent the tus of water in plant tissues. In isothermal
amounts of water that are strongly bound, conditions, air acts as an osmotic mem-
weakly bound and sorbed in multimolecular brane and equilibrium is often slow and
water clusters, respectively. For a tissue that dependent on temperature. An increase in
is in equilibrium with a given aw, the temperature generally results in a decrease
02 Dessication - Chap 2 18/3/02 1:54 pm Page 63

Methods for Studying Water Relations Under Stress 63

in equilibrium water content of plant tis- event. A high negative H value at low
sues at a given RH (i.e. water activity) or an water content suggests the strong affinity of
increase in equilibrium water activity at adjacent water molecules toward ionic sites
constant tissue water content. The shift of and/or other polar sites of the substrate. As
water activity at the constant water content water content increases, the H becomes
by temperature is mainly due to the change less negative (Fig. 2.4b). The primary hydra-
in water binding, dissociation of water, tion process (i.e. strong and weak binding
physical state of water or increase in solu- sites) is considered to be completed when
bility of solute in water. Tensile strength of the differential enthalpy of hydration (H)
water, the pressure holding molecules approaches zero (Luscher-Mattli and Ruegg,
together, increases by 81.6 mbars on aver- 1982; Rupley et al., 1983; Bruni and
age for a reduction of 1°C. Temperature Leopold, 1991). The change of S reflects
dependence of isotherm shift is described the relative degree of order, and the S peak
by the Clausius–Clapeyron equation: is presumably associated with the saturation
of all primary hydration sites. It should be
aw2 q +
w 1
ln = =  −  (15) clearly noted that the relationships of
aw1 R T
 2 T1
H/WC, G/WC and S/WC describe ther-
where q is the excess heat of sorption;
w is modynamic interactions between water and
the latent heat of vaporization for water biomaterials, but not necessarily the func-
(44.0 kJ kg1 at 25°C); R is the gas constant; tions of water and biological structures in
aw1 and aw2 are water activities for a given physiological processes. A possible associa-
equilibrium water content at temperature tion between water sorption behaviours and
T1 and T2, respectively. The plot of ln aw desiccation tolerance of plant tissues was
against 1/T at any given tissue water con- discussed in a number of studies (Vertucci
tent is a straight line and its slope gives (q and Leopold, 1987b; Farrant et al., 1988;

w)/R, from which the excess heat of Pritchard, 1991; Sakurai et al., 1995; Eira et
sorption, q, can be derived (Fig. 2.5a). al., 1999; Sun, 2000). No consistent differ-
In practice, some thermodynamic quan- ence in water sorption characteristics has
tities of tissue hydration can be calculated been found between desiccation-sensitive
according to isotherms at two different (recalcitrant) and desiccation-tolerant
temperatures. The aw1 and aw2 for a given (orthodox) seed tissues (Sun, 2000).
equilibrium water content at two tempera- The van’t Hoff relationship provides
tures can be taken from water sorption another convenient means to analyse tem-
curves or calculated from fitted sorption perature dependence of sorption isotherm.
equations (Fig. 2.5b and Fig. 2.7a). The van’t Hoff equation and the
Differential enthalpy of hydration (H, Clausius–Clapeyron equation are essen-
including q and
w), differential free tially the same in theory, but different in
energy of hydration (G) and differential their mathematical treatment of experimen-
entropy of hydration (S) are given by: tal data. The Clausius–Clapeyron equation
handles two temperature points, whereas
RT1T2 aw1
H ln (16) the van’t Hoff equation can handle a series
T2  T1 aw2
of temperature points at once. The van’t
G RT ln (aw) (17) Hoff equation expresses the relationship of
the equilibrium water activity (aw) for a
S H  G (18)
T given water content against the tempera-
ture (1/T) (Fig. 2.5b), and is written in its
These thermodynamic quantities are the differential mathematical form as:
functions of water content in tissues. The
relationships of H/WC, G/WC and ∂ ln aw  R ∂(1/T) (19)
S/WC provide important information with
regard to the hydration properties of tissues where T is absolute temperature in kelvin,
(Fig. 2.4b). Water sorption is an exothermic and R is the gas constant. The H is the
02 Dessication - Chap 2 18/3/02 1:54 pm Page 64

64 W.Q. Sun

differential enthalpy of water sorption. It is require the storage of desiccation-sensitive

important to note that the relationship seeds and other tissues in a refrigerated
between ln(aw) and (1/T) is not necessarily condition or at liquid nitrogen tempera-
a straight line. Within a relatively narrow ture. When the extrapolation is used, the
range of temperature, linear approximation non-linear nature of the relationship
may be used to calculate H accurately. between ln(aw) and (1/T) needs to be taken
However, there is considerable interest in into consideration. The study on tempera-
studying water sorption properties of bio- ture dependence of water sorption using
logical tissues at a much wider range of the van’t Hoff equation (Fig. 2.5a and b) is
temperature. For example, long-term used to establish the theoretic framework
preservation of genetic resources may for the optimization of germplasm preser-

(a) 0.0
0.09 g g–1 dw

ln (aw or RH/100)

–2.0 0.04 g g–1 dw


–4.0 0.02 g g–1 dw

3.3 3.4 3.5 3.6

1/Temperature (K,  10–3)

Water content (g g–1 dw)


0.10 50% RH



10% RH

0 5 10 15 20 25
Temperature ( C)

Fig. 2.5. (a) Temperature dependence of water sorption for the same seed material at different water
contents (i.e. the van’t Hoff plot). Drawn with data from Eira et al. (1999). (b) Equilibrium water content at
specific water activities as a function of temperature for whole seeds of Coffea arabica cv. Mundo Nova.
This relationship is called ‘isopleth’.
02 Dessication - Chap 2 18/3/02 1:54 pm Page 65

Methods for Studying Water Relations Under Stress 65

vation protocols (Vertucci et al., 1994, polar hydration sites. A recent study using
1995; Eira et al., 1999). the D’Arcy–Watt model suggested that
water redistribution among different types
of hydration sites might be related to the Monolayer hydration and water-
rapid loss of seed viability during storage
clustering function
after osmotic priming and drying back (Sun
The monolayer hydration values of plant et al., 1997).
tissues, the amount of water bound to spe- Water clustering in binding sites is
cific polar sites, can be easily determined, another important hydration event that is
using BET or GAB isotherm models. For of significance to desiccation tolerance of
most plant tissues and their major chemi- plant tissues and the survival of tissue in
cal components, the monolayer value at the dried state. Clustering formation is
ambient temperature is estimated to be related to a number of transport phenom-
between 0.04 and 0.09 g g1 dw using the ena. For example, clustering reduces the
BET or GAB model (Rahman and Labuza, effective mobility of water by increasing
1999). The BET monolayer value of many the size of the diffusing molecular group or
orthodox seeds was also found to be in this by increasing the tortuosity of diffusion
range (Vertucci and Leopold, 1987a,b; paths (Stannett et al., 1982). The range of
Bruni and Leopold, 1991; Vertucci and water activity where the self-association of
Roos, 1993; Sun et al., 1997). The mono- water takes place can be examined by the
layer value of Typha pollen was much less clustering function (Lugue et al., 1995;
than that of orthodox seeds (Buitink et al., Dominguez and Heredia, 1999). The clus-
1998b). The monolayer hydration is gener- tering function is written as:
ally complete at a water activity of
G11/V1 V2[(aw/V1)/aw]  1 (20)
0.20–0.30 (i.e. 150 to 250 MPa). It is
important to note that the monolayer value where G11/V1 is the clustering function, V1
decreases rapidly as temperature increases, is the volume fraction of water, V2 is the
and increases as temperature declines. The volume fraction of biopolymers, and aw is
monolayer water is of great importance for water activity (Zimm and Lundberg, 1956).
the survival of many dry organisms (e.g. The subscript ‘11’ in G11/V1 denotes the
spores, pollen grains and seeds) during water–water interaction as a function of
storage. In food science, the water activity water content (component 1). The cluster-
at the monolayer value is defined as the ing function can be applied to an isotherm
critical water activity. At a water activity sorption model such as the GAB equation
above 0.20–0.30, the rate of chemical reac- with some modifications. The GAB equa-
tions begins to increase significantly tion needs to be rewritten in terms of vol-
because of the greater solubility and mobil- ume fraction instead of weight fraction.
ity of the reactants. At water contents The GAB equation can be rewritten as:
below the monolayer value, the rate of MmCKaw
lipid oxidation and associated free radical Mw V1p1/V2p2 (21)
(1Kaw)(1Kaw CKaw)
damage increases. The presence of mono-
layer water inhibits the undesirable inter-
actions between polar groups on adjacent
carbohydrate or protein molecules, thereby (1Kaw)(1Kaw CKaw)
preserving their rehydration ability and aw/V1 (22)
biological functions (Rahman and Labuza,
1999). where p1 and p2 are the density of water
There is no defined monolayer parame- and biopolymers. The density of sorbed
ter in the D’Arcy–Watt model. However, water is assumed to be equal to 1.0 g cm3.
the first term of the D’Arcy–Watt equation Substituting aw/V1 in Equation (20) with
may be used as an approximation, as it rep- Equation (22), the clustering function can
resents water that is bound strongly to be expressed as:
02 Dessication - Chap 2 18/3/02 1:54 pm Page 66

66 W.Q. Sun

(2KCKMmCK) (2CK22K2)aw expressed as the percentage of the corre-

G11/V1 (23)
MmCKp2 sponding maximum value in the fully
hydrated tissues (Fig. 2.7b). Therefore, the
According to Equation (23), G11/V1 is pro-
occupancy relationship indicates the
portionally related to aw and the reciprocal
degree of hydration for different types of
of polymer density (p2) function. G11/V1
hydration sites during desiccation. Figure
can be solved easily by the substitution of
2.7b shows that the occupancy for three
Mm, C and K constants from the GAB equa-
types of hydration sites changes as the
tion. Figure 2.6 shows a plot of the water-
water content of Q. rubra seed tissues
clustering function of soybean axes. The
decreases during desiccation. Desiccation
clustering plot is basically a straight line
of seed tissues to 0.30 g g1 dw (the critical
against water activity. When G11/V1 is
water content) removed about 90% of mul-
greater than 1, water is expected to clus-
tilayer molecular sorption water, but only
ter (Zimm and Lundberg, 1956). The auto-
about 10% of water molecules attached to
association (clustering) of water in a few
the weak hydration sites in seed tissues.
desiccation-tolerant seeds is observed to
The removal of water from weak hydration
occur at water activity ranging from 0.55 to
sites appears to be related to desiccation
0.60 (W.Q. Sun, unpublished data).
damage in Q. rubra seeds (Sun, 1999). The
critical water content of Q. robur axes also Occupancy of water-binding sites corresponds to the amount of matrix-bound
water (Pritchard and Manger, 1998).
The D’Arcy–Watt model can be used to However, the question of whether the
examine the occupancy of water-binding water-binding or sorption behaviour in
sites as a function of water content accord- seed tissues is related to their desiccation
ing to Luscher-Mattli and Ruegg (1982). tolerance remains unresolved. The loss of
The occupancy represents the amount of viability in many recalcitrant seeds occurs
water attached to certain hydration sites, at a water content that is much higher than

Clustering function




0.0 0.2 0.4 0.6 0.8 1.0

Water activity

Fig. 2.6. Water-clustering function showing the waterwater association in soybean seed axes as a function
of equilibrium water activity. Apparent water clusters first appear at a water activity of 0.58 (arrow). The
water-clustering function Equation (23) was solved through the study on biopolymer volumetric change
during hydration [i.e. P2= f (V1)] by applying water sorption analysis. See text for further explanation.
02 Dessication - Chap 2 18/3/02 1:54 pm Page 67

Methods for Studying Water Relations Under Stress 67

that of ‘bound’ water (Pammenter et al., recently released manual by Bell and
1991; Berjak et al., 1992). Clearly, more Labuza (2000). This book generally dis-
comprehensive studies are needed. cusses water activity in food materials, but
Readers who wish to know more about the principles are also applicable to plant
water sorption analysis may refer to a desiccation tolerance studies. Practical
(a) 0.8

5 C

4.974 /  0.0219 / 
0.6 WC = + 0.0673 /  +
1 + 90.2 /  1 – 0.990 / 
Water content (g g–1 dw)

25 C
1.279 /  0.026 / 
0.4 WC = + 0.0373 /  +
1 + 27.4 /  1 – 0.986 / 



0.0 0.2 0.4 0.6 0.8 1.0

Water activity


80 5 C
Sorption sites occupied (%)

25 C

Strong binding site

40 Weak binding site

Multilayer sorption

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Water content (g g–1 dw)

Fig. 2.7. (a) The interpretation of desorption isotherms of Quercus rubra cotyledonary tissues, using the
D’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. rubra
cotyledonary tissues at different water contents. The occupancy is based on the percentage of the
corresponding maximum values in the fully hydrated state (i.e. full turgor). The change of occupancy reveals
how and when water is removed in different types of hydration sites during dehydration.
02 Dessication - Chap 2 18/3/02 1:54 pm Page 68

68 W.Q. Sun

examples are provided to elucidate how to curves are biphasic. During the first drying
solve many equations. phase, the loss of water follows a simple
exponential function. During the second
phase, water content does not decrease
2.5. Measurement of Drying Rate and much because the tissue is very much
Desiccation Stress closer to achieving equilibrium with the
air. Because water loss during the first
2.5.1. Driving force for water loss and phase is described by an exponential func-
expression of drying rate tion, the rate constant () of water loss can
be used as an expression of drying rate.
The loss of water from tissues depends on
two factors: the gradient in water potential
between tissue surface and external air or 2.5.2. Quantification of desiccation stress
solution, and the hydraulic conductivity of
the tissue. The volume flow of water from The response of plant tissues to desiccation
the tissue to air can be described by: is significantly affected by dehydration con-
ditions, such as drying rate (see Chapter 3).
Vw ALp (o  i) (24)
Under slow-drying conditions, plant tissues
where Vw is the volume flow of water per stay longer at intermediate water contents.
unit time (m3 s1), A is the surface area of Fast drying is often reported to improve
the tissue (m2) and Lp is the hydraulic con- desiccation tolerance of recalcitrant plant
ductivity of the tissue (m s1 Pa1). The o seeds (reviewed by Pammenter and Berjak,
and i are water potentials of external air 1999). There is no doubt that the level of
and the tissue, respectively. The difference desiccation stress would vary with drying
in water potential (o  i) is a measure of rate, and the questions are: (i) how desicca-
the driving force for dehydration. i is a tion stress can be quantified; and (ii) how
function of time that describes the decrease drying rate affects the level of desiccation
in tissue water potential during drying. The stress. The change in chemical potential of
hydraulic conductivity of the tissue (Lp) is a cellular water is a good measure for the
measure of the diffusional resistance of the degree of desiccation stress. When the
water transport pathway within the tissue. chemical potential of water is compared,
A good measure for the drying rate is *w and mwgh in Equation (5) cancel out.
essential for comparative studies on desic- The difference in chemical potential of cel-
cation tolerance. According to Equation lular water between the dehydrated state
(24), the rate of water loss from the tissue is (Dw) and the hydrated state (Hw) is:
time-dependent. Under constant RH and –
HwDw RT(ln aHwln aDw) Vw(PHwPDw) (26)
temperature, the water content of the tissue
is expected to decrease exponentially over According to Equation (26), the degree of
time until i reaches o (Fig. 2.8a). The desiccation stress is proportional to
curve of water loss can be described by: changes in osmotic potential and hydrosta-
tic pressure (P) in cells. Therefore, the
WC =  exp(t) (25)
change of water potential, d/dt, can be
where  is the initial water content,  is the used to quantify the level of desiccation
rate constant of water loss, and t is time of stress. Figure 2.8b shows the plots of tissue
drying. This relationship was first used by water potential against drying time. Under
Tompsett and Pritchard (1998) to compare the conditions of constant temperature and
the dehydration rate of A. hippocastanum relative humidity, such plots are straight
seeds. Drying curves of other seed tissues lines down to the fraction of apoplastic
have been examined under a wide range of water. Water potential of the tissue
desiccation conditions, and they conform decreases faster and deviates away from
to Equation (25) (Li and Sun, 1999; Liang the straight line when the apoplastic water
and Sun, 2000). Typically, water content is lost (see Fig. 2.3b, the low-RWC break
02 Dessication - Chap 2 18/3/02 1:54 pm Page 69

Methods for Studying Water Relations Under Stress 69

point). The slope of each straight line por- mathematical evaluation of this linear rela-
tion (d/dt) represents the degree of direct tionship will not be presented here.
physical stress under different desiccation If the plant tissue is viewed as a viscoelas-
conditions. The relationship between tic system, the mechanical stress caused by
d/dt and the rate constant () of water water loss can be considered as a simple
loss (drying rate) is linear (Fig. 2.9). A stress–strain response. The physico-chemical


33% RH
88% RH
Water content (g g–1 dw)

94% RH


0.8  = 0.00502

 = 0.0211

0.0  = 0.103

0 80 160 240 320 400

Drying time (h)

33% RH
88% RH
–5 94% RH
Water potential (MPa)


d/dt = –0.032
d/dt = –0.160

d/dt = –0.689

0 80 160 240 320 400

Drying time (h)

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 with
exponential functions (WC =  exp(t)), and the rate constants of water loss, , are shown near each
curve. (b) Plots of tissue water potential against drying time. The mechanical stress on tissue caused by the
water loss can be considered as a simple stress–strain response. The slope of /time plot, d/dt, is directly
related to the intensity of desiccation stress. Data from Liang and Sun (2000).
02 Dessication - Chap 2 18/3/02 1:54 pm Page 70

70 W.Q. Sun


Dehydration rate (–d/dt)




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 relative
humidities ranging from 6 to 94% to achieve different drying rates and stress conditions. Drawn using data
from Liang and Sun (2000).

aspect of desiccation stress can be assessed by treats biological systems as fully reversible
integrating the function of tissue water poten- ones and does not give much considera-
tial over time (Fig. 2.8b). Figure 2.10a and b tion to the term time, one of the most
shows an example of the quantitative analysis important factors in any biological
of desiccation stress. The cumulative water response. This limitation is particularly
stress during desiccation at three different RHs relevant to the study of desiccation. The
was plotted against drying time and water application of thermodynamics is gener-
content, respectively. Under the slow-drying ally sufficient in many cases for fully
condition (94% RH), the mechanical stress hydrated tissues. However, at intermediate
(d/dt) is small (Fig. 2.8b); however, the or low moisture levels, the non-equilib-
cumulative physico-chemical stress is remark- rium, kinetic principles play a more
ably high because the time to dry to the same important role. During desiccation, the
water content increases exponentially as the biological system basically shifts from a
drying rate decreases (Fig. 2.10a and b). The thermodynamic state to a non-equilibrium
quantitative analysis of mechanical and kinetic state (Leopold et al., 1994; Sun et
physico-chemical aspects of desiccation stress al., 1994; Sun, 1997, 1998). The thermo-
has led to an understanding of the physiologi- dynamic approach does not sufficiently
cal basis of the optimal drying rate to achieve address the kinetics of various reactions
the maximum desiccation tolerance of and processes in intermediate- to low-
Theobroma cacao axes (Liang and Sun, 2000). moisture systems.
The kinetic and functional approach to
cellular water relations focuses on how the
2.6. Water Relations – the Kinetic and interactions between water and other cellu-
Functional Approach lar components can influence the struc-
tures and biological properties of each
The thermodynamic approach to water other. In this chapter, principles of the
relations has its limitations, because it kinetic approach and the interactions
02 Dessication - Chap 2 18/3/02 1:54 pm Page 71

Methods for Studying Water Relations Under Stress 71


Cumulative water stress (MPa  h)



33% RH

500 88% RH
94% RH

0 80 160 240 320 400

Drying time (h)

33% RH
Cumulative water stress (MPa  h)

88% RH
94% RH



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 water
stress is calculated by integrating the /time function (see text for details). (b) Cumulative water stress as the
function of tissue water content under different desiccation conditions. Cumulative stress is much higher in
slow-drying conditions because the dehydration time increases exponentially as drying rate decreases.

between water and many other biomole- Instead, a general account will be offered,
cules will not be discussed in detail. These so that readers can be confident about
topics will be covered in other chapters on choosing the appropriate method to quan-
desiccation damage and mechanisms of tify particular water properties in studies
desiccation tolerance (see Chapters 9–12). on desiccation tolerance.
02 Dessication - Chap 2 18/3/02 1:54 pm Page 72

72 W.Q. Sun

2.6.1. General considerations the study on water in skeletal muscle as an

example, fast techniques (e.g. laser Raman Time scale spectroscopy, infrared spectroscopy and
dielectric relaxation) could not find any
Any study on the state of water by a bio- intramolecular differences in hydrogen
physical technique involves measuring bond lengths, angles or strength between
parameters of time scale directly or indi- muscle water and pure water (Beall, 1981).
rectly. Biophysical techniques often use the However, slower techniques, such as
diffusional correlation time as the time nuclear magnetic resonance (NMR) (Fung
scale to make comparisons of the measure- and McGaughy, 1974), electron paramag-
ments on water. Many of the physical prop- netic resonance (EPR) (Belagyi, 1975) (see
erties of water are theoretically related to Chapter 4), fluorescence polarization
the diffusional correlation time (D) of (Knight and Wiggins, 1979) and freezing
water, the average time between jumps in behaviour (Rustgi et al., 1978), showed a
position for water molecules in the system. restricted motion of at least a portion of cell
Two critical numbers on this time scale (D) water. This situation is identical to pho-
are 105 s and 1011 s. The D of water in ice tographing moving objects with different
is 105 s and in pure liquid is 1011 s, one shutter speeds. If the shutter speed is very
million times faster. Under normal condi- high relative to the velocities of two moving
tions, water in biological systems exists in a objects (e.g. 1/800 s), the photo will proba-
state somewhere between the solid state of bly not record any information as to
crystalline ice and the liquid hydrogen- whether one object is moving faster than
bonded lattice of pure water. In low- the other. On the other hand, if the shutter
moisture systems, however, the D of water speed is too slow (e.g. 1/2 s), the images of
molecules in the intracellular glasses would both moving objects will be blurred and no
be much slower than 105 s. Readers may meaningful information can be obtained
refer to a series of studies on molecular from such a photo. Only with a proper
mobility in seeds and pollen by Buitink et shutter speed can the photo reflect the dif-
al. (1998a, 1999, 2000a,b,c; Chapter 10 of ference in the velocity between the two
this volume). moving objects.
The ability of a biophysical technique to
yield useful information about the physical Structural complexity and dynamics
state of water largely depends on how fast a
of molecular ordering
measurement can be made. Slow tech-
niques which require measurement times Hydration of protein is a good example,
greater than the diffusional correlation time illustrating the complexity of structures
yield an average over all molecules in the and functions for water in biological sys-
population with a kinetic contribution from tems. When a mole of lysozyme is hydrated
diffusion, whereas fast techniques can yield by 60 moles of water (~ 0.07 g g1 dry pro-
instantaneous information about intramole- tein), water is primarily located to charged
cular factors such as H–O bond lengths and groups, and its mobility is reduced at least
hydrogen bond angles (geometric factors). by 100 times relative to pure water. At this
To choose the appropriate technique, one low hydration level, no structural differ-
has to bear in mind that the type of prop- ence is observed in the protein. As hydra-
erty or structure that a technique can probe tion increases to 220 moles of water per
is related to the time scale. Generally speak- mole of protein (~ 0.25 g g1 dry protein),
ing, fast techniques would probably pro- water begins to form clusters of various
duce data of instantaneous structures of sizes and arrangements around the charged
water and other biomolecules, but slow and polar sites of the protein. Internal pro-
techniques would provide more informa- tein motion (H exchange) increases by 1000
tion about the interactions between water times to be comparable to that of solution,
molecules and their environment. Taking while the protein sample is still a solid. At
02 Dessication - Chap 2 18/3/02 1:54 pm Page 73

Methods for Studying Water Relations Under Stress 73

a hydration level of 300 moles of water per tions that a model allows, have to be taken
mole of protein (~ 0.38 g g1 dry protein), into consideration for experimental design
enzymatic activity, mobility of bound lig- and implementation. If possible, additional
and and fast water motion become easily experiments should be conducted to con-
detectable. Dielectric relaxation measure- firm the results and to examine whether the
ments show two water relaxation times, assumptions are satisfied. Unfortunately,
one of 2  1011 s, close to that of bulky biophysical models or equations have fre-
pure water, and the other of 109 s, indicat- quently been used to analyse the actual
ing the heterogeneous nature of water data without checking their assumptions.
behaviours and functions (Rupley et al., Worse still, sometimes a model was
1983). At the hydration level of 0.38 g g1 selected after the entire experiment was
dry protein, each water molecule covers, on completed (Beall, 1983).
average, 20 Å2 of protein surface, which is Conceptual models have been used for
twice the effective area of a water molecule. the interpretation of the data on water in
Yet several populations of water molecules biological systems, with many pitfalls. For
are observed at such low hydration. example, the ‘two-fraction fast-exchange
The surfaces of membranes, proteins model’ (Zimmermann and Brittin, 1957)
and other macromolecules impose geomet- assumes that there is a small fraction of
ric limitations on the possible arrange- highly immobilized cell water on the sur-
ments of hydration water. Interfacial water face of macromolecules (ice-like) and a
molecules, being part of the network of bio- large fraction of cell water that behaves like
logical interfaces, are dynamically oriented bulk water. Rapid exchange between the
and exhibit restricted motion (i.e. are two populations yields reduced average
‘bound’). The ordering of molecules on var- properties. This two-fraction model can be
ious biological surfaces is strictly local, written as:
and may fluctuate rapidly between possible 1 X (1  X)
arrangements. Ideally, a biophysical tech- (27)
T * Tslow TH2O
nique used to study the state of water in
biological systems should have the resolu- where T * is measured (average) relaxation
tion to differentiate closely related struc- time; X and (1X) are the fraction of
tures or populations. However, as discussed immobilized water and the fraction of cell
earlier, kinetic measurements reflect only water that is like bulk water, respectively;
average or time-average properties over all Tslow and TH2O are the relaxation times of
molecules in the system and, in many the slow fraction and bulk water. In this
cases, do not provide definitive answers to equation, there is only one measured para-
the questions of interest. To interpret the meter (T *), but three unknown quantities
data of kinetic measurements, the investiga- (X, Tslow and TH2O). To estimate X, Tslow
tor must impose a conceptual model, which and TH2O must be arbitrarily assigned. This
may be controversial (Beall, 1983). Such model represents a simplistic view on the
studies are often misunderstood and misin- dynamics of water in biological systems,
terpreted by readers who are less familiar which is still in use by some workers. If
with the biophysical techniques used. one intends to solve Tslow, then X must be
estimated through other methods. When X
is equated to the ‘non-freezable fraction’ or The model-dependent interpretation:
‘osmotically inactive fraction’, additional
the pitfalls
assumptions are made. By redefining X as
The selection of a theoretic model or the an adjustable parameter in different sys-
development of a new model is an impor- tems, a new model is established (Beall,
tant step in any kinetic study, which 1983). This example clearly shows the
should be done before actual measure- uncertainty of biophysical interpretation.
ments are made. The assumptions that a Simply because the model is easy to use
model contains, and the specific predic- and fits the data well it does not necessarily
02 Dessication - Chap 2 18/3/02 1:54 pm Page 74

74 W.Q. Sun

mean that it represents the true state of measurements is briefly summarized in

water in a system. Of course, all models are Table 2.1. Readers are advised to consult
open to interpretation. other references, including those cited
However, it is possible to measure above, and Chapter 4 in this volume. In this
changes in the properties of water in living chapter, only a brief introduction will be
systems that correlate with physiological provided on several techniques that have
functions (Clegg et al., 1982; Clegg, 1986; been increasingly used in recent years.
Bruni et al., 1989). Changes in dynamic
properties of water at different hydration Differential scanning calorimetry
levels indicate the existence of different
fractions of water, which may vary in struc- Differential scanning calorimetry (DSC) is
ture and property and presumably play dif- probably the most commonly used thermal
ferent biological roles. Studies have analysis technique. It has been used by a
identified the existence of at least four or number of workers to study the possible
five fractions of water, presumably relating relationship between freezing, desiccation
to different interactions between water and tolerance and water properties in plant tis-
cellular constituents (Clegg, 1986; sues (Williams and Leopold, 1989;
Ratkovic, 1987; Vertucci, 1990; Pissis et al., Vertucci, 1990; Pammenter et al., 1991;
1996; Sun, 2000). Hydration levels corre- Berjak et al., 1992; Sun et al., 1994; Vertucci
sponding to these fractions of cellular et al., 1994, 1995; Buitink et al., 1998b;
water are associated with the onset of vari- Pritchard and Manger, 1998; Sun and
ous metabolic activities in organisms Davidson, 1998; Sun, 1999). DSC measures
(Clegg, 1986). the heat flow of plant tissues associated
with various thermal events during cooling
and/or heating scans. Such thermal events
2.6.2. Biophysical techniques include phase transitions (e.g. freezing,
(see also Chapter 4) melting, glass transition, etc.), polymor-
phism, thermochemistry and the kinetics
Kinetic properties and functions of water for a variety of complex reactions (e.g. in
have been studied, using calorimetry vivo protein denaturation). The key idea
(Ruegg et al., 1975; Bakradze and Balla, involved in DSC measurement of water sta-
1983; Vertucci, 1990; Sun, 1999), infrared tus is that thermal changes of water and
(IR) and Raman spectroscopy (Careri et al., their corresponding quantities of energy
1979; Cameron et al., 1988), NMR spec- are greatly affected by the presence of other
troscopy (Fung and McGaughy, 1974; biomaterials in plant tissues, and that ther-
Mathur-de Vre, 1979; Seewaldt et al., 1981; mal behaviours of other biomaterials in
Rorschach and Hazlewood, 1986; Ratkovic, plant tissues are affected by water content.
1987), quasi-elastic neutron-scattering For example, as water content decreases,
spectroscopy (Lehmann, 1984; Trantham et the onset freezing and melting temperature
al., 1984) and dielectric relaxation tech- of water decreases due to solute concentra-
niques (Harvey and Hoekstra, 1972; tion, while at the same time glass transition
Kamiyoshi and Kudo, 1978; Clegg et al., temperature of the tissue increases due to
1982; Pissis et al., 1987, 1996; Bruni and the reduced plasticization effect by water.
Leopold, 1992). These techniques differ By analysing thermal behaviours of water
greatly in how and what they measure with and biomaterials as a function of water
respect to the dynamic properties and content, temperature and time, the status of
structures of water and other biomolecules. water in plant tissues can be studied and
A great deal of confusion over the physical the water status correlated to its biological
state of water in biological systems has functions.
resulted from the separation of information Technically, DSC is really a quite simple
obtained with diverse techniques applied method. There are two cells in the DSC
to similar systems. The nature of different detector, one reference cell and one sample
02 Dessication - Chap 2 18/3/02 1:54 pm Page 75

Methods for Studying Water Relations Under Stress 75

cell. An empty crucible is placed into the Thermally stimulated current (TSC)
reference cell and a crucible containing the method
tissue sample is placed into the sample
Different dielectric relaxation techniques
cell. During a DSC experiment, both refer-
had been used previously to study the
ence cell and sample cell are cooled and/or
properties of water in biological systems
heated at a constant rate over a range of
(Harvey and Hoekstra, 1972; Kamiyoshi
temperatures. When a thermal event occurs
and Kudo, 1978; Clegg et al., 1982; Careri
in the tissue, it releases or absorbs heat and Giansanti, 1984). More recently, the
energy (i.e. heat flow). A plot of heat flow TSC technique has been employed to study
as a function of temperature is called a the mode of hydration and water organiza-
thermogram, from which the thermal tion in plant tissues (Pissis et al., 1987,
behaviour of the tissue can be deduced. 1996; Bruni and Leopold, 1992; Sun et al.,
Glass transition is usually marked by a 1994; Sun, 2000). This technique is capable
stepwise shift in the baseline of a thermo- of providing information concerning the
gram. This distinguishes it from freezing mobility and rotational freedom of hydra-
and melting transitions, which produce tion water, hydration sites and mechanisms
peaks. A melting event is accompanied by (Mascarenhas, 1980; Pissis et al., 1987,
an endothermic peak and a freezing event 1996; Pissis, 1990; Bruni and Leopold,
is accompanied by an exothermic peak. 1992). The TSC technique is based upon:
The area under the particular peak repre- (i) the dependence of the microdynamics of
sents the total heat energy or enthalpy water dielectric relaxation on their sur-
change (H) for the event. DSC is a highly roundings resulting in different dielectric
informative tool for analysing biological relaxation times for water in different frac-
materials. Other information such as transi- tions; and (ii) the influence of water on the
tion temperature and heat capacity change dielectric relaxation mechanisms of other
(Cp) of the tissue upon cooling or heating biomolecules (similar to those used for
can also be calculated from a thermogram. DSC measurements).
The difficulty in analysing DSC thermo- The TSC method measures the tiny
grams lies in the correct identification of current generated by the thermally acti-
origin for thermal events in heterogeneous vated release of stored dielectric polariza-
biological samples. For example, lipid tran- tion during controlled heating and
sition in seeds can mask the actual glass basically consists of three steps: (i) the
transition (Williams and Leopold, 1989) polarization of a sample by a strong d.c.
and interfere with the accurate calculation electric field at a particular temperature;
of freezing and melting enthalpies (Sun, (ii) ‘freeze-in’ the polarization by cooling
1999). down to a sufficiently low temperature

Table 2.1. Biophysical techniques used to study the dynamic and structural properties of water and
macromolecules 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) 104 ~100 + (+) (+) +
Relaxation (NMR, EPR, dielectric) 1011 ~100 + + (+)
Ultrasonic absorption 1010 ~105 + + +
Quasi-elastic neutron scattering 1013 ~107 + + +
Infrared and Raman spectroscopy 1016 ~1012 + + + +

DSC, differential scanning calorimetry; DTA, differential thermal analysis; NMR, nuclear magnetic resonance;
EPR, electron paragmagnetic resonance.
02 Dessication - Chap 2 18/3/02 1:54 pm Page 76

76 W.Q. Sun

(e.g. liquid nitrogen temperature) while magnetic field at the nucleus.) Since the
the field is still on; and (iii) the measure- electron density around each nucleus in a
ment of the TSC spectrum during heating molecule varies according to the type of
after the d.c. field is disconnected (Bruni nuclei and its molecular environment, the
and Leopold, 1992). When a polarized tis- opposing field and thus the effective field
sue reaches a temperature at which dipole at each nucleus will differ, which is called
molecules (such as water) relax (lose their ‘chemical shift’. The chemical shift of a
fixed orientation), a tiny current is gener- nucleus is the difference between the reso-
ated and recorded. From a TSC spectrum, nance frequency of the nucleus and a stan-
several important physical parameters can dard (relative to the standard, expressed in
be obtained, including the intensity of p.p.m., ). The chemical shift is a very pre-
depolarization charge (peak size, related cise measure of the chemical environment
to the size of the water pool), depolariza- around a nucleus.
tion temperature, its activation energy and The major frustration for many biolo-
static permittivity. The measurement of gists wishing to understand and to use
dielectric relaxation properties of water NMR is the complexity of the subject.
and water-plasticized biomolecules offers However, as with other physical tech-
valuable insight into the organization of niques used in studies of biological sys-
water in plant tissues and the molecular tems, NMR may be used in an ‘empirical’
interactions between water and other bio- mode, simply examining the variation of
molecules during desiccation (Bruni and an NMR parameter with the change of
Leopold, 1992; Sun, 2000). experimental variable (e.g. water content)
(James, 1993). Figure 2.11 shows 1H-NMR
spectra of mung bean seeds at three water Nuclear magnetic resonance (NMR)
contents. The 1H-NMR spectra were broad,
NMR spectroscopy studies the interaction with the line width in the order of 103 Hz.
of electromagnetic radiation with matter. It Two NMR peaks were easily identifiable.
is a powerful tool for the studies of kinetic The peak of water in the immobile fraction
motion of water in tissues and of macro- had a peak maximum  (chemical shift)
molecule/water or membrane/water inter- value of 4.3 p.p.m. relative to the proton in
actions. Solid-state NMR can be used to D2O, which was used as a standard refer-
determine the molecular structure of solid ence. The peak of water in the mobile frac-
tissue samples. Solid-state H-NMR is often tion had a peak maximum  value at the
used to investigate the relaxation character- same place as the proton in D2O (i.e.  = 0
istics of the protons of water molecules in p.p.m.). At a water content of 0.07 g g1
low-moisture biological systems (Mathur- dw, water appeared to exist primarily in
de Vre, 1979; Seewaldt et al., 1981; the immobile fraction. The very small pro-
Rorschach and Hazlewood, 1986; Ratkovic, portion of water in the mobile fraction
1987; Chapter 4). The basic principle of H- appeared as a shoulder in the spectrum.
NMR is that each of two hydrogen nuclei The amount of water in the mobile fraction
in a water molecule possesses a single spin increased rapidly as water content
proton, which will cause the nucleus to increased. At 0.24 g g1 dw, two peaks
produce an NMR signal. When an atom is merged almost completely as one peak,
placed in a magnetic field, the spin of its centred at  = 0 p.p.m. The relative propor-
electrons will orient toward the direction tion of the immobile and mobile water frac-
of the applied magnetic field. This orienta- tions may be estimated using the standard
tion produces a small local magnetic field signal processing techniques.
at the nucleus that opposes the externally Two important spin relaxation parame-
applied field, resulting in a smaller mag- ters are T1, the spin–lattice relaxation, and
netic field (i.e. effective field) at the T2, the spin–spin relaxation time. The
nucleus than the applied field. (Note that spin–lattice relaxation (T1) involves the
in some cases it might also enhance the exchange of energy with the environment
02 Dessication - Chap 2 18/3/02 1:54 pm Page 77

Methods for Studying Water Relations Under Stress 77

(the lattice), and is caused by fluctuating come into resonance with monochromatic
local magnetic fields arising from the radiation. The magnetic field of most com-
motion of the molecules. The spin–spin mercial ESR spectrometers is about 0.3 T,
relaxation (T2) characterizes interactions corresponding to resonance with an elec-
between spins and is related to the width tromagnetic frequency of ~10 GHz and
of the NMR peak (Fig. 2.11). T1 and T2 can wavelength of ~3 cm. Therefore, the range
be used to study chemical kinetics and of applicability of ESR is narrower than
rotational and conformational motion of that of NMR. ESR is basically a microwave
molecules. technique, and is one of the fastest-growing
areas in analytical instrumentation because
of recent and remarkable achievements in Electron spin resonance
microwave technology. ESR consists of a
(see also Chapter 4)
microwave source, a cavity, a microwave
Electron spin resonance (ESR) or electron detector and an electromagnet. The sample
paramagnetic resonance (EPR) is related to is placed in a glass or quartz tube, which is
NMR. It is the study of molecules with inserted into the cavity. The ESR spectrum
unpaired electrons (free radicals, transition is obtained by measuring the microwave
metal complexes, triplet states, etc.) by absorption as the magnetic field strength is
observing the magnetic fields at which they continuously changed. This method

Signal amplitude

0.24 g g–1

0.14 g g–1

0.07 g g–1

–16 –8 0 8 16 24
Chemical shift (, p.p.m.)

Fig. 2.11. The 1H-NMR spectra of water in mung bean seeds at three water contents. The width of all
spectra was 4000 Hz. D2O was used as a standard reference. The inset shows the assignment of 1H-NMR
signal into two different water fractions: mobile water (fraction 1) and immobile water (fraction 2). The
spectrum was recorded with FX90QNMR (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-establish
the equilibrium via spin–lattice and spin–spin relaxation before the next scan (W.Q. Sun, unpublished data).
02 Dessication - Chap 2 18/3/02 1:54 pm Page 78

78 W.Q. Sun

detects the number of ‘unpaired spins’ of cals) is commercially available. By incor-

electronic charges. The ‘strange’ ESR spec- porating some probes such as nitroxide
trum is the first derivative of the derivatives into tissues before drying,
microwave energy absorption (Fig. 2.12). detailed studies may be undertaken on the
The hyperfine structure (splitting of indi- changes in the aqueous and non-aqueous
vidual resonance lines into components) of intracellular environments upon desicca-
an ESR spectrum is a fingerprint that helps tion. The ESR technique provides a fairly
to identify free radical species in the sam- direct measurement of the change in cyto-
ple and characterize their environments. plasmic viscosity, which probably plays
The ESR technique does not directly an important role in metabolic down-
measure water properties in tissues; how- regulation in desiccation tolerance (see
ever, it can be used to study many ques- Chapter 10).
tions that are related to desiccation. Using
an ESR spin-labelling technique, Belagyi
(1975) reported that a portion of cell water 2.7. Concluding Remarks
in muscles exhibited a restricted motion. In
recent years, Hoekstra and his co-workers Comparative studies play a key role in
have used this technique to study mem- understanding the mechanisms or strate-
brane behaviours, molecular mobility, cyto- gies of various organisms in the survival of
plasmic viscosity and partitioning of desiccation. The water status of tissues in
amphiphilic molecules of desiccation-tol- desiccation tolerance studies should be
erant and desiccation-intolerant plant tis- expressed precisely by preferred thermody-
sues upon desiccation (Golovina et al., namic parameters to permit the compari-
1998; Buitink et al., 1999, 2000a,b,c; son of data from different biological
Leprince et al., 1999; Chaper 4). A variety systems. The commonly used parameter,
of molecular spin probes (stable free radi- water content, is not adequate for the
Microwave absorption (A)


First derivative (dA/dB)

Magnetic field (B)

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 of
absorption corresponds to the point where the first derivative passes through zero (dashed lines). (See
Figures in Chapter 4.)
02 Dessication - Chap 2 18/3/02 1:54 pm Page 79

Methods for Studying Water Relations Under Stress 79

expression of tissue water status in most of desiccation stresses would certainly

cases. Whenever possible, the researcher improve the mechanistic studies of desic-
should first study the components of the cation tolerance. Water plays an important
water relations of cells or tissues and role in maintaining the structural integrity
obtain important reference parameters of biological systems. Although the kinetic
about their water status. The Höfler dia- properties of water in many biological sys-
gram and PV curve can be applied to most tems have been extensively studied, the
well-hydrated plant tissues, whereas the organization of cellular water and its rela-
isothermal sorption study can be applied to tion to desiccation tolerance or desiccation
intermediate- to low-moisture systems. damage are not fully understood. Further
Drying rate and desiccation stress can be studies on the interactions between water
quantified by introducing thermodynamic and macromolecular structures by biophys-
concepts into the study of water-loss ical techniques are essential to identify
dynamics during desiccation (drying fundamental cellular or metabolic compo-
curve). The quantitative (instead of qualita- nents that are associated with desiccation
tive) analysis of physico-chemical aspects damage or desiccation tolerance.

2.8. References

Bakradze, N.G. and Balla, Y.I. (1983) Crystallization of intracellular water in plant tissues. Biophysics
28, 125–128.
Beall, P.T. (1981) The water for life. The Sciences January 1, 6–29.
Beall, P.T. (1983) States of water in biological systems. Cryobiology 20, 324–334.
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.
Belagyi, J. (1975) Water structure in striated muscle by spin labeling techniques. Acta Biochimica et
Biophysica; Academiae Scientiarum Hungaricae 10, 63–70.
Bell, L.N. and Labuza, T.P. (2000) Moisture Sorption: Practical Aspects of Isotherm Measurement and
Use. Eagan Press, Eagan, Minnesota, 123 pp.
Berjak, P. and Pammenter, N.W. (1994) Recalcitrance is not an all-or-nothing situation. Seed Science
Research 4, 263–264.
Berjak, P., Pammenter, N.W. and Vertucci, C. (1992) Homoiohydrous (recalcitrant) seeds: develop-
mental status, desiccation sensitivity and the state of water in axes of Landolphia kirkii Dyer.
Planta 186, 249–261.
Brunauer, S., Emmett, P.H. and Teller, E. (1938) Adsorption of gases in multimolecular layers. Journal
of American Chemical Society 60, 309–319.
Brunauer, S., Deming, L.S., Deming, W.E. and Teller, E. (1940) On a theory of the van der Waals
absorption of gasses. Journal of American Chemical Society 62, 1723.
Bruni, F. and Leopold, A.C. (1991) Hydration, protons and onset of physiological activities in maize
seeds. Physiologia Plantarum 81, 359–366.
Bruni, F. and Leopold, A.C. (1992) Pools of water in anhydrobiotic organisms: a thermally stimulated
depolarization current study. Biophysical Journal 63, 663–672.
Bruni, F., Careri, G. and Clegg, J.S. (1989) Dielectric properties of Artemia cysts at low water con-
tents: evidence for a percolative transition. Biophysical Journal 55, 331–338.
Buitink, J., Claessens, M.M.A.E., Hemminga, M.A. and Hoekstra, F.A. (1998a) Influence of water con-
tent and temperature on molecular mobility and intracellular glasses in seed and pollen. Plant
Physiology 118, 531–541.
Buitink, J., Walters, C., Hoekstra, F.A. and Crane, J. (1998b) Storage behaviour of Typha latifolia
pollen at low water contents: interpretation on the basis of water activity and glass concepts.
Physiologia Plantarum 103, 145–153.
Buitink, J., Hemminga, M.A. and Hoekstra, F.A. (1999) Characterization of molecular mobility in seed
tissues: an EPR spin probe study. Biophysical Journal 76, 3315–3322.
02 Dessication - Chap 2 18/3/02 1:54 pm Page 80

80 W.Q. Sun

Buitink, J., Leprince, O., Hemminga, M.A. and Hoekstra, F.A. (2000a) Molecular mobility in the cyto-
plasm: an approach to describe and predict lifespan of dry germplasm. Proceedings of the
National Academy of Sciences, USA 97, 2385–2390.
Buitink, J., Leprince, O., Hemminga, M.A. and Hoekstra, F.A. (2000b) The effects of moisture and
temperature on the aging kinetics of pollen: interpretation in terms of cytoplasmic mobility.
Plant Cell and Environment 23, 967–974.
Buitink, J., Leprince, O. and Hoekstra, F.A. (2000c) Dehydration-induced redistribution of
amphiphilic molecules between cytoplasm and lipids is associated with desiccation tolerance in
seeds. Plant Physiology 124, 1413–1426.
Cameron, I.L., Ord, V.A. and Fullerton, G.D. (1988) Water of hydration in the intra- and extra-cellular
environment of human erythrocyte. Biochemistry and Cell Biology 66, 1186–1199.
Careri, G. and Giansanti, A. (1984) Deuterium effect in the dielectric losses of wheat seeds. Lett
Nuovo Cimento 40, 193–196.
Careri, G., Giansanti, A. and Gratton, E. (1979) Lysozyme film hydration events: an IR and gravimet-
ric study. Biopolymers 18, 1187–1203.
Chirife, J. and Buera, M.D.P. (1996) Water activity, water glass dynamics, and the control of microbio-
logical growth in foods. Critical Reviews in Food Science and Nutrition 36, 465–513.
Clegg, J.S. (1986) The physical properties and metabolic status of Artemia cysts at low water con-
tents: the water replacement hypothesis. In: Leopold, A.C. (ed.) Membranes, Metabolism and
Dry Organisms. Cornell University Press, Ithaca, New York, pp. 169–185.
Clegg, J.S., Szwarnowski, S., McClean, V.E.R., Sheppard, R.J. and Grant, E.H. (1982)
Interrelationships between water and cell metabolism in Artemia cysts. X. Microwave dielectric
studies. Biochimica et Biophysica Acta 721, 458–468.
Crafts, A.S., Currier, H.S. and Stocking, C.R. (1949) Water in the Physiology of Plants. Chronica
Botanica, Waltham, Massachusetts.
D’Arcy, R.L. and Watt, I.C. (1970) Analysis of sorption isotherms of non-homogeneous sorbents.
Transactions of the Faraday Society 66, 1236–1245.
Dominguez, E. and Heredia, A. (1999) Water hydration in cutinized cell wall: a physico-chemical
analysis. Biochimica et Biophysica Acta 1426, 168–176.
Eira, M.T.S., Walters, C. and Caldas, L.S. (1999) Water sorption properties in Coffea spp. seeds and
embryos. Seed Science Research 9, 321–330.
Ellis, R.H., Hong, T.D. and Roberts, E.H. (1990) An intermediate category of seed storage behaviour.
I. Coffee. Journal of Experimental Botany 41, 1167–1174.
Ellis, R.H., Hong, T.D. and Roberts, E.H. (1991) An intermediate category of seed storage behaviour.
II. Effects of provenance, immaturity, and imbibition on desiccation-tolerance in coffee. Journal
of Experimental Botany 42, 653–657.
Farrant, J.M., Pammenter, N.W. and Berjak, P. (1988) Recalcitrance – a current assessment. Seed
Science and Technology 16, 155–156.
Fung, B.M. and McGaughy, T.W. (1974) The state of water in muscle as studied by pulsed NMR.
Biochimica et Biophysica Acta 343, 663–673.
Golovina, E.A., Hoekstra, F.A. and Hemminga, M.A. (1998) Drying increases intracellular partitioning
of amphiphilic substances into the lipid phase. Plant Physiology 118, 975–986.
Harvey, S.C. and Hoekstra, P. (1972) Dielectric relaxation spectra of water adsorbed on lysozyme.
Journal of Physical Chemistry 76, 2981–2994.
Holmstrup, M. and Zachariassen, K.E. (1996) Physiology of cold hardiness in earthworms.
Comparative Biochemistry and Physiology 115A, 91–101.
Honegger, R. (1995) Experimental studies with foliose macrolichens: fungal responses to spatial dis-
turbance at the organismic level and to spatial problems at the cellular level during drought
stress events. Canadian Journal of Botany 73, s569–s578.
Hüsken, D., Steudle, E. and Zimmermann, U. (1978) Pressure probe technique for measuring water
relations of cells in higher plants. Plant Physiology 61, 158–163.
International Seed Testing Association (1993) International rules for seed testing, rules 1993. Seed
Science and Technology 21 (suppl.), 1–75.
James, T.L. (1993) Fundamentals of NMR. In: Gorenstein, D. (ed.) Nuclear Magnetic Resonance
(NMR), online textbook (
Kamiyoshi, K. and Kudo, A. (1978) Dielectric relaxation of water contained in plant tissues. Japanese
Journal of Applied Physics 17, 1531–1536.
02 Dessication - Chap 2 18/3/02 1:54 pm Page 81

Methods for Studying Water Relations Under Stress 81

Knight, V.A. and Wiggins, P.M. (1979) A possible role for water in performance of cellular work. II.
Measurements of scattering of light by actomyosin. Bioelectrochemistry and Bioenergetics 6,
Lehmann, M.S. (1984) Probing the protein-bound water with other small molecules using neutron
small angle scattering. Journal of Physics: Colloids C7, 235–239.
Leopold, A.C., Sun, W.Q. and Bernal-Lugo, I. (1994) The glassy state in seeds: analysis and function.
Seed Science Research 4, 267–274.
Leprince, O., Buitink, J. and Hoekstra, F.A. (1999) Axes and cotyledons of recalcitrant seeds of
Castanea sativa Mill exhibited contrasting responses of respiration to drying in relation to desic-
cation sensitivity. Journal of Experimental Botany 338, 1515–1524.
Li, C.R. and Sun, W.Q. (1999) Desiccation sensitivity and activities of free radical-scavenging
enzymes in recalcitrant Theobroma cacao seeds. Seed Science Research 9, 209–217.
Liang, Y.H. and Sun, W.Q. (2000) Desiccation tolerance of recalcitrant Theobroma cacao embryonic
axes: the optimal drying rate and its physiological basis. Journal of Experimental Botany 51,
Lugue, P., Gavara, R. and Heredia, A. (1995) A study of the hydration process of isolated cuticular
membranes. New Phytologist 129, 283–288.
Luscher-Mattli, M. and Ruegg, M. (1982) Thermodynamic functions of biopolymer hydration. I. Their
determination by vapor pressure studies, discussed in an analysis of the primary hydration
process. Biopolymers 21, 403–418.
Mascarenhas, S. (1980) Biolectrets: electrets in biomaterials and biopolymers. In: Sessler, G.M. (ed.)
Electrets. Springer-Verlag, Berlin, pp. 321–346.
Mathur-de Vre, R. (1979) The NMR studies of water in biological systems. Progress in Biophysics and
Molecular Biology 35, 103–134.
Meidner, H. and Sheriff, D.W. (1976) Water and Plants. John Wiley & Sons, New York.
Milburn, J.A. (1970) Cavitation and osmotic potential of Sordaria ascospores. New Phytologist 69,
Oertli, J.J. (1989) The plant cell’s response to consequences of negative turgor presure. In: Kreeb,
K.H., Richter, H. and Hinckley, T.M. (eds) Structural and Functional Responses to
Environmental Stress: Water Shortage. SPB Academic, The Hague, The Netherlands, pp. 73–78.
Pammenter, N.W. and Berjak, P. (1999) A review of recalcitrant seed physiology in relation to desic-
cation-tolerance mechanisms. Seed Science Research 9, 13–37.
Pammenter, N.W., Vertucci, C.W. and Berjak, P. (1991) Homeoiohydrous (recalcitrant) seeds: dehydra-
tion, the state of water and viability characteristics in Landolphia kirkii. Plant Physiology 96,
Pissis, P. (1990) The dielectric relaxation of water in plant tissues. Journal of Experimental Botany
41, 677–684.
Pissis, P., Anagnostopoulou-Konsta, A. and Apekis, L. (1987) A dielectric study of the state of water
in plant stems. Journal of Experimental Botany 38, 1528–1540.
Pissis, P., Konsta, A.A., Ratkovic, S., Todorovic, S. and Laudat, J. (1996) Temperature and hydration-
dependence of molecular mobility in seeds. Journal of Thermal Analysis 47, 1463–1483.
Potts, M. (1994) Desiccation tolerance of prokaryotes. Microbiological Reviews 58, 755–805.
Poulsen, K.M. and Eriksen, E.N. (1992) Physiological aspect of recalcitrance in embryonic axes of
Quercus robur L. Seed Science Research 2, 215–221.
Pritchard, H.W. (1991) Water potential and embryonic axis viability in recalcitrant seeds of Quercus
rubra. Annals of Botany 67, 43–49.
Pritchard, H.W. and Manger, K.R. (1998) A calorimetric perspective on desiccation stress during
preservation procedures with recalcitrant seeds of Quercus robur L. CryoLetters 19 (suppl.),
Proctor, M.C.F. (1999) Water-relations parameters of some bryophytes evaluated by thermocouple
psychrometry. Journal of Bryology 21, 263–270.
Proctor, M.C.F., Nagy, Z., Csintalan, Z.S. and Takács, Z. (1998) Water content components in
bryophytes: analysis of pressure–volume curve. Journal of Experimental Botany 49, 1845–1854.
Rahman, M.S. and Labuza, T.P. (1999) Water activity and food preservation. In: Shafiur Rahman, M.
(ed.) Handbook of Food Preservation. Marcel Dekker, New York, pp. 339–382.
Ratkovic, S. (1987) Proton NMR of maize seed water: the relationship between spin-lattice relaxation
time and water content. Seed Science and Technology 15, 147–154.
02 Dessication - Chap 2 18/3/02 1:54 pm Page 82

82 W.Q. Sun

Rorschach, H.E. and Hazlewood, C.F. (1986) Protein dynamics and the NMR relaxation time T1 of
water in biological systems. Journal of Magnetic Resonance 70, 79–88.
Ruegg, M., Moor, U. and Blanc, B.H. (1975) Hydration and thermal denaturation of ß-lactoglobulin:
calorimetric study. Biochimica et Biophysica Acta 400, 334–342.
Rupley, J.A., Gratton, E. and Careri, G. (1983) Water and globular proteins. Trends in Biochemical
Sciences 8, 18–22.
Rustgi, S.N., Peemoeller, H., Thompson, R.T., Kydon, D.W. and Pintar, M.M. (1978) A study of molec-
ular dynamics and freezing phase transition in tissues by proton spin relaxation. Biophysical
Journal 22, 439–452.
Sakurai, M., Kawai, K., Inoue, Y., Hino, A. and Kobayashi, S. (1995) Effects of trehalose on the water
structure in yeast cells as studied by in vivo 1H NMR spectroscopy. Bulletin of Chemical Society
of Japan 68, 3621–3627.
Scheidegger, C., Schroeter, B. and Frey, B. (1995) Structural and functional processes during water
vapour uptake and desiccation in selected lichens with green algal photobionts. Planta 197,
Seewaldt, V., Priestley, D.A., Leopold, A.C., Feigenson, W. and Goodsaid-Zalduondo, F. (1981)
Membrane organization in soybean seeds during hydration. Planta 52, 19–23.
Sinclair, T.R. and Ludlow, M.M. (1985) Who taught plants thermodynamics? The unfulfilled poten-
tial of plant water potential. Australian Journal of Plant Physiology 12, 213–217.
Stadelmann, E.J. (1984) The derivation of the cell wall elasticity function from the cell turgor poten-
tial. Journal of Experimental Botany 35, 859–868.
Stannett, V.T., Ranade, G.R. and Koros, W.J. (1982) Characterization of water vapor transport in glassy
polyacrylonitrile by combined permeation and sorption techniques. Journal of Membrance
Sciences 10, 219–233.
Steudle, E., Zimmermann, U. and Luttge, U. (1977) Effect of turgor pressure and cell size on the wall
elasticity of plant cells. Plant Physiology 59, 285–289.
Sun, W.Q. (1997) Glassy state and seed storage stability: the WLF kinetics of seed viability loss at
T > Tg and the plasticization effect of water on seed storage stability. Annals of Botany 79,
Sun, W.Q. (1998) Function of the glassy state in seed storage stability. In: Taylor, A.G. and Huang,
X.L. (eds) Progress in Seed Research. New York State Agricultural Experiment Station, Cornell
University, Geneva, New York, pp. 169–179.
Sun, W.Q. (1999) State and phase transition behaviors of Quercus rubra seed axes and cotyledonary
tissues: relevance to the desiccation sensitivity and cryopreservation of recalcitrant seeds.
Cryobiology 38, 372–385.
Sun, W.Q. (2000) Dielectric relaxation of water and water-plasticized biomolecules in relation to cel-
lular water organization, cytoplasmic viscosity and desiccation tolerance in recalcitrant seed tis-
sues. Plant Physiology 124, 1203–1215.
Sun, W.Q. and Davidson, P. (1998) Protein stability in the amorphous carbohydrate matrix: relevance
to anhydrobiosis. Biochimica et Biophysica Acta 1425, 245–254.
Sun, W.Q. and Gouk, S.S. (1999) Preferred parameters and methods for studying moisture content of
recalcitrant seeds. In: Marzalina, M., Khoo, K.C., Jayanthi, N., Tsan, F.Y. and Krishnapillay, T.M.
(eds) Recalcitrant Seeds: Proceedings of the IUFRO Seed Symposium. Forest Research Institute
of Malaysia, Kuala Lumpur, pp. 403–430.
Sun, W.Q., Irving, T.C. and Leopold, A.C. (1994) The role of sugar, vitrification and membrane phase
transition in seed desiccation tolerance. Physiologia Plantarum 90, 621–628.
Sun, W.Q., Koh, D.C.Y. and Ong, C.M. (1997) Correlation of modified water sorption properties with
the decline of storage stability of osmotically-primed seeds of Vigna radiata (L.) Wikzek. Seed
Science Research 7, 391–397.
Thomson, W.W. and Platt, K.A. (1997) Conservation of cell order in desiccation mesophyll of
Selaginella lepidophylla ([Hook and Grev.] Spring). Annals of Botany 79, 439–447.
Tompsett, P.B. and Pritchard, H.W. (1998) The effect of chilling and moisture status on the germina-
tion, desiccation tolerance and longevity of Aesculus hippocastarum L. seeds. Annals of Botany
82, 249–261.
Trantham, E.C., Rorschach, H.E., Clegg, J.S., Hazlewood, C.F., Nicklow, R.M. and Wakabayashi, N.
(1984) The diffusive properties of water in Artemia cells determined by quasi-electron neutron
scattering. Biophysical Journal 45, 927–938.
02 Dessication - Chap 2 18/3/02 1:54 pm Page 83

Methods for Studying Water Relations Under Stress 83

Vertucci, C.W. (1990) Calorimetric studies of the state of water in seed tissues. Biophysical Journal
58, 1463–1471.
Vertucci, C.W. and Leopold, A.C. (1986) Physiological activities associated with hydration level in
seeds. In: Leopold, A.C. (ed.) Membranes, Metabolism and Dry Organisms. Cornell University
Press, Ithaca, New York, pp. 35–49.
Vertucci, C.W. and Leopold, A.C. (1987a) Water binding in legume seeds. Plant Physiology 85,
Vertucci, C.W. and Leopold, A.C. (1987b) The relationship between water binding and desiccation
tolerance in tissues. Plant Physiology 85, 232–238.
Vertucci, C.W. and Roos, E.E. (1993) Theoretical basis of protocols for seed storage. II. The influence
of temperature on optimal moisture levels. Seed Science Research 3, 201–213.
Vertucci, C.W., Crane, J., Porter, R.A. and Oelke, E.A. (1994) Physical properties of water in Zizania
embryos in relation to maturity status, water content and temperature. Seed Science Research 4,
Vertucci, C.W., Crane, J., Porter, R.A. and Oelke, E.A. (1995) Survival of Zizania embryos in relation
to water content, temperature and maturity status. Seed Science Research 5, 31–40.
Vicre, M., Sherwin, H.W., Driouich, A., Jaffer, M.A. and Farrant, J.M. (1999) Cell wall characteristics
and structure of hydrated and dry leaves of the resurrection plant Craterostigma wilmsii, a
microscopical study. Journal of Plant Physiology 155, 719–726.
Walters, C. (1998a) Understanding the mechanisms and kinetics of seed aging. Seed Science
Research 8, 223–244.
Walters, C. (1998b) Water activity, bad habits die hard: a response. CryoLetters 19, 265–266.
Williams, R.J. and Leopold, A.C. (1989) The vitreous state in maize embyos. Plant Physiology 89,
Wolfe, J. and Leopold, A.C. (1986) A spectrum of desiccation. In: Leopold, A.C. (ed.) Membranes,
Metabolism and Dry Organisms. Cornell University Press, Ithaca, New York, p. 1.
Zimm, B.H. and Lundberg, J.L. (1956) Sorption of vapors by high polymers. Journal of Physical
Chemistry 60, 425–428.
Zimmermann, J.R. and Brittin, W.E. (1957) Nuclear magnetic resonance studies in multiple phase
systems: lifetime of a water molecule in an absorbency phase on silica gel. Journal of Physical
Chemistry 61, 1328–1333.
02 Dessication - Chap 2 18/3/02 1:54 pm Page 84

84 W.Q. Sun

Appendix: Solutions for Controlled are generally accurate within  2%. RHs of
Dehydration and Rehydration commonly used salt solutions between 5
and 40°C are compiled in Table A2.2. RHs
Saturated salt solutions of additional salt solutions at 25°C are
given in Table A2.3. These data are taken
In a closed container, a saturated salt solu- from earlier works of Rockland (1960),
tion (with excess salt present) produces a Winston and Bates (1960) and Young
constant water vapour pressure at a given (1967), tested and corrected by the author.
temperature. Relative humidity (RH) in the Users are advised to avoid salts that release
container is calculated by: salt vapours into the atmosphere. When
autoclaving is required, the decomposition
RH A exp(B/T) (1)
temperature of a salt should be checked.
where A and B are constants, and T is the After autoclaving, the closed container
temperature in kelvin (Wexler, 1997). The should be allowed sufficient time to equili-
values of A and B as well as the valid tem- brate (i.e. avoiding condensation and
perature range are given in Table A2.1 for supersaturation). The supersaturation in
various salts. For example, RH of saturated the liquid phase and the condensation of
KCl solution at 25°C is equal to 49.38  water on the wall in the container affect
exp(159/298) = 84.2 (%). Calculated values RH significantly.

Table A2.1. Commonly used salts, their vapour pressure constants A and B, and the valid temperature
range. Data are taken from Wexler (1997).
Temperature range Relative humidity
Compound (°C) at 25°C A B
NaOH.H2O 15–60 6 5.48 27
LiBr.2H2O 10–30 6 0.23 996
ZnBr2.2H2O 5–30 8 1.69 455
KOH.2H2O 5–30 9 0.014 1924
LiCl.H2O 20–65 11 14.53 75
CaBr2.6H2O 11–22 16 0.17 1360
LiI.3H2O 15–65 18 0.15 1424
CaCl2.6H2O 15–25 29 0.11 1653
MgCl2.6H2O 5–45 33 29.26 34
NaI.2H2O 5–45 38 3.62 702
Ca(NO3) 2.4H2O 10–30 51 1.89 981
Mg(NO3) 2.6H22O 5–35 53 25.28 220
NaBr.2H2O 0–35 58 20.49 308
NH4NO3 10–40 62 3.54 853
KI 5–30 69 29.35 254
SrCl2.6H2O 5 –30 71 31.58 241
NaNO3 10–40 74 26.94 302
NaCl 10–40 75 69.20 25
NH4Cl 10–40 79 35.67 235
KBr 5–25 81 40.98 203
(NH4) 2SO4 10–40 81 62.06 79
KCl 5–25 84 49.38 159
Sr(NO3) 2.4H2O 5–25 85 28.34 328
BaCl2.2H2O 5–25 90 69.99 75
CsI 5–25 91 70.77 75
KNO3 0–50 92 43.22 225
K2SO4 10–50 97 86.75 34
02 Dessication - Chap 2 18/3/02 1:54 pm Page 85

Methods for Studying Water Relations Under Stress 85

Table A2.2. Equilibrium relative humidities of saturated salt solutions at different temperatures. Data are
taken from Rockland (1960), Winston and Bates (1960) and Young (1967).
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 1.0
ZnCl2 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 11.0
CaBr2 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 23.0
MgBr2 32.0 31.0 31.5 31.0 30.5 30.0 30.0 30.0
MgCl2 34.0 33.5 33.5 33.0 32.5 32.0 32.5 32.0
CaCl2 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 40.0
NaI 43.5 – 38.0 38.5 38.0 36.0 34.0 32.5
Zn(NO3) 2 45.0 43.0 40.7 40.0 32.5 24.0 21.0 19.0
KCNS 54.0 52.0 50.0 47.0 46.5 43.5 41.5 41.0
Mg(NO3) 2 55.0 53.0 53.7 53.0 52.5 52.0 50.5 51.0
Na2Cr2O7.H2O 59.5 60.0 56.5 54.5 53.0 52.5 51.0 50.0
NaBr 60.5 58.0 58.0 57.8 57.2 57.0 57.0 57.0
Ca(NO3) 2 61.0 66.0 58.0 56.0 52.2 51.0 45.5 46.0
NaBr.2H2O 63.0 61.0 59.0 57.5 56.0 54.5 53.0 51.5
CuCl2 66.7 68.0 68.0 68.5 67.0 67.0 67.0 67.0
LiAc 72.0 72.0 71.0 70.0 68.0 66.0 65.0 64.0
NH4NO3 73.0 75.0 70.0 65.5 62.5 59.5 56.8 53.0
NaCl 76.0 75.8 75.5 75.3 75.1 75.0 75.0 75.0
NaNO3 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 79.0
NH4Cl 82.0 79.0 79.5 79.0 78.0 77.5 75.5 74.0
Li2SO4 84.0 84.0 84.0 85.0 85.0 85.0 85.0 81.0
KBr 85.0 86.0 85.0 83.5 83.0 82.0 81.0 80.0
KCl 87.8 86.7 86.0 85.3 85.0 83.5 83.0 83.0
K2CrO4 89.0 89.0 88.0 88.0 87.0 86.0 84.0 82.0
BaCl2 95.0 93.0 92.0 90.7 90.0 89.0 88.0 87.0
ZnSO4 95.0 93.0 92.0 90.0 88.0 86.0 85.0 84.0
KNO3 96.5 95.5 95.0 94.0 92.5 91.5 89.5 88.5
K2SO4 98.0 97.0 97.0 97.0 97.0 97.0 96.0 96.0
Na2HPO4 98.0 98.0 98.0 98.0 97.0 96.0 93.0 91.0
Pb(NO3) 3 99.0 98.5 98.5 98.5 96.2 95.5 95.2 94.7

Non-saturated salt solutions during equilibration, because the equilibra-

tion between the tissue, vapour phase and
Non-saturated salt solutions can also be solution phase results in a slight decrease or
used. This method allows for the creation of increase in salt concentration. This problem
a precise and evenly graded series of RHs (or can be minimized using high mass ratios
water potentials) with the same salt. The dis- between the solution and the sample. A
advantage of using non-saturated salt solu- mass ratio of 150–200 (i.e. 150–200 g solu-
tions is that they have limited buffering tion g1 tissue) is sufficient. Water potentials
capacity relative to saturated salt solutions. of NaCl solutions at different concentrations
RHs inside the container are not constant and temperatures between 5 and 40°C are
02 Dessication - Chap 2 18/3/02 1:54 pm Page 86

86 W.Q. Sun

Table A2.3. A list of saturated salt solutions (with the presence of excess salt) and their equilibrium
relative humidities (RHs) at 25°C.
Saturated salt solution RH (%) Saturated salt solution RH (%)
ZnBr2 8.6 SrBr2.6H2O 58.5
H3PO4 9.0 FeCl2.4H2O 60.0
CaAc2.H2O + sucrose 13.0 NaMnO4.3H2O 61.5
CaAc2.H2O 17.0 NH4NO3+ AgNO3 61.5
Ca(CNS) 2.3H2O 17.5 CuBr2 62.5
LiI.3H2O 18.0 CoCl2 64.0
KHCO2 (Formate) 21.5 NaNO2 64.2
KAc.1.5H2O 22.2 K2S2O3 66.0
NiBr2.3H2O 27.0 CuCl2.2H2O 67.7
MgBr2.6H2O 31.5 NaCl + NaNO3 69.0
Sr(CNS) 2.3H2O 31.5 SrCl2.6H2O 71.0
SrI2.6H2O 33.0 SrCl2 71.0
MnBr2.6H2O 34.5 NH4Cl + KNO3 71.2
Cu(NO3) 2.6H2O 35.0 NaCl + KCl 71.5
Ca(MnO4) 2.4H2O 37.5 NaAc.3H2O 73.0
FeBr2.6H2O 39.0 NaCl + Na2SO4.7H2O 74.0
NaI.2H2O 39.2 BaBr2 74.5
Mg(ClO4) 2.6H2O 41.0 K tartrate 75.0
CoBr2 41.5 NH4Br2 75.0
CrCl3 42.5 Zn(CNS) 2 80.5
BaI2.2H2O 43.0 NaH2PO4 81.0
K2CO3.2H2O 43.0 AgNO3 82.0
CeCl3 45.5 KCl + KClO3 85.0
LiNO3.3H2O 47.0 KNa tartrate 87.0
Mg(CNS) 2 47.5 Na2CO3.10H2O 87.0
KNO2 48.1 MgSO4.7H2O 89.0
K4P4O7.3H2O 49.5 BaCl2.2H2O 90.3
Co(NO3) 2.6H2O 49.8 Na tartrate 92.0
NH4NO3 + NaNO3 50.0 (NH4)H2PO4 92.7
KBr + urea 51.0 NH4HPO4 93.0
Zn(MnO4) 2.6H2O 51.0 CaH4(PO4) 2.H2O 96.0
NiCl2.6H2O 53.0 KH2PO4 96.0
Na2Cr2O7.2H2O 53.7 CaHPO4.2H2O 97.0
Ba(CNS) 2.2H2O 54.5 CuSO4.5H2O 97.2
Pb(NO3) 2 + NH4NO3 55.0 K2Cr2O7 98.0
MnCl2.4H2O 56.0 KClO3 98.0

given in Table A2.4. Besides NaCl solutions, tial or RH are often not available. PEG is
CaCl2 and KCl solutions offer good RH con- inexpensive and not corrosive, whereas
trol. Water potentials of CaCl2 and KCl solu- many salt solutions are corrosive and
tions are listed in Table A2.5. volatile. Effects of PEG concentration and
temperature on water potential were studied
by Michel and Kaufmann (1973). An empiri-
Polyethylene glycol (PEG) solutions cal equation has been derived to calculate
the water potential of PEG solutions at given
PEG solutions are widely used in controlled concentrations and temperatures:
dehydration and rehydration. PEG solutions
have several advantages over salt solutions.  (1.18  103C) – (1.18  105 C2)
Salt solutions at high specific water poten- (2.67  105CT) (8.39  108C 2T) (2)
02 Dessication - Chap 2 18/3/02 1:54 pm Page 87

Methods for Studying Water Relations Under Stress 87

Table A2.4. Water potentials of sodium chloride (NaCl) solutions at different concentrations and
temperatures. Mass (%); g solute per 100 g solution.
Water potential (MPa)
Molality Mass
(mol kg1) (%) 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 0.25
0.1 0.58 0.43 0.44 0.45 0.45 0.46 0.47 0.48 0.49
0.2 1.16 0.85 0.87 0.88 0.90 0.92 0.93 0.95 0.96
0.3 1.72 1.27 1.30 1.32 1.34 1.37 1.39 1.42 1.44
0.4 2.28 1.69 1.73 1.76 1.79 1.82 1.86 1.89 1.92
0.5 2.84 2.12 2.16 2.20 2.24 2.28 2.32 2.36 2.40
0.6 3.39 2.54 2.59 2.64 2.69 2.74 2.79 2.84 2.89
0.7 3.93 2.97 3.03 3.09 3.15 3.21 3.27 3.33 3.39
0.8 4.47 3.40 3.47 3.54 3.61 3.68 3.75 3.82 3.89
0.9 5.00 3.83 3.92 4.00 4.08 4.16 4.23 4.31 4.39
1.0 5.52 4.27 4.37 4.46 4.55 4.64 4.73 4.82 4.90
1.1 6.04 4.71 4.82 4.92 5.03 5.13 5.23 5.32 5.42
1.2 6.55 5.16 5.28 5.39 5.51 5.62 5.73 5.84 5.94
1.3 7.06 5.61 5.74 5.87 5.99 6.12 6.24 6.35 6.47
1.4 7.56 6.07 6.21 6.35 6.49 6.62 6.75 6.88 7.01
1.5 8.06 6.53 6.68 6.84 6.99 7.13 7.28 7.41 7.55
1.6 8.55 7.00 7.16 7.33 7.49 7.65 7.81 7.95 8.11
1.7 9.04 7.46 7.64 7.82 8.00 8.17 8.33 8.49 8.65
1.8 9.52 7.94 8.13 8.33 8.52 8.70 8.88 9.04 9.21
1.9 9.99 8.43 8.63 8.84 9.04 9.24 9.43 9.60 9.78
2.0 10.46 8.92 9.13 9.36 9.57 9.78 9.98 10.16 10.35

where C is the concentration of PEG (molec- water potential is calculated with Equation
ular weight 6000) in g kg1 water and T is (2). The PEG solution can be dried at 105°C
temperature (°C). Water potential of PEG in an oven to a constant dry weight. The
solutions is curvilinearly related to the con- problem with submerging the tissue in a
centration and increases linearly with tem- PEG solution is that PEG can enter the inter-
perature. The error of calculated water cellular spaces. PEG is considered a non-
potential is generally within 0.03 MPa in penetrating polymer because it does not
comparison with the psychrometric mea- cross the membrane! Our calorimetric study
surements. Water potentials of PEG-6000 reveals a PEG melting peak in submerged
solutions at concentrations ranging from 10 tissues, even after extensive washing.
to 400 g kg1 water and at temperatures
between 5 and 40°C are given in Table A2.6.
PEG solutions are very viscous, especially at Glycerol solutions
high concentrations; hence it takes a longer
time to achieve the equilibrium between the Glycerol can also be used to create a pre-
PEG solution and the tissue. Caution is cise and evenly graded series of RHs
needed to prevent bacterial and fungal cont- between 30 and 98%. Glycerol solutions
amination during the study. The equilibra- are safe to use and less corrosive (except
tion can be accelerated by placing closed for tin) than salt solutions. Microbial
containers in a gently shaking incubator and growth can be effectively inhibited by
in some cases submerging the tissue in solu- adding four drops of saturated CuSO4 solu-
tion. The change in PEG concentration after tion to each 100 ml of glycerol solution
the experiment can be determined by the (ASTM, 1983). The CuSO4-treated solution
gravimetric method, and the equilibrium can be used repeatedly after the required
02 Dessication - Chap 2 18/3/02 1:54 pm Page 88

88 W.Q. Sun

Table A2.5. Water potentials of potassium chloride (KCl) and calcium

chloride (CaCl2) solutions at 20 and 25°C. Mass (%): g solute per 100
g solution.
KCl CaCl2
Mass (%) 20°Ca 25°Cb 20°Ca 25°Cb
0.5 0.28 0.31 0.27 0.28
1 0.56 0.61 0.54 0.57
2 1.12 1.22 1.07 1.16
3 1.68 1.85 1.62 1.81
4 2.26 2.48 2.22 2.50
5 2.83 3.13 2.87 3.24
6 3.42 3.80 3.58 4.02
7 4.02 4.48 4.36 4.89
8 4.64 5.18 5.23 5.77
9 5.25 5.90 6.15 6.73
10 5.87 6.65 7.16 7.76
12 7.18 8.21 9.40 10.09
14 9.89 12.00 12.85
16 11.68 14.99 16.13
18 13.62 18.45 20.02
20 15.69 22.34 24.60
22 26.50
24 20.34 30.89 36.20
26 36.26
28 42.37 51.67
30 50.06
32 60.68 71.78
36 97.33
40 129.12
a Derived from Handbook of Chemistry and Physics, 78th edn

(Lide, 1997). Water potential is calculated according to the

freezing-point depression.
b Data were derived from Robinson and Stokes (1959).

concentration adjustment. The composi- The ASTM’s method uses the refractive
tion of glycerol solutions can be accurately index at 25°C to express glycerol concen-
determined using specific gravity or the tration. The relationship between RH,
refractive index. Equilibrium RHs of glyc- refractive index and temperature is
erol solutions at 24°C were reported by described by the following equations:
Braun and Braun (1958). Using the same
(R0 A)2 (100 A)2 A2  (RH A)2 (3)
set of data, Forney and Brandl (1992)
derived an empirical equation to calculate A 25.6  0.195T 0.0008T 2 (4)
equilibrium RHs of glycerol solutions.
R 1.3333 (1.398  103 R0) (5)
Equilibrium RHs and water potentials of
glycerol solutions at 20°C were derived where RH is the desired relative humidity
according to the freezing-point depression. (%), R is the refractive index of the glycerol
These data are listed in Table A2.7. One solution, and T is temperature (°C). The
can calculate the required concentration of value of A is calculated using Equation (4).
a glycerol solution for a desired RH at a Ro is calculated by substituting A and RH
given temperature, using the ASTM’s stan- in Equation (3). The refractive index at
dard recommended practice (ASTM, 1983). 25°C is calculated using Equation (5). For a
02 Dessication - Chap 2 18/3/02 1:54 pm Page 89

Methods for Studying Water Relations Under Stress 89

glycerol solution of the known refractive where A is defined by Equation (4), and Ro
index (R), equilibrium RH at different tem- is calculated from Equation (5). The mea-
peratures can be calculated by rearranging surement of refractive index is quite
Equations (3), (4) and (5) (Sun and Gouk, tedious. The concentration (mass %) of the
1999). The relationship is: desired glycerol solution has been derived
from concentrative properties of aqueous
RH = (100 + A)2 + A2 − (R0 + A)2 − A (6) glycerol solutions (Lide, 1997) by the

Table A2.6. Water potentials of polyethylene glycol (MW 6000) solutions. Data were derived according
to Michel and Kaufmann (1973).
Water potential (MPa) at different temperatures
(g kg1 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 0.002
20 0.025 0.023 0.020 0.017 0.014 0.011 0.008 0.006
30 0.042 0.037 0.033 0.028 0.024 0.020 0.015 0.011
40 0.060 0.054 0.048 0.042 0.036 0.030 0.024 0.018
50 0.081 0.073 0.065 0.058 0.050 0.042 0.034 0.027
60 0.104 0.094 0.085 0.075 0.066 0.056 0.047 0.037
70 0.129 0.118 0.106 0.095 0.083 0.072 0.061 0.049
80 0.157 0.143 0.130 0.116 0.103 0.090 0.076 0.063
90 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 0.096
110 0.25 0.23 0.21 0.194 0.174 0.154 0.134 0.114
120 0.29 0.27 0.25 0.22 0.20 0.179 0.157 0.135
130 0.33 0.30 0.28 0.26 0.23 0.21 0.182 0.157
140 0.37 0.34 0.32 0.29 0.26 0.24 0.21 0.181
150 0.41 0.38 0.35 0.32 0.30 0.27 0.24 0.21
160 0.46 0.43 0.39 0.36 0.33 0.30 0.27 0.23
170 0.51 0.47 0.44 0.40 0.37 0.33 0.30 0.26
180 0.56 0.52 0.48 0.44 0.41 0.37 0.33 0.29
190 0.61 0.57 0.53 0.49 0.45 0.41 0.37 0.33
200 0.66 0.62 0.58 0.53 0.49 0.45 0.40 0.36
210 0.72 0.68 0.63 0.58 0.54 0.49 0.44 0.40
220 0.78 0.73 0.68 0.63 0.58 0.53 0.48 0.43
230 0.84 0.79 0.74 0.68 0.63 0.58 0.53 0.47
240 0.91 0.85 0.79 0.74 0.68 0.63 0.57 0.51
250 0.97 0.91 0.85 0.79 0.73 0.67 0.62 0.56
260 1.04 0.98 0.92 0.85 0.79 0.73 0.66 0.60
270 1.11 1.05 0.98 0.91 0.85 0.78 0.71 0.65
280 1.19 1.11 1.04 0.97 0.90 0.83 0.76 0.69
290 1.26 1.19 1.11 1.04 0.96 0.89 0.82 0.74
300 1.34 1.26 1.18 1.10 1.03 0.95 0.87 0.79
310 1.42 1.34 1.25 1.17 1.09 1.01 0.93 0.85
320 1.50 1.41 1.33 1.24 1.16 1.07 0.99 0.90
330 1.58 1.49 1.41 1.32 1.23 1.14 1.05 0.96
340 1.67 1.58 1.48 1.39 1.30 1.20 1.11 1.01
350 1.76 1.66 1.56 1.47 1.37 1.27 1.17 1.07
360 1.85 1.75 1.65 1.54 1.44 1.34 1.24 1.13
370 1.95 1.84 1.73 1.62 1.52 1.41 1.30 1.20
380 2.04 1.93 1.82 1.71 1.60 1.48 1.37 1.26
390 2.14 2.02 1.91 1.79 1.68 1.56 1.44 1.33
400 2.24 2.12 2.00 1.88 1.76 1.64 1.52 1.40
02 Dessication - Chap 2 18/3/02 1:54 pm Page 90

90 W.Q. Sun

Table A2.7. Equilibrium relative humidity (RH) and water potential of glycerol solutions at 20°C and
24°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 2.77
12 1.0262 97.4 3.45 97.5 3.47
14 1.0311 96.9 4.16 97.0 4.18
16 1.0360 96.4 4.89 96.5 4.89
18 1.0409 95.8 5.69 95.9 5.74
20 1.0459 95.2 6.52 95.4 6.46
24 1.0561 93.9 8.35 94.1 8.34
28 1.0664 92.4 10.42 92.6 10.55
32 1.0770 90.7 12.71 91.0 12.94
36 1.0876 88.9 15.28 89.2 15.68
40 1.0984 86.9 18.19 87.2 18.79
44 1.1092 84.9 22.46
48 1.1200 82.5 26.39
52 1.1308 79.7 31.13
56 1.1419 76.6 36.57
60 1.1530 73.2 42.78
64 1.1643 69.4 50.11
68 1.1755 65.2 58.68
72 1.1866 60.6 68.71
76 1.1976 55.5 80.77
80 1.2085 49.8 95.64
84 1.2192 43.6 113.88
88 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 solutions
at other concentrations.

author. RHs of glycerol solutions at concen- ASTM’s method is 0.2% at 25°C, and
trations between 10 and 92% (mass %) and increases as temperature deviates from
at temperatures between 5 and 35°C has 25°C (ASTM, 1983). Data in Table A2.8 are
been calculated according to the ASTM’s consistent with those in Table A2.7, which
method (Table A2.8). The accuracy of were derived by different methods.
02 Dessication - Chap 2 18/3/02 1:54 pm Page 91

Methods for Studying Water Relations Under Stress 91

Table A2.8. Equilibrium relative humidity (RH) of glycerol solutions at different

temperatures. 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 98.3
12 97.6 97.7 97.7 97.8 97.8 97.9 97.9
14 97.1 97.2 97.2 97.3 97.4 97.4 97.5
16 96.6 96.6 96.7 96.8 96.9 96.9 97.0
18 96.0 96.1 96.1 96.2 96.3 96.4 96.4
20 95.4 95.5 95.5 95.6 95.7 95.8 95.9
24 94.0 94.1 94.2 94.3 94.4 94.5 94.6
28 92.5 92.6 92.8 92.9 93.0 93.1 93.2
32 90.8 91.0 91.1 91.3 91.4 91.5 91.7
36 88.9 89.1 89.3 89.4 89.6 89.7 89.9
40 86.8 87.0 87.2 87.4 87.6 87.7 87.9
44 84.5 84.7 84.9 85.1 85.3 85.5 85.7
48 82.0 82.2 82.5 82.7 82.9 83.1 83.3
52 79.2 79.5 79.7 80.0 80.2 80.4 80.6
56 76.2 76.5 76.7 77.0 77.2 77.4 77.7
60 72.8 73.0 73.3 73.6 73.8 74.1 74.3
64 69.0 69.3 69.6 69.9 70.1 70.4 70.6
68 64.9 65.2 65.5 65.8 66.1 66.3 66.6
72 60.3 60.6 60.9 61.2 61.5 61.8 62.1
76 55.2 55.6 55.9 56.2 56.5 56.8 57.1
80 49.5 49.8 50.2 50.5 50.8 51.1 51.4
84 43.0 43.4 43.7 44.0 44.4 44.7 45.0
88 35.5 35.9 36.2 36.5 36.9 37.2 37.5


ASTM (1983) Maintaining constant relative humidity by means of aqueous solutions. In: 1983
Annual 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 water
for humidity control. Corrosion 14, 117–118.
Forney, C.F. and Brandl, D.G. (1992) Control of humidity in small controlled environment chambers
using 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,
Michel, B.E. and Kaufmann, M.R. (1973) The osmotic potential of polyethylene glycol 6000. Plant
Physiology 51, 914–916.
Robinson, R.A. and Stokes, R.H. (1959) Electrolyte Solutions, 2nd edn. Butterworths Scientific
Publications, London, 559 pp.
Rockland, L.B. (1960) Saturated salt solutions for static control of relative humidity between 5 and
40°C. Analytical Chemistry 32, 1375–1376.
Sun, W.Q. and Gouk, S.S. (1999) Preferred parameters and methods for studying moisture content of
recalcitrant 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 Institute
Malaysia, Kuala Lumpur, pp. 404–430.
Wexler, A. (1997) Constant humidity solutions. In: Lide, D.R. (ed.) Handbook of Chemistry and
Physics, 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 biological
research. Ecology 41, 232–237.
Young, J.F. (1967) Humidity control in the laboratory using salt solutions – a review. Journal of
Applied Chemistry 17, 241–245.
02 Dessication - Chap 2 18/3/02 1:54 pm Page 92
Dessication - Chap 03 18/3/02 1:55 pm Page 93

3 Experimental Aspects of Drying and


Norman W. Pammenter,1 Patricia Berjak,1 James Wesley-Smith1 and

Clare 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 94
3.2. Drying Rate 94
3.2.1. Commonly employed drying techniques 94 Seed material 95 Vegetative tissue 97
3.2.2. Quantification and modelling of drying rates 98
3.2.3. Effects of different drying rates 99 Desiccation-sensitive tissue 99 Desiccation-tolerant tissue 100
3.2.4. ‘Ultradrying’ of desiccation-tolerant material 101
3.3. Influence of Rehydration Technique 102
3.4. Length of Time in the Partially Dehydrated State 102
3.5. Methods of Assessing Response to Rehydration 103
3.5.1. ‘Germination’ 103
3.5.2. Resurrection plants 104
3.5.3. Electrolyte leakage 104
3.5.4. Tetrazolium test 104
3.5.5. Other responses 104
3.6. Expression of Water Content Data 105
3.6.1. Mass basis 105
3.6.2. Water potential 105
3.6.3. Relative water content 106
3.7. Conclusion 106
3.8. References 106

© CAB International 2002. Desiccation and Survival in Plants: Drying Without Dying
(eds M. Black and H.W. Pritchard) 93
Dessication - Chap 03 18/3/02 1:55 pm Page 94

94 N.W. Pammenter et al.

3.1. Introduction dration may have adverse metabolic and

other deleterious consequences unrelated
When plant material is subjected to dehy- to drying.
dration, the observed response (in terms of 3. The rate of air movement across the tis-
damage accumulation and survival) can sue; this affects the thickness of the bound-
vary with the techniques used to assess the ary layer and the ability of water vapour to
response. This has the potential to cause diffuse through it. Forced ventilation will
confusion in the interpretation of experi- lead to faster drying than will still air.
mental data. It is the objective of this chap- 4. The size and shape of the tissue; this
ter to outline the range of techniques that affects the surface area-to-volume ratio and
have been used, to note the effects each can the distance water must diffuse from the
have on the observed response and, as far interior of the tissue to the surface. Small
as possible, to suggest underlying causes of tissue pieces will dry faster than large
these effects. ones, and ‘flat’ tissue will dry faster than
Experimental or technical aspects that ‘bulky’ tissue.
can influence the response to dehydration 5. The chemical and physical composition
include drying rate, the length of time the of the outer layer of the tissue; this will
tissue is maintained in the (partially) dry affect the permeability of the tissue to
state, the rehydration and recovery tech- water, in either the liquid or vapour form.
niques, and the method used to assess the Tissues with lignified, suberized or waxy
response. The developmental status of the outer layers will dry more slowly than
tissue may also influence the response to those without such layers.
drying. The information presented in this 6. The amount of material to be dried; this
chapter is derived almost exclusively from will affect factors such as surface area-to-
studies on seeds and vegetative tissue (vas- volume ratios and boundary layers.
cular and non-vascular species). The dis- Generally, small quantities of material will
cussion has not been extended to pollen dry faster than will a large mass.
and spores, largely because their size is
These factors are, to a greater or lesser
such that they normally dehydrate rapidly
extent, under the control of the experi-
and so effects of drying conditions are less
menter, who can thus alter (but not control
frequently studied (see Chapter 6).
with precision) the rate at which the mater-
ial dries. It should be noted that the terms
‘rapid’, ‘intermediate’ and ‘slow’ are com-
3.2. Drying Rate parative only within a single study; there
are no absolute boundaries to these terms.
During drying, water molecules diffuse A drying rate that in one study might be
from the tissue to the surrounding air. described as ‘rapid’ might be considered to
There are a number of factors that can be ‘slow’ in another.
affect the rate at which this diffusion
occurs and hence the rate at which the tis-
sue will dry. Some of these factors are:
3.2.1. Commonly employed drying
1. The vapour pressure of the surrounding techniques
air; this affects the difference in free energy
of water between the tissue and the air, and Commonly used drying techniques vary in
hence the drying rate. Tissue will obvi- the degree to which temperature and
ously dehydrate faster in dry than in humidity are controlled and the extent of
humid air. air movement across the material being
2. Temperature; this will also influence the dried. In the case of seeds they also vary in
water free energy difference, which will be whether or not outer coverings are
greater at higher temperatures. However, removed. The choice of method is often
use of elevated temperatures during dehy- influenced by the amount of material that
Dessication - Chap 03 18/3/02 1:55 pm Page 95

Experimental Aspects of Drying and Recovery 95

is being dried, the facilities available and will vary amongst species. Cromarty et al.
the reason for which the material is being (1985) have given details of the design of
dried. Some of the methods are specific for seed bank facilities for orthodox seeds,
seeds, and some for resurrection plants, but including detailed consideration of drying
many can be used for any small piece or protocols. Drying times using these proto-
pieces of tissue. Commonly used tech- cols are of the order of days.
niques are summarized in Table 3.1. As 4. Air flow in a laminar flow cabinet
drying conditions can have marked effects (Grout et al., 1983; Normah et al., 1986;
on the response of tissue to drying (Section Pence, 1992; Chandel et al., 1995). This
3.2.3), it is critical that the drying condi- technique can be used for small seeds and
tions (e.g. temperature, relative humidity parts of seeds (excised embryos or embry-
(RH), light conditions, air flow) used in any onic axes) and has the advantages that it
investigation are reported. introduces an air flow over the material
(forced ventilation) and reduces the intro-
duction of further microbial contaminants. Seed material However, the temperature and RH of the air
Techniques that have been used to dry seed are not controlled, and will vary with
material are outlined below: locality and between seasons, but drying
rates of the order of hours can be achieved
1. Air drying of seeds or fruits in sun or with small tissue pieces.
shade (e.g. Albrecht, 1993). Of all the tech- 5. Drying seeds by burying them in acti-
niques used this one offers the least control vated silica gel (Pammenter et al., 1998).
of the various factors affecting drying, but This will yield the most rapid rate of dry-
it is commonly used in the field where ing in the absence of forced ventilation,
facilities are limited, samples are large and and has been adopted as the standard tech-
drying shortly after collection is desired. nique by the IPGRI–Danida Forest Seed
When drying seeds, the usual practice is to Centre sponsored project on the handling
spread them in an even layer, but, if the and storage of recalcitrant tropical tree
layer is more than one seed deep, the sam- seeds (IPGRI/DFSC, 1996).
ple must be turned frequently to prevent 6. Rapid air flow over material in a small
uneven drying. A similar practice is chamber, i.e. flash drying. A common prac-
employed for the whole fruits, for particu- tice is to place the material on a grid in a
lar species. Drying using this technique can small container and pass air into the bot-
take several days. tom, over the sample, and vent from the
2. Drying under ambient laboratory condi- top of the chamber (Berjak et al., 1990;
tions (e.g. Wu et al., 1998) has the same Pammenter et al., 1991). The air may or
disadvantages as the first method, except may not be dried. If air from a gas cylinder
that ambient conditions probably vary less or a compressor is used, it should be dry.
in the laboratory than outside. Drying (As a word of warning, not all compressors
times will be similar to those achieved in are properly maintained, and a compressed
the first method. air line to a laboratory bench may have
3. Walk-in chambers with temperature droplets of water in it and is obviously
and RH control. These facilities are nor- unsuitable.) An improved system designed
mally available only in large-scale com- by one of us (J.W.-S.) in which air is circu-
mercial or research organizations and are lated through silica gel and over excised
generally used to dry substantial quanti- embryonic axes is illustrated in Fig. 3.1;
ties of material. Recommended practice is this equipment yields the fastest drying
to dry at 10–25°C and 10–15% RH rates of all the techniques we have used
(International Plant Genetic Resources (several minutes to hours).
Institute (IPGRI), 1994). This approach 7. Drying excised axes under partial vac-
permits standard and repeatable drying uum (Fu et al., 1993). Although this tech-
conditions, but the drying rate achieved nique yielded rapid drying (faster than
Dessication - Chap 03

Table 3.1. Summary of drying methods and approximate drying times used in studies on responses to desiccation.
Quantity of Drying

Drying method material time Reference

Seeds Sun drying Kilograms Days Albrecht, 1993
Still air at controlled temperature and RH Days to weeks Cromarty et al., 1985; IPGRI, 1994;
Wu et al., 1998
1:55 pm

Forced ventilation at controlled temperature and RH Monolayer Hours to days Ntuli et al., 1997
Buried in silica gel Tens to hundreds Grout et al., 1983; Pammenter et al.,
of seeds 1998
Excised axes Laminar flow hood Monolayer Hours Grout et al., 1983; Normah et al., 1986
Page 96

Flash-drying – forced ventilation with or without Tens of minutes Pammenter et al., 1991
silica gel to hours
Vegetative tissue Withdrawal of watering from whole plant Whole tracheophytes Days Gaff et al., 1992; Vander Willigen, et al.,
N.W. Pammenter et al.

Excised plant parts or moss clumps on bench top Segments of leaves, Hours Bartels et al., 1990; Farrant et al., 1999
twigs, moss clumps
Small chambers at controlled temperature and RH Hetherington and Smillie, 1982;
Seel et al., 1992
Forced ventilation in laminar flow hood or small Bochicchio et al., 1998; Farrant et al.,
chambers 1999
Dessication - Chap 03 18/3/02 1:55 pm Page 97

Experimental Aspects of Drying and Recovery 97

storage above silica gel), none of the axes axes excised from seeds (Grout et al., 1983;
survived the treatment. Consequently, this Normah et al., 1986; Pritchard and
technique is not recommended without Prendergast, 1986; Berjak et al., 1990). This
further investigation. has the advantage of permitting drying
8. Drying to equilibrium at constant RH. rates of the order of tens of minutes to a
This is generally done by placing the mate- few hours, but can be very laborious if
rial over saturated solutions of salts, within large numbers of axes are required. If this
a small chamber. The air can be still or may approach is adopted, it is important to
be stirred by a fan in the chamber (Ntuli et minimize damage whilst excising the axes.
al., 1997). The advantage of this technique
is that the equivalent water potential Vegetative tissue
at equilibrium is known ( =
(RT/V)*ln(RH/100), where R is the univer- A similar wide range of drying techniques
sal gas constant, T the absolute tempera- has been applied to vegetative material,
ture, V molal volume of water and RH although in this case the drying is gener-
relative humidity). However, under differ- ally for experimental purposes and only
ent RH conditions the material will dry at small quantities are dried. One of the few
different rates and will reach equilibrium examples of drying large quantities con-
cerns the alga Porphyra (Ooshua, 1993). As
after different times, ranging up to several
is the case with seeds, the various tech-
niques differ in the degree of control and
Whatever drying method is adopted, the the drying rates achieved.
rate of drying is often ultimately deter- In experiments on tracheophytes, a com-
mined by the size of the tissue; large seeds mon method is to induce desiccation by the
will always dry more slowly than small simple expedient of withholding water (e.g.
ones. To overcome this limitation, it has Gaff et al., 1992; Reynolds and Bewley,
become common practice to dry embryonic 1993; Quartacci et al., 1997; Vander

Embryonic axes

Plastic mesh


Silica gel

Fig. 3.1. The improved apparatus for flash-drying of excised embryonic axes. A computer fan circulates air
through the silica gel and over the axes.
Dessication - Chap 03 18/3/02 1:55 pm Page 98

98 N.W. Pammenter et al.

Willigen et al., 2001). This technique mim- 3.2.2. Quantification and modelling of
ics conditions and drying rates (usually drying rates
days to weeks) occurring in the natural
habitat of the plants, most drying appearing The factors determining drying rate are
to occur only after the soil has lost virtually manifold, many of them are difficult to
all its water (Sherwin et al., 1998; Norwood quantify and they change during the drying
et al., 1999). The size and nature of the process. Consequently, mechanistic model-
material (younger leaves enclosed or cov- ling of the drying process is extremely diffi-
ered by older ones) often results in uneven cult. During the drying process, water is
drying within a single plant. converted from the liquid form in the tissue
Faster drying rates (hours to days) have to the vapour phase in the surrounding air.
been achieved by using excised tissues The driving force is the difference in the
(twigs, leaves or callus) on the laboratory free energy of water between the tissue and
bench top, with forced ventilation in a lam- the air. As the tissue dries the free energy of
inar flow cabinet, or rapid air flow in a the water decreases, leading to a decrease in
small chamber (e.g. Bartels et al., 1990; the free energy differential and so to non-
Reynolds and Bewley, 1993; Quartacci et linear drying kinetics. There are a number
al., 1997; Farrant et al., 1999). Drying in of resistances retarding the diffusion of
small chambers over silica gel or saturated water from the tissue to the air. These
salt solutions, with or without stirring the include the boundary layer (the layer of still
air, has also been employed (Hetherington air in immediate contact with the tissue),
and Smillie, 1982; Bochicchio et al., 1998; the outer layer of the tissue (the nature and
Farrant et al., 1999). Non-vascular plants permeability of which vary) and the resis-
dry in the field within a few hours tance to the movement of water from the
(although this rate could be retarded by the interior of the tissue to the surface. The site
clumped nature of the growth form). of evaporation is not known, and so it is
Laboratory drying techniques are often unclear whether water moves from the inte-
chosen to simulate natural rates, within rior to the tissue surface in the liquid or
small enclosed chambers at RHs ranging vapour phase. The boundary layer will be
from 50 to 85% at constant temperature influenced by the speed of the air moving
(e.g. Seel et al., 1992; Oliver et al., 1993; over the tissue and by the size and shape of
Tuba et al., 1996). Very rapid drying has the tissue. With forced ventilation, the air
been achieved by the use of silica gel or of flow is probably turbulent, rather than lami-
a lyophilizer (Oliver and Bewley, 1984; nar, and so difficult to model. The size and
Oliver et al., 1998). shape of seeds and excised axes are often
An extremely important experimental irregular and variable, and so their effect on
aspect of drying vegetative tissue is the the boundary layer is also difficult to model.
light conditions during dehydration, as During the drying process, as the tissue
photo-oxidation can be an important com- dehydrates, the resistance to the transfer of
ponent of desiccation damage in photosyn- water from the interior to the surface will
thetic tissue (Seel et al., 1992; Sherwin and probably change, complicating the analysis.
Farrant, 1998; Tuba et al., 1998). Although All this complexity means that mechanistic
many seeds have green cotyledons that pre- modelling of drying rates must, by necessity,
sumably contain chlorophyll, the light con- be based on simplifying assumptions.
ditions during drying have not attracted Cromarty et al. (1985) have suggested a
the attention of seed biologists. It is also model for use in seed banks, based on satu-
possible that temperature could affect the ration vapour pressure at seed temperature,
response; seeds of Zizania palustris are velocity of air over the seed lot, seed mass
more tolerant of desiccation when dried at and oil content. The model assumes a thin
25°C than at lower (Kovach and Bradford, layer of seeds and a spherical shape.
1992; Berjak et al., 1994; Ntuli et al., 1997) Empirical models of drying rates are also
or higher (Ntuli et al., 1997) temperatures. not always successful. Depending on the
Dessication - Chap 03 18/3/02 1:55 pm Page 99

Experimental Aspects of Drying and Recovery 99

methods used, and the drying rates heterophyllus (J. Wesley-Smith, unpub-
obtained, different factors could be deter- lished data), C. australe (Govindasamy,
mining the rate of drying, and the rate-deter- 1997) and T. dregeana (Govindasamy,
mining factor could be changing during the 1997; Pammenter et al., 1999) were dried at
drying process. Although exponential rela- 96% RH (slowly), drying was exponential;
tionships sometimes can be fitted to the data when axes of these species were dried over
from a drying time course, particularly for silica gel (rapidly), the initial drying was
seeds or excised axes, it is our experience faster than predicted by an exponential
that in many cases the same form of equa- rate. Similarly, when seeds of Ekebergia
tion cannot be used to describe the data for capensis were dried slowly (seeds with
different drying rates (e.g. Pammenter et al., endocarp buried in silica gel; dehydration
1998). As a generalization, if tissue is dried over 10 days), drying was exponential;
relatively slowly, the relationship between when seeds were dried more rapidly (seeds
water content and drying time is exponen- without endocarp buried in silica gel;
tial. However, for material dried rapidly, the dehydration over 1 day), initial drying was
initial water loss is considerably faster than faster than predicted by an exponential
that predicted by an exponential relation- relationship (Fig. 3.2; Pammenter et al.,
ship. It must be re-emphasized that the 1998). These authors suggested that the
terms ‘slowly’ and ‘rapidly’ are relative. drying kinetics indicated that uneven dry-
There is no drying rate common across ing of the tissue might be occurring under
species where drying changes from faster rapidly dehydrating conditions. It does
than exponential to exponential; a fast dry- appear, however, that excised axes of
ing rate in one experiment could be the Theobroma cacao show exponential drying
equivalent of slow in another. over a range of drying rates (see Chapter 2).
For example, during ‘slow’ drying of
whole seeds of Landolphia kirkii
(Pammenter et al., 1991), and of Camellia 3.2.3. Effects of different drying rates
sinensis (Berjak et al., 1993), the water con-
tent of the axes within the seeds followed It is beyond the scope of this chapter to
an exponential relationship with time, but discuss in detail the consequences of dehy-
‘rapid’ drying of excised axes did not drating plant tissue. However, reference
(curve-fitting exercises were not reported must be made to the subject to understand
in the original publications; the data have the influence of drying rate on the response
been re-analysed). Similarly, initial faster- to dehydration. The effect appears to
than-exponential drying rates have been depend on whether the tissue is inherently
observed in rapidly dried excised axes of desiccation-sensitive or -tolerant.
Syzigium guiniense, Castanospermum aus-
trale, Trichilia dregeana, Artocarpus het- Desiccation-sensitive tissue
erophyllus, Azadirachta indica, and the
radicle tips of axes of Podocarpus henkelii. Certainly with desiccation-sensitive (recal-
By way of contrast, axes of Avicennia citrant) seeds, or embryonic axes excised
marina and entire axes of P. henkelii (the from these seeds, material that is dried
embryonic axes of both species are rela- rapidly (of the order of tens of minutes to
tively large) show exponential drying hours) can survive to lower water contents
(N.W. Pammenter, P. Berjak and J. Wesley- before viability is lost than material that is
Smith, unpublished observations). It might dried slowly (over a period of days)
be argued that the same relationship can- (Normah et al., 1986; Pritchard and
not be used to describe ‘slow’ and ‘rapid’ Prendergast, 1986; Farrant et al., 1989;
drying because different material is often Pammenter et al., 1991, 1998; Pritchard,
used to obtain different drying rates: seeds 1991; Berjak et al., 1993; Pritchard and
for slow drying and excised axes for rapid Manger, 1998). The rapid drying is not
drying. However, when excised axes of A. actually increasing desiccation tolerance as
Dessication - Chap 03 18/3/02 1:55 pm Page 100

100 N.W. Pammenter et al.

Axis water content (g water g–1 dry mass)

(a) (b)




0 2 4 6 8 10 0 10 20 30 40 50
Time (days) Time (h)

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

such; it is simply that, if the tissue is dried state, it rapidly loses viability (Walters et
fast enough, low water contents can be al., 2001).
achieved before sufficient time elapses for Studies on the effects of dehydration of
it to die. It has been suggested that some of desiccation-sensitive vegetative tissue are
the processes leading to viability loss are more limited. However, if much of the vol-
aqueous-based and so occur at relatively ume of the cells is occupied by fluid-filled
high (intermediate) water contents, of the vacuoles, mechanical damage associated
order of 1.0–0.3 g water g1 dry mass, cor- with volume reduction consequent upon
responding to water potentials of about drying might be important (Iljin, 1957;
1.5 to 14 MPa (Vertucci and Farrant, Vertucci and Farrant, 1995; Farrant et al.,
1995; Farrant et al., 1997; Pammenter and 1997), in which case drying rate might
Berjak, 1999; Walters et al., 2001). Material have little effect.
that is dried very rapidly passes through
these intermediate water contents so fast Desiccation-tolerant tissue
that the damage caused by the deleterious
processes does not have time to accumu- In tissue that is, or becomes, desiccation-
late; thus viability loss does not occur as a tolerant, the effect of drying rate depends
consequence (Pammenter et al., 1998; upon the stage of development and/or the
Pammenter and Berjak, 1999). Although nature of the tolerance mechanisms.
rapid drying does permit viability retention Developing orthodox seeds start to
to low water contents, those tolerated by acquire desiccation tolerance concomitant
desiccation-sensitive seeds or axes (mini- with, or slightly preceding, reserve accu-
mum of about 0.2 g water g1 dry mass) are mulation, and the population as a whole
never as low as water contents usually is generally tolerant by the end of this
occurring naturally in dry desiccation-tol- stage (e.g. Bewley and Black, 1994). The
erant (orthodox) seeds (< 0.05 g water g1 response to rate of drying of orthodox
dry mass). Also, survival of these low seeds that are still in the desiccation-sen-
water contents by embryonic axes of recal- sitive stage of development is in almost
citrant seeds is apparent only if the mater- direct contrast to the response of recalci-
ial is assessed for survival immediately trant seeds. If, after histodifferentiation,
after drying. If the material is maintained prior to the acquisition of desiccation tol-
(at room temperature) in the partially dry erance, a developing orthodox seed is
Dessication - Chap 03 18/3/02 1:55 pm Page 101

Experimental Aspects of Drying and Recovery 101

dried rapidly, it will not survive; if it is chlorophyll during drying, whereas

dried slowly, it will (Kermode and Bewley, species that are poikilochlorophyllous lose
1985; Bewley and Black, 1994). This is chlorophyll and dismantle thylakoid mem-
probably because, during slow drying, suf- branes (Hetherington and Smillie, 1982;
ficient time elapses for the development of Sherwin and Farrant, 1998), although it is
the tolerance mechanisms. possible that some of the observed abnor-
The response of desiccation-tolerant malities are a consequence of the fixation
vegetative tissue (‘resurrection’ plants) to method (see Platt et al., 1997).
drying rate appears to vary with the Poikilochlorophylly appears to preclude
nature of the tolerance mechanism rapid drying rates and, although some
(reviewed by Oliver and Bewley, 1997). In homoiochlorophyllous plants can survive
non-vascular resurrection plants, toler- faster drying rates, there are other species
ance seems to be achieved predominantly that cannot (Farrant et al., 1999).
by an ability to repair damage caused by Excluding old leaves, which would natu-
desiccation, and is thus primarily based rally senesce, all tissues of most resurrec-
on constitutive mechanisms (Oliver and tion angiosperms are tolerant; however,
Bewley, 1997). Drying rate appears to have there are species in which only the young,
little influence on ultimate survival, but immature tissues are tolerant (Gaff and
recovery takes longer in rapidly dried tis- Ellis, 1974; Vander Willigen et al., 2001),
sue, perhaps suggesting the existence of suggesting that developmental stage is
some inducible protection mechanisms another compounding factor. Leaf tissues
(Schonbeck and Bewley, 1981a). It has been of some species show tolerance whether
observed that non-vascular resurrection attached or detached from the parent
plants tend to become less desiccation- plant, whereas others survive only
tolerant if kept in the hydrated state for attached, or after an initial drying phase
extended periods compared with daily on the parent plant, during which they
dehydration/rehydration ‘hardening’ cycles presumably acquire the necessary signals
(Schonbeck and Norton, 1979; Schonbeck for tolerance (Gaff and Loveys, 1992). A
and Bewley, 1981b). detailed discussion of these responses,
Resurrection tracheophytes have been and their underlying causes, is beyond the
classified as ‘modified desiccation-tolerant scope of this chapter. Suffice it to say that
plants’, in comparison with non-vascular these complexities must be borne in mind
resurrection plants (‘fully desiccation-tol- by the investigator when designing and
erant plants’), because their ability to sur- interpreting the results of experiments.
vive desiccation is rate-dependent (Oliver
and Bewley, 1997). The responses of resur-
rection angiosperms to drying technique 3.2.4. ‘Ultradrying’ of desiccation-tolerant
appears to be complex. Rapid desiccation material
is generally lethal, although Bochicchio et
al. (1998) have shown that it is water con- There has been recent interest in, and
tent, rather than drying rate, that affects lively debate on, the effects of storing
survival of detached leaves of the resurrec- orthodox seeds at water contents below
tion plant Boea hygroscopica. The general those normally used in gene banks. It is not
effect of drying rate on the response of res- intended to review that debate here, but
urrection tracheophytes is a consequence simply to point out that, even in desicca-
of an inducible desiccation tolerance, this tion-tolerant tissue, the experimental tech-
tolerance being based on protection during nique – the extent to which the material is
desiccation, rather than repair on rehydra- dried – may have an influence on the
tion. This group can be further subdivided response to drying (and subsequent stor-
into two, based on the strategy to prevent age). For more information, the reader is
light-associated damage on drying. The referred to Ellis (1998), Walters (1998) and
homoiochlorophyllous species retain Walters and Engels (1998).
Dessication - Chap 03 18/3/02 1:55 pm Page 102

102 N.W. Pammenter et al.

3.3. Influence of Rehydration Technique or more slowly in misting chambers (Seel

et al., 1992, Tuba et al., 1996). As with
It is well known that if dry biological mate- dehydration, lighting and temperature
rial is immersed in water a number of sub- must be carefully controlled.
stances of low molecular weight will leak
from the tissue (discussed by Hoekstra et
al., 1999). If the tissue is desiccation-toler- 3.4. Length of Time in the Partially
ant, this leakage will subside with rehydra- Dehydrated State
tion, although, if the tissue is initially very
dry, leakage can be extensive and could be When desiccation-sensitive tissue is dehy-
damaging, this damage being exacerbated drated, it is subjected to a number of
by imbibition at low temperatures (Pollock, stresses as it dries. The type of damage that
1969; Hobbs and Obendorf, 1972). Because potentially can occur will change as the
of this, it is common in studies on seeds or water content decreases (see Chapter 9); as
excised axes to ‘prehumidify’ tissue by the intensity of the stress increases, the
maintaining it in a saturated atmosphere or effect on the tissue generally becomes more
placing on damp paper, before immersion severe. However, the effect of a stress, par-
in water. However, many recalcitrant seeds ticularly a mild stress, is not instantaneous.
are damaged at water contents far in excess If a stress induces a metabolic disorder, it
of those at which ‘imbibitional damage’ takes time for the damage consequent upon
occurs, and there is little evidence to sug- that disorder to accumulate. Thus, the
gest that such damage upon imbibition is effect of a stress depends not only on its
an important factor in desiccation-sensitive intensity, but also on the time for which the
seed material. Kovach and Bradford (1992) stress is applied. It is this concept of ‘inten-
initially ascribed the response to desicca- sity’ versus ‘duration’ of a stress that under-
tion of seeds of Z. palustris to imbibitional lies the confusion that has obscured the
damage, although Vertucci et al. (1995) interpretation of the effects of drying rates
suggested that the damage was a direct on desiccation-sensitive seed material.
result of desiccation, rather than imbibi- To unravel the confounding issues of
tion. Sacandé et al. (1998) have demon- water content and time in drying experi-
strated imbibitional damge exacerbated by ments, it is necessary to dry tissue almost
low-temperature imbibition and increased instantaneously to a range of water con-
storage time in seeds of neem (A. indica) at tents and then to maintain the material at
water contents < 8% (fresh mass basis), but these water contents. In practice, this is not
this is a water content considerably lower a simple experimental achievement. Most
than most recalcitrant seeds will survive. seeds are too large to dry rapidly and, if
In vegetative tissues, rehydration tech- isolated embryonic axes are dried, it is dif-
niques are generally even less well ficult to ‘store’ them in the partially dehy-
described and assessed than are the dehy- drated state without some other deleterious
dration techniques, and consequently the conditions (such as anoxia or microbial
effects of various methods of rehydration proliferation) occurring. Maintaining vege-
are relatively unknown. In the tracheo- tative tissue in the partially dehydrated
phytes, whole plants are generally rewa- state is probably even more difficult and no
tered to field capacity with (Norwood et experiments are known where this has
al., 1999) and with or without (Sherwin been attempted.
and Farrant, 1998) additional aerial spray- Despite these difficulties, some informa-
ing. Dehydrated excised leaves are rehy- tion is available. Walters et al. (2001) have
drated by floating on or in water (Gaff and shown that partially dehydrated embryonic
Loveys, 1992; Reynolds and Bewley, 1993; axes of tea lose viability within a few days,
Bochicchio et al., 1998; Dace et al., 1998). and that the rate at which viability is lost
Mosses are rehydrated by immersion in depends on the water content. Similarly,
distilled water (Oliver and Bewley, 1984) isolated axes of T. dregeana dried over sil-
Dessication - Chap 03 18/3/02 1:55 pm Page 103

Experimental Aspects of Drying and Recovery 103

ica gel lost viability as the water content 3.5.1. ’Germination’

reached the level in equilibrium with the
desiccant, whilst axes dried at 96% RH lost With seeds, the most common assessment
viability some days after the tissue had method is to set them out to germinate.
reached equilibrium (Pammenter et al., However, as pointed out by Hong and Ellis
1999). Similarly, in the relatively long-lived (1996), it is possible that the treatment may
seeds of Araucaria huntsteinii, longevity at induce a dormancy and that seeds that do
6°C was reduced as water content was not germinate may not actually be dead.
reduced below about 45% (Pritchard et al., Those authors recommended that the dura-
1995). As an aside, this raises questions tion of germination tests be extended until
concerning the concept of ‘degree of recal- all non-germinated seeds have been posi-
citrance’. Would this be assessed on the tively identified as dead by the fact that they
basis of the minimum water content to rot. Another problem is that a seed may pro-
which the seed (or embryonic axis) can be duce a radicle, and so be scored as having
dried without loss of viability, or on the germinated, but be so damaged as to be
time for which it survives in equilibrium unable to establish a viable seedling (Fu et
with some predetermined water potential? al., 1993; Berjak et al., 1999). The precision
The concept of ‘intensity’ versus ‘duration’ of a germination test can be increased by fol-
of a stress has practical applications. It has lowing the time course of germination. An
been suggested that, in the case of recalci- increased lag before the first seed germi-
trant seeds that germinate in storage, partial nates or a decrease in the rate of germina-
dehydration may prevent this and so tion (increased time to 50% germination)
increase storage life span (Hong and Ellis, may indicate damage that is repaired during
1996). However, it appears that even a mild the lag phase. A simple assessment of final
water stress applied to seeds of the tropical germination would not reveal this damage.
species T. dregeana is deleterious (Drew et A number of suggestions have been made
al., 2000), and relatively mild partial drying concerning fitting equations to germination
of the temperate seeds of A. huntsteinii data (Brown and Mayer, 1988), but they gen-
(Pritchard et al., 1995) and Aesculus hip- erally require more samples than are often
pocastanum (Tompsett and Pritchard, 1998) available when undertaking studies on
can reduce longevity. Thus ‘sub-imbibed recalcitrant seeds from wild species.
storage’ should be approached with caution When experiments are conducted on
as it can actually reduce life span. excised embryonic axes, ‘germination’ can
In passing, it should be noted that even be assessed by placing the axes on damp
desiccation-tolerant organisms do not have paper in an enclosed chamber (such as a
an infinite life span in the dehydrated Petri dish), although it is common to use in
state; they accumulate damage in this state, vitro growth media. As many recalcitrant
and so desiccation could be considered to seeds, particularly from the tropics, har-
be a stress, even in tolerant tissues. bour fungal propagules (Berjak, 1996;
Calistru et al., 2000), their removal by suit-
able pretreatment is essential under these
3.5. Methods of Assessing Response to conditions. As with seeds, care must be
Dehydration taken in assessing ‘germination’. Swelling
and/or greening of an axis suggests that it
A variety of techniques have been used to is not dead (although a dehydrated axis
assess damage in response to drying. will swell on rehydration), but it does not
Different techniques measure different phe- necessarily imply that it is capable of pro-
nomena (membrane characterisics, respira- ducing an independent plantlet.
tory competence, photosynthetic activity), Assessment can also be complicated by
but the ultimate test is whether an indepen- choice of the medium, as this may have an
dent functioning organ(ism) can be re-estab- influence on the ‘growth’ of the tissue (as it
lished after dehydration and rehydration. does in normal in vitro multiplication and
Dessication - Chap 03 18/3/02 1:55 pm Page 104

104 N.W. Pammenter et al.

propagation). Material that has been dam- damage associated with imbibition. With
aged, but not killed, by the dehydration seeds or excised axes there is generally
treatment may take a considerable time to good agreement between leakage character-
show signs of growth, and so care should istics and other signs of damage such as
be taken not to discard it too early. loss of vigour or viability (McKersie and
Tomes, 1980; Pammenter et al., 1991;
Berjak et al., 1992, 1993). Older techniques
3.5.2. Resurrection plants to assess membrane integrity involve the
use of vital dyes, which leak from damaged
With whole resurrection plants, the criteria cells (Gaff and Loveys, 1992).
for determining whether the organism is
‘functional’ may vary. This may simply be
on physical appearance (particularly the 3.5.4. Tetrazolium test
greening of poikilochlorophyllous tissue),
measurement of chlorophyll fluorescence The reduction of colourless tetrazolium
characteristics (Fv/Fm), which indicates the chloride (2,3,5-triphenyl tetrazolium chlo-
photochemical efficiency of photosystem ride (TTZ)) to a pink/red formazan dye is
II), or assessment of the ability to assimi- taken as a measure of respiratory activity, as
late carbon dioxide photosynthetically, or TTZ is reduced by components of the mito-
to respire. chondrial electron transport chain.
Although the tetrazolium test is used exten-
sively to assess the quality of orthodox
3.5.3. Electrolyte leakage seeds, its use in the study of desiccation
response has been limited. Ntuli et al.
A technique that is commonly employed (1997) showed that considerable differences
with small pieces of tissue (small seeds, occurred between the ability to germinate
excised axes, leaves of resurrection tra- and apparent viability as a result of tetra-
cheophytes, ‘pieces’ of non-vascular zolium tests, when investigating the desic-
plants) is to measure leakage of electrolytes cation response of Z. palustris, indicating
or of specific ions such as K+. An advan- that the two tests were not equivalent or not
tage of this technique is its simplicity and necessarily measuring the same thing. A
rapidity, especially using multiple-cell further caveat in using the TTZ test as a via-
electrical conductivity meters, which are bility assay is that a dead seed supporting a
commercially available. The extent of elec- vigorous mycelium internally will test posi-
trolyte leakage is considered to assess the tive as a result of fungal respiration.
degree of membrane damage (Bramlage et
al., 1978; McKersie and Tomes, 1980). As
the rate of leakage over the first few min- 3.5.5. Other responses
utes is often higher than the steady rate
established later, the steady-state rate of A number of biochemical and biophysical
leakage is often taken as an indication of responses to dehydration have been
membrane damage (McKersie and Stinson, assessed by a variety of workers. Examples
1980). An alternative approach is to assess include the activities of antioxidants
leakage (or rate of leakage) after a given (Tommasi et al., 1999), accumulation of late
time as a proportion of total leakage (or embryogenesis abundant proteins (Finch-
maximum rate), which occurs when all Savage et al., 1994; Gee et al., 1994), ethyl-
membranes are fully disrupted by treat- ene production, respiration and protein
ments such as homogenizing, boiling, auto- synthesis (Salmen Espindola et al., 1994),
claving or repeated freeze/thaw cycles. It is partitioning of amphipathic molecules
common practice to prehumidify tissue in between cytosol and membranes (Golovina
a saturated atmosphere or on damp filter et al., 1998) and changes in cytoplasmic vis-
paper prior to immersion to reduce any cosity (Leprince and Hoekstra, 1998). These
Dessication - Chap 03 18/3/02 1:55 pm Page 105

Experimental Aspects of Drying and Recovery 105

are not covered here because often it was change in ‘water content’ reflects the pro-
the details of these responses that were the portional change in the amount of water in
objectives of the investigations, and so they the tissue; if the water content changes
do not fall within the scope of a discussion from 1.0 to 0.5 g water g1 dry weight, the
of general experimental approaches. tissue has lost half its water. If the data are
expressed on a fresh mass basis, for tissue
at 1.0 g water g1 dry mass that loses half
3.6. Expression of Water Content Data its water, water content on a fresh mass
(see also Chapter 2) basis changes from 50% to 33.3%.

3.6.1. Mass basis

3.6.2. Water potential
The amount of water in plant tissue has
been expressed in a number of different The responses of tissue to drying are deter-
ways, but generally most commonly on mined by the processes that occur during
some form of ‘mass’ basis. This is probably drying. The processes that occur at any
because it is the simplest measure to water content are influenced by the free
obtain; the hydrated/partially hydrated tis- energy status of the water, and so it is bio-
sue is weighed, dried for some predeter- logically more meaningful to express tissue
mined time, and reweighed. The water in terms of water potential rather
International Seed Testing Association rec- than water content. Water potential of
ommends drying at 103°C for 17 h (ISTA, small pieces of tissue can be measured by
1999); an alternative approach, particularly thermocouple psychrometry, but this is
if using small amounts of tissue, is to dry at limited to higher potentials (above about
a lower temperature (to reduce loss of non- 5 MPa). However, recent technical
water volatile material) to constant weight. advances permit dewpoint psychrometric
The data can then be expressed on a fresh measurements to much lower water poten-
mass basis (mass of water per unit fresh tials. Sorption isotherms (equilibrating tis-
mass, often presented as a percentage); this sue at known fixed RH to constant water
indicates the proportion of the hydrated or content) can be used to establish the rela-
partially dehydrated tissue that is water. tionship between water content and water
Alternatively, the data can be expressed on potential (Vertucci et al., 1994), although
a dry mass basis (mass of water per unit this is difficult at high water potentials
dry mass). Strictly speaking, when express- because the relationship between RH and
ing the data on a mass basis, the term water potential changes rapidly in this
‘water content’ is incorrect; this should be region. Soaking tissue in solutions of
reserved for expressing the absolute known concentrations (and hence known
amount of water in the tissue, irrespective water potentials) of non-penetrating solutes
of the quantity of tissue. What is generally such as polyethylene glycol (PEG) 8000 has
described as ‘water content’ is actually a been used to assess the water
‘water concentration’ (the amount of water content/water potential relationship at
per unit amount of fresh or dry tissue). high water potentials (Vertucci et al., 1994;
Thus, water content on a dry mass basis Pritchard et al., 1995; Tompsett and
should be termed ‘dry mass-specific water Pritchard, 1998). A complication of estab-
concentration’. However, use of the term lishing sorption isotherms is the phenome-
‘water content’ to describe a ‘water concen- non of hysteresis; the equilibrium water
tration’ is so deeply entrenched that it is content at any RH depends on whether dry
unlikely to change. We prefer data to be tissue is being hydrated or hydrated tissue
expressed on a dry mass basis. In this case, is losing water (for a discussion, see Eira et
the basis to which values are being normal- al., 1999). There is an additional problem
ized does not change as the amount of when working with recalcitrant seeds or
water changes, and the proportional axes of tropical species. This material gen-
Dessication - Chap 03 18/3/02 1:55 pm Page 106

106 N.W. Pammenter et al.

erally harbours very high levels of fungal complication when calculating RWC is the
propagules, and fungal proliferation almost estimate of water content at ‘full turgor’. If
invariably accompanies attempts to equili- recalcitrant seeds are immersed in water,
brate tissue with atmospheres of RH above they will take up water as they germinate,
about 80%; it is therefore impossible to and so full turgor cannot be equated with
discriminate between the contribution of water content of tissue imbibed in water.
the seed material and the fungal mycelium Data could be expressed relative to the
to the derived isotherm. Under these con- water content at shedding, but this can be
ditions, use of concentrated solutions of very variable among seeds within a harvest,
PEG 8000 is advised. as well as between collections. With vegeta-
tive tissue it is possible to over-hydrate tis-
sue such that liquid water occupies some of
3.6.3. Relative water content the intercellular air spaces in leaves, or
exists as intercellular or surface water in
Relative water content, RWC (amount of non-vascular plants (Beckett, 1997). These
water in the tissue/amount of water at full effects lead to overestimates of water con-
turgor), has also been used to express the tent at full turgor.
water status of tissue (see Grange and Finch-
Savage (1992) for seed tissue, and Vander
Willigen et al. (2001) for vegetative tissue). 3.7. Conclusion
It is more meaningful than simple water
contents, although relative cell volume When investigating the response of tissue
(which is based on symplastic water only) is to dehydration, a range of techniques are
probably a better measure of direct stress to available. However, the observed response
which the tissue is subjected. To assess the is likely to depend on factors such as the
proportions of apoplastic and symplastic drying method, and possibly the rehydra-
water in tissue requires the construction of tion technique and method of assessment.
pressure–volume curves. Not only is this The techniques adopted will depend on
difficult (because of the difficulty of measur- the size or amount of tissue being dried,
ing water potential at low water contents), the facilities available and, importantly, the
but it is possible that the assumptions purpose of the investigation. The investiga-
underlying the analysis of pressure–volume tor should be aware of the technical com-
curves (Tyree and Hammel, 1972) may not plications when assessing and interpreting
hold at low water contents. An additional the 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 plant
Craterostigma 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-
Dessication - Chap 03 18/3/02 1:55 pm Page 107

Experimental Aspects of Drying and Recovery 107

tion rate on characteristics of water and desiccation-sensitivity in recalcitrant seeds of Camellia

sinensis. Seed Science Research 3, 155–166.
Berjak, P., Bradford, K.J., Kovach, D.A. and Pammenter, N.W. (1994) Differential effects of tempera-
ture on ultrastructural responses to dehydration in seeds of Zizania palustris. Seed Science
Research 4, 111–121.
Berjak, P., Walker, M., Watt, M.P. and Mycock, D.J. (1999) Experimental parameters underlying fail-
ure or success in germplasm cryopreservation: a case study on zygotic axes of Quercus robur L.
Cryo-Letters 20, 251–262.
Bewley, J.D. and Black, M. (1994) Seeds: Physiology of Development and Germination, 2nd edn.
Plenum Press, New York.
Bochicchio, A., Vazzana, C., Puliga, S., Alberti, A., Cinganelli, S. and Vermieri, P. (1998) Moisture
content of the dried leaf is critical to desiccation tolerance in detached leaves of the resurrection
plant Boea hygroscopica. Plant Growth Regulation 24, 163–170.
Bramlage, W.J., Leopold, A.C. and Parrish, D.J. (1978) Chilling stress to soybeans during imbibition.
Plant Physiology 61, 525–529.
Brown, R.F. and Mayer, D.G. (1988) Representing cumulative germination. 2. The use of the Weibull
function and other empirically derived curves. Annals of Botany 61, 127–138.
Calistru, C., McLean, M., Pammenter, N.W. and Berjak, P. (2000) The effects of mycofloral infection
on the viability and ultrastructure of wet-stored recalcitrant seeds of Avicennia marina (Forssk.)
Vierh. Seed Science Research 10, 341–353.
Chandel, K.P.S., Chaudhury, R., Radhamani, J. and Malik, S.K. (1995) Desiccation and freezing sensi-
tivity in recalcitrant seeds of tea, cocoa and jackfruit. Annals of Botany 76, 443–450.
Cromarty, A.S., Ellis, R.H. and Roberts, E.H. (1985) Design of Seed Storage Facilities for Genetic
Conservation. International Board for Plant Genetic Resources (now IPGRI), Rome.
Dace, H., Sherwin, H.W., Illing, N. and Farrant, J.M. (1998) Use of metabolic inhibitors to elucidate
mechanisms of recovery from desiccation stress in the resurrection plant Xerophyta humilis.
Plant Growth Regulation 24, 171–177.
Drew, P.J., Pammenter, N.W. and Berjak, P. (2000) ‘Sub-imbibed’ storage is not an option for extend-
ing longevity of recalcitrant seeds of the tropical species, Trichilia dregeana Sond. Seed Science
Research 10, 355–363.
Eira, M.T.S., Walters, C. and Caldas, L.S. (1999) Water sorption properties in Coffea spp. seeds and
embryos. Seed Science Research 9, 321–330.
Ellis, R.H. (1998) Longevity of seeds stored hermetically at low moisture contents. Seed Science
Research 8 (Suppl. 1), 9–10.
Farrant, J.M., Pammenter, N.W. and Berjak, P. (1989) Germination-associated events and the desicca-
tion sensitivity of recalcitrant seeds – a study on three unrelated species. Planta 178, 189–198.
Farrant, J.M., Pammenter, N.W., Berjak, P. and Walters, C. (1997) Subcellular organization and meta-
bolic activity during the development of seeds that attain different levels of desiccation toler-
ance. Seed Science Research 7, 135–144.
Farrant, J.M., Cooper, K., Kruger, L.A. and Sherwin, H.W. (1999) The effect of drying rate on the sur-
vival of three desiccation-tolerant angiosperm species. Annals of Botany 84, 371–379.
Finch-Savage, W.E., Pramanik, S.K. and Bewley, J.D. (1994) The expression of dehydrin proteins in
desiccation-sensitive (recalcitrant) seeds of temperate trees. Planta 193, 478–485.
Fu, J.R., Xia, Q.H. and Tang, L.F. (1993) Effects of desiccation on excised embryonic axes of three
recalcitrant seeds and studies on cryopreservation. Seed Science and Technology 21, 85–95.
Gaff, D.F. and Ellis, R.P. (1974) Southern African grasses with foliage that revives after dehydration.
Bothalia 11, 305–308.
Gaff, D.F. and Loveys, B.R. (1992) Abscisic acid levels in drying plants of a resurrection grass.
Transactions of Malaysian Society of Plant Physiology 3, 286–287.
Gaff, D.F., Bartels, D., Gaff, J.L. and Schneider, K. (1992) Gene expression at low RWC in two hardy
tropical grasses. Transactions of Malaysian Society of Plant Physiology 3, 238–240.
Gee, O.H., Probert, R.J. and Coomber, S.A. (1994) ‘Dehydrin-like’ proteins and desiccation tolerance
in seeds. Seed Science Research 4, 135–141.
Golovina, E.A., Hoekstra, F.A. and Hemminga, M.A. (1998) Drying increases intercellular partioning
of amphiphilic substances into the lipid phase: impact on membrane permeability and signifi-
cance for desiccation tolerance. Plant Physiology 118, 975–986.
Dessication - Chap 03 18/3/02 1:55 pm Page 108

108 N.W. Pammenter et al.

Govindasamy, R. (1997) Desiccation rate, desiccation response and damage accumulation: can desic-
cation sensitivity be quantified? Unpublished BSc (Hons) thesis, University of Natal, Durban,
South Africa, 63 pp.
Grange, R.I. and Finch-Savage, W.E. (1992) Embryo water status and development of the recalcitrant
species Quercus robur L.: determination of water relations parameters by pressure–volume
analysis. Journal of Experimental Botany 43, 657–662.
Grout, B.W.W., Shelton, K. and Pritchard, H.W. (1983) Orthodox behaviour of oil palm seed and cryo-
preservation of the excised embryo for genetic conservation. Annals of Botany 52, 381–384.
Hetherington, S.E. and Smillie, R.M. (1982) Humidity-sensitive degreening and regreening of leaves
of Borya nitida Labill. as followed by changes in chlorophyll fluorescence. Australian Journal of
Plant Physiology 9, 587–599.
Hobbs, P.R. and Obendorf, R.L. (1972) Interaction of initial seed moisture and imbibitional tempera-
ture on germination and productivity of soybean. Crop Science 12, 664–667.
Hoekstra, F.A., Golovina, E.A., van Aelst, A.C. and Hemminga, M.A. (1999) Imbibitional leakage from
anhydrobiotes revisited. Plant Cell and Environment 22, 1121–1131.
Hong, T.D. and Ellis, R.H. (1996) A Protocol to Determine Seed Storage Behaviour. IPGRI, Rome, 64
Iljin, W.S. (1957) Drought resistance in plants and physiological processes. Annual Review of Plant
Physiology 3, 341–363.
IPGRI (International Plant Genetic Resources Institute) (1994) Genebank Standards. FAO/IPGRI,
Rome, 13 pp.
IPGRI/DFSC (1996) The project on handling and storage of recalcitrant and intermediate tropical for-
est tree seeds. Newsletter 1, July 1996.
ISTA (International Seed Testing Association) (1999) Rule 9.5.8, Low constant temperature oven
method. Seed Science and Technology 27 (Suppl. International Rules for Seed Testing, Rules
1999), 49.
Kermode, A.R. and Bewley, J.D. (1985) The role of maturation drying in the transition from seed
development to germination. 1. Acquisition of desiccation-tolerance and germinability during
development of Ricinus communis L. seeds. Journal of Experimental Botany 36, 1906–1915.
Kovach, D.A. and Bradford, K.J. (1992) Imbibitional damage and desiccation tolerance of wild rice
(Zizania palustris) seeds. Journal of Experimental Botany 43, 747–757.
Leprince, O. and Hoekstra, F.A. (1998) The responses of cytochrome redox state and energy metabo-
lism to dehydration support a role for cytoplasmic viscosity in desiccation tolerance. Plant
Physiology 118, 1253–1264.
McKersie, B.D. and Stinson, R.H. (1980) Effect of dehydration on leakage and membrane structure in
Lotus corniculatus L. seeds. Plant Physiology 66, 316–320.
McKersie, B.D. and Tomes, D.T. (1980) Effects of dehydration treatments on germination, seedling
vigour, and cytoplasmic leakage in wild oats and birdsfoot trefoil. Canadian Journal of Botany
58, 471–476.
Normah, M.N., Chin, H.F. and Hor, Y.L. (1986) Desiccation and cryopreservation of embryonic axes
of Hevea brasiliensis Muell.-Arg. Pertanika 9, 299–303.
Norwood, M., Truesdale, M.R., Richter, A. and Scott, P. (1999) Metabolic changes in leaves and roots
during dehydration of the resurrection plant Craterostigma plantagineum (Hochst). South
African Journal of Botany 65, 421–427.
Ntuli, T.M., Berjak, P., Pammenter, N.W. and Smith, M.T. (1997) Effects of temperature on the desic-
cation responses of seeds of Zizania palustris. Seed Science Research 7, 145–160.
Oliver, M.J. and Bewley, J.D. (1984) Plant desiccation and protein synthesis. IV. RNA synthesis, sta-
bility, and recruitment of RNA into protein synthesis during desiccation and rehydration of the
desiccation-tolerant moss, Tortula ruralis. Plant Physiology 74, 21–25.
Oliver, M.J. and Bewley, J.D. (1997) Desiccation-tolerance of plant tissues: a mechanistic overview.
Horticultural Reviews 18, 171–213.
Oliver, M.J., Mischler, B.D. and Quisenberry, J.E. (1993) Comparative measures of desiccation-toler-
ance in the Tortula ruralis complex. I. Variation in damage control and repair. American Journal
of Botany 80, 127–136.
Oliver, M.J., Wood, A.J. and O’Mahony, P. (1998) ‘To dryness and beyond’ – preparation for the dried
state and rehydration in vegetative desiccation-tolerant plants. Plant Growth Regulation 24,
Dessication - Chap 03 18/3/02 1:55 pm Page 109

Experimental Aspects of Drying and Recovery 109

Ooshua, T. (1993) The cultivation of Porphyra ‘Nori’. In: Ohno, M. and Critchley, A.T. (eds) Seaweed
Cultivation and Marine Ranching. Japan International Co-operation Agency, Nagai, Japan, pp. 57–74.
Pammenter, N.W. and Berjak, P. (1999) A review of recalcitrant seed physiology in relation to desic-
cation-tolerance mechanisms. Seed Science Research 9, 13–37.
Pammenter, N.W., Vertucci, C.W. and Berjak, P. (1991) Homeohydrous (recalcitrant) seeds: dehydra-
tion, the state of water and viability characteristics in Landolphia kirkii. Plant Physiology 96,
Pammenter, N.W., Greggains, V., Kioko, J.I., Wesley-Smith, J., Berjak, P. and Finch-Savage, W.E. (1998)
Effects of differential drying rates on viability retention of Ekebergia capensis. Seed Science
Research 8, 463–471.
Pammenter, N.W., Berjak, P. and Walters, C. (1999) The effect of drying rate, and processes leading to
viability loss in recalcitrant seeds. In: Marzalina, M., Khoo, K.C., Jayanthi, N., Tsan, F.Y. and
Krishnapillay, B. (eds) IUFRO Seed Symposium 1998 Recalcitrant Seeds. Forestry Research
Institute Malaysia, Kuala Lumpur, Malaysia, pp. 14–24.
Pence, V.C. (1992) Desiccation and the survival of Aesculus, Castanea and Quercus embryo axes
through cryopreservation. Cryobiology 29, 391–399.
Platt, K.A., Oliver, M.J. and Thomson, W.W. (1997) Importance of fixative for reliable ultrastructural
preservation of poikilohydric plant tissues. Observations on dry, partially, and fully hydrated
tissues of Selaginella lepidophylla. Annals of Botany 80, 599–610.
Pollock, B.M. (1969) Imbibitional temperature sensitivity of Lima bean seeds controlled by initial
seed moisture. Plant Physiology 44, 907–911.
Pritchard, H.W. (1991) Water potential and embryonic axis viability in recalcitrant seeds of Quercus
rubra. Annals of Botany 67, 43–49.
Pritchard, H.W. and Manger, K.R. (1998) A calorimetric perspective on desiccation stress during
preservation procedures with recalcitrant seeds of Quercus robur L. Cryo-Letters 19 (Suppl. 1),
Pritchard, H.W. and Prendergast, F.G. (1986) Effects of desiccation and cryopreservation on the in
vitro viability of embryos of the recalcitrant seed species Araucaria huntsteinii K. Schum.
Journal of Experimental Botany 37, 1388–1397.
Pritchard, H.W., Tompsett, P.B., Manger, K. and Smidt, W.J. (1995) The effect of moisture content on
the low temperature responses of Araucaria huntsteinii seed and embryos. Annals of Botany 76,
Quartacci, M.F., Forli, M., Rascio, N., Dalla Vecchia, F., Bochicchio, A. and Navari-Izzo, F. (1997)
Desiccation-tolerant Sporobolus staphiainus: lipid composition and cellular ultrastructure dur-
ing dehydration and rehydration. Journal of Experimental Botany 48, 1269–1279.
Reynolds, T.L. and Bewley, J.D. (1993) Characterization of protein synthetic changes in a desiccation-
tolerant fern, Polypodium virginianum. Comparison of the effects of drying, rehydration and
abscisic 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 the longevity of neem (Azadirachta indica) seeds. Seed Science Research 8,
Salmen Espindola, L., Noin, M., Corbineau, F. and Côme, D. (1994) Cellular and metabolic damage
induced by desiccation in recalcitrant Araucaria angustifolia embryos. Seed Science Research 4,
Schonbeck, M.W. and Bewley, J.D. (1981a) Responses of the moss Tortula ruralis to desiccation treat-
ments. I. Effects of minimum water content and rates of dehydration and rehydration. Canadian
Journal of Botany 59, 2698–2706.
Schonbeck, M.W. and Bewley, J.D. (1981b) Responses of the moss Tortula ruralis to desiccation treat-
ments. II. Variations in desiccation tolerance. Canadian Journal of Botany 59, 2707–2712.
Schonbeck, M.W. and Norton, T.A. (1979) Drought-hardening in the upper-shore seaweeds Fucus spi-
ralis and Pelvetia canaliculata. Journal of Ecology 67, 687–696.
Seel, W.E., Hendry, G.A.F. and Lee, J.A. (1992) The combined effect of desiccation and irradiance on
mosses from xeric and hydric habitats. Journal of Experimental Botany 43, 1023–1030.
Sherwin, H.W. and Farrant, J.M. (1998) Protection mechanisms against excess light in the resurrec-
tion plants Craterostigma wilmsii and Xerophyta viscosa. Plant Growth Regulation 24, 203–210.
Sherwin, H.W., Pammenter, N.W., February, E., Vander Willigen, C. and Farrant, J.M. (1998) Xylem
Dessication - Chap 03 18/3/02 1:55 pm Page 110

110 N.W. Pammenter et al.

hydraulic characteristics, water relations and wood anatomy of the resurrection plant
Myrothamnus flabellifolius Welw. Annals of Botany 81, 567–575.
Tommasi, F., Paciolla, C. and Arrigoni, O. (1999) The ascorbate system in recalcitrant and orthodox
seeds. Physiologia Plantarum 105, 193–198.
Tompsett, P.B. and Pritchard, H.W. (1998) The effect of chilling and moisture status on the germina-
tion, desiccation tolerance and longevity of Aesculus hippocastanum L. seed. Annals of Botany
82, 249–261.
Tuba, Z., Csintalan, Z. and Proctor, M.C.F. (1996) Photosynthetic responses of a moss, Tortula ruralis,
ssp. ruralis, and the lichens Cladonia convoluta and C. furcata to water deficit and short periods
of dessication, and their ecophysiological significance: a baseline study at present-day CO2 con-
centration. New Phytologist 133, 353–361.
Tuba, Z., Proctor, M.C.F. and Csintalan, Z. (1998) Ecophysiological responses of homoiochlorophyl-
lous and poikilochlorophyllous desiccation tolerant plants: a comparison and an ecological per-
spective. Plant Growth Regulation 24, 211–217.
Tyree, M.T. and Hammel, H.T. (1972) The measurement of the turgor pressure and water relations of
plants by the pressure-bomb technique. Journal of Experimental Botany 23, 267–282.
Vander Willigen, C., Pammenter, N.W., Mundree, S.G. and Farrant, J.M. (2001) Some physiological
comparisons between the resurrection grass, Eragrostis nindensis, and the related desiccation-
sensitive species, E. curvula. Plant Growth Regulation 35, 121–129.
Vertucci, C.W. and Farrant, J.M. (1995) Acquisition and loss of desiccation tolerance. In: Kigel, J. and
Galili, G. (eds) Seed Development and Germination. Marcel Dekker, New York, pp. 237–271.
Vertucci, C.W., Crane, J., Porter, R.A. and Oelke, E.A. (1994) Physical properties of water in Zizania
embryos in relation to maturity status, water content and temperature. Seed Science Research 4,
Vertucci, C.W., Crane, J., Porter, R.A. and Oelke, E.A. (1995) Survival of Zizania embryos in relation
to water content, temperature and maturity status. Seed Science Research 5, 31–40.
Walters, C. (1998) Ultra-dry technology: perspective from the National Seed Laboratory, USA. Seed
Science Research 8 (Suppl. 1), 11–14.
Walters, C. and Engels, J. (1998) The effects of storing seeds under extremely dry conditions. Seed
Science Research 8 (Suppl. 1), 3–8.
Walters, C., Pammenter, N.W., Berjak, P. and Crane, J. (2001) Desiccation damage, accelerated ageing
and respiration in desiccation tolerant and sensitive seeds. Seed Science Research 11, 135–148.
Wu, X.-M., Wu, N.-F., Qian, X.-Z., Li, R.-G., Huang, F.-H. and Zhu, L. (1998) Phenotypic and geno-
typic changes in rapeseed after 18 years of storage and regeneration. Seed Science Research 8
(Suppl. 1), 55–64.
Dessication - Chap 04 18/3/02 1:55 pm Page 111

4 Biochemical and Biophysical Methods for

Quantifying Desiccation Phenomena in Seeds
and Vegetative Tissues

Olivier Leprince1 and Elena A. Golovina2,3

1UMR Physiologie Moléculaire des Semences, Institut National d’Horticulture, 16 Bd
Lavoisier, F49045, Angers, France; 2Laboratory of Plant Physiology, Department of
Plant Sciences, University of Wageningen, Arboretumlaan 4, 6703 BD Wageningen,
The Netherlands; 3Timiryazev Institute of Plant Physiology, Botanicheskaya 35,
Moscow 127276, Russia

4.1. Introduction 112

4.2. Caveats: the Consequences of Being Dry 112
4.3. How to Study Biochemical Responses to Drying 114
4.3.1. Responses of gas exchange and volatile emission to drying 114 Headspace analysis 114 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 116
4.3.4. Oxidative stress and anhydrobiosis 116
4.4. Spectroscopy Techniques 119
4.4.1. Electron paramagnetic resonance (EPR) 119 General description 119 Applications of EPR methods 120
4.4.2. Nuclear magnetic resonance 127 General description 127 The NMR study of water in living systems 128 NMR imaging 130 High-resolution multinuclear NMR spectroscopy 131 Structure and dynamics of cellular membranes 133
4.4.3. Fourier transform infrared (FTIR) spectroscopy 134 General description of infrared spectroscopy 134 Biological applications 134
4.5. Additional Techniques to Study Biochemical and Biophysical
Aspects of Desiccation Tolerance 136
4.5.1. Differential scanning calorimetry (DSC) 136
4.5.2. Electron microscopy 136
4.6. Acknowledgements 137
4.7. References 137
© CAB International 2002. Desiccation and Survival in Plants: Drying Without Dying
(eds M. Black and H.W. Pritchard) 111
Dessication - Chap 04 18/3/02 1:55 pm Page 112

112 O. Leprince and E.A. Golovina

4.1. Introduction resort to non-invasive techniques that

enable the investigator to assess any bio-
In recent years, the development of physi- chemical phenomenon in drying tissues
cal techniques has brought substantial without introducing water during the
insights into the physical state of water and analysis. The few techniques available are
cellular components of desiccated systems. briefly presented; and (ii) characterize
These techniques cover a large range of mutants and/or transgenic plants the phe-
spectroscopic techniques such as nuclear notypic traits of which can be associated
magnetic resonance (NMR), electron para- both with a particular biochemical path-
magnetic resonance (EPR, also referred to way and the level of desiccation tolerance
as electron spin resonance (ESR)), Fourier (Chapter 12; Wolkers et al., 1998a; Shiota
transform infrared (FTIR) spectroscopy as and Kamada, 2000; Weber et al., 2000;
well as dielectric measurements and Wehmeyer and Vierling, 2000). For exam-
calorimetry. The success of these tech- ple, the antisense inhibition of ADP-glu-
niques and spectroscopy in particular orig- cose pyrophosphorylase, a key enzyme in
inated in their versatility and in their starch synthesis, was found to increase the
ability to assess the physical state of desic- sucrose and nitrogen content of mature
cated systems by non-invasive means. seeds of transgenic Vicia narbonensis.
Below, the resourcefulness and limitations Also, the decrease in ADP-glucose
of the physical techniques are reviewed. pyrophosphorylase activity altered the cel-
In contrast to physical aspects, most of lular volume and water relations during
the fundamental questions pertaining to the seed-filling phase (Weber et al., 2000).
the biochemical aspects of desiccation These observations show that a specific
tolerance in anhydrobiotes remain to be alteration in carbon metabolism has
answered. The lack of non-destructive pleiotropic effects on seed development
and sensitive techniques has greatly and illustrate the potential of molecular
impeded our understanding of the role of biology to assess non-destructively the role
metabolism in desiccation tolerance. of various biochemical and biophysical phe-
Furthermore, all of our biochemical assays nomena related to desiccation tolerance.
and isolation of organelles have been set
up in dilute solutions using water or
organic compounds as solvents. However, 4.2. Caveats: the Consequences of Being
they have been indiscriminately applied to Dry
drying and desiccated specimens. Here it
is argued that the experimental approach Since water acts as a solvent and substrate
of grinding dry or nearly dried specimens in the cell in a variety of ways, its reduced
in aqueous buffers and measuring meta- availability in dried tissues will induce a
bolic activities and biological markers of set of physical and biochemical responses
oxidative stress in vitro in dilute solutions that may disappear during an invasive
is unlikely to reflect the in vivo situation. measurement, thereby confusing the inter-
This methodology has complicated the pretation of the data. Before ascertaining a
interpretation of data regarding biochemi- cause–effect relationship between desicca-
cal activities associated with different tion tolerance and a specific biochemical
hydration levels (Lynch and Clegg, 1986; and biophysical process, the following
Vertucci and Leopold, 1986). Hoekstra and remarks should be taken into account:
van Roekel (1983) have clearly illustrated
how isolation-inflicting injury of isolated 1. To adequately link the response of
mitochondria in germinating pollen can metabolism to drying with desiccation tol-
confuse the interpretation of results erance, it is necessary to map metabolic
obtained in vitro. To partially overcome activities as a function of water content or
this technical bottleneck, two strategies water potential of the drying cell and not
can be adopted: (i) whenever possible, as a function of time of drying (Vertucci
Dessication - Chap 04 18/3/02 1:55 pm Page 113

Methods for Quantifying Desiccation Phenomena 113

and Leopold, 1986; Leprince et al., 1999, oxidative reactions) occurring during nat-
2000). This necessity is more acute when ural ageing (i.e. below Tg) are compared
accumulation of dry matter occurs during with those occurring during accelerated
development. Often a sensitive microbal- ageing (between Tc and Tg (for example,
ance is needed to determine fresh and dry 75–85% relative humidity (RH) and tem-
weights of milligram quantities of samples. peratures between 35 and 50°C) or above
Furthermore, fast measuring techniques Tc (100% RH, 41°C)). This is illustrated in
should be preferred over time-consuming the study of Lievonen et al. (1998) on non-
assays so that the rates of water loss match enzymatic browning reaction rates around
the time necessary to acquire the data. the Tg of mixtures made of water, glycerol
Furthermore, Hendry (1993) argued that and maltodextrin. It is also clearly illus-
attempts to characterize desiccation phe- trated in the kinetics of seed and pollen
nomena in drying tissues should be set ageing (see, for example, Buitink et al.,
against a range of desiccation-induced 2000g and references therein).
damage and not only against percentages of 3. To be quantified and characterized,
survival after drying. metabolites, proteins, DNA, organelles,
2. It is important to recognize that during etc., must be extracted and purified
drying the cytoplasmic viscosity increases beforehand. This procedure strongly
dramatically until glass formation depends on the tissue water content. This
(Leprince and Hoekstra, 1998; Buitink et is valid for both water-soluble compounds
al., 2000e; Chapters 2 and 10). Unless using aqueous extraction and lipid-solu-
enzymatic activities are assessed in an ble compounds using organic solvents.
environment similar to that found in the For lipid extraction and separation using
drying cells, we do not know how the rise the Folch’s method, Hamilton et al. (1992)
in viscosity during drying will affect meta- recommended that the amount of water
bolic rates and/or pathways. We can present in the tissues should be calculated
already predict that O2-processing systems and taken into account during the differ-
will be altered by the loss of water since ent washing and partitioning procedures.
O2 solubility is known to decrease with For aqueous extraction, we do not know
rising viscosity (Gros et al., 1992; Leprince whether the extraction and purification of
and Hoekstra, 1998). Biochemical events water-soluble metabolites or proteins dif-
during drying should obey different laws fer quantitatively and qualitatively when
of diffusion since the cytoplasm will tissues are ground either fresh or dried.
undergo a physical transformation from a This point is particularly relevant for
liquid state to a solid-like state (i.e a glass). amphiphilic molecules such as phenolic
The moisture contents at which these compounds, which are likely to partition
changes in diffusion characteristics occur into the membranes and/or oil bodies dur-
during drying should preferably be deter- ing drying and vice versa during rehydra-
mined. It has been suggested that this tion (Chapter 10; Golovina et al., 1998;
moisture content corresponds to the glass Buitink et al., 2000e). Thus, the desicca-
formation that is measured by the tempera- tion-induced changes in metabolite con-
ture at which the drying cytoplasm forms a centrations and in protein conformation
glass (Tg). Below Tg, solid state should be interpreted with caution if these
physics/chemistry prevails (Chapter 10). changes are assessed from crude extracts.
However, Buitink et al. (2000f) suggested 4. Most of the methods used so far are
that the most important change in the averaging techniques. However, it is
physical properties of the cytoplasm likely that there is a gradient of water
occurs 50°C above Tg, at a temperature cor- within seed tissues during dehydration
responding to the so-called collapse tem- and rehydration. Therefore, a qualitative
perature (Tc). A distinction between liquid and quantitative gradient of responses
state and solid state should be made when within the tissues submitted to drying
chemical and biochemical events (such as might be expected.
Dessication - Chap 04 18/3/02 1:55 pm Page 114

114 O. Leprince and E.A. Golovina

4.3. How to Study Biochemical ter (Rogerson and Matthews, 1977; Vertucci
Responses to Drying and Leopold, 1986, 1987). In these tech-
niques, the gas to be analysed has to accu-
Two technically different strategies are mulate over time in a closed environment
available to probe the responses of metabo- before taking the measurement (i.e. static
lism to drying by non-invasive means: the headspace analysis). To reach a detectable
detection and analysis of gases that concentration, the gas accumulation may
emanate from, or are absorbed by, the dry- take some time, particularly in drying sam-
ing tissues (so-called headspace analysis) ples in which the metabolism and gas diffu-
and the resort to in vitro or in vivo NMR. In sion are greatly reduced by the lack of
photosynthetic anhydrobiotes, fluorescence water. Consequently, the assay may be too
spectroscopy is also a convenient tool to slow in comparison with the rate of water
characterize energy metabolism during dry- loss. Furthermore, an additional problem is
ing (see Chapter 7). The methods that reliably maintaining the specimen at the
assess free-radical-induced damage in des- same water content during the measure-
iccation tolerance are also reviewed. ment. Thus, unless the kinetics of water
loss matches the time frame needed to
assess the metabolic rates, the relation
4.3.1. Responses of gas exchange and between hydration levels and metabolic
volatile emission to drying activities in drying tissues may not be accu-
rate. Therefore, it is best to adapt a flow- Headspace analysis through system coupled to an active
trapping system (i.e. dynamic headspace
A wide variety of gaseous metabolites can analysis). A flow of dry or humidified air
be studied in drying tissues: O2 and CO2 passes over the sample acting as both a gas
exchange as markers of respiration or photo- carrier and dehydrating agent. The volatiles
synthesis, ethanol and acetaldehyde as are then absorbed by a compound located
markers of fermentation, alkanes, alkones, in the exit flow and analysed by GC (Wilson
alkenes, aldehydes (volatile) as markers of and McDonald, 1986; Zhang et al., 1994).
oxidative stress. Several instruments such For dried tissues that are in a glassy state,
as infrared CO2 analysers, gas-phase O2 elec- the release of volatiles may take several
trodes or O2 analysers can detect and days or weeks because of the slow diffusion
analyse CO2 and O2 exchanges. These types of molecules. To overcome this problem,
of analysers have mainly been used to several authors have used a thermal desorp-
describe the effects of desiccation on photo- tion technique consisting of heating the dry
synthetic activities of resurrection plants specimens to at least 60°C. This procedure
(Schwab et al., 1989) and photosymbiotic is thought to purge the volatiles that are
lichens (Nash et al., 1990; Scheidegger et trapped in the glassy matrix (Wilson and
al., 1995). However, they are not always McDonald, 1986; Hailstones and Smith,
suited to assaying low respiration rates from 1989; Zhang et al., 1994; Degoussée et al.,
nearly dried material because of their low 1995). However, it is not always possible to
sensitivity (O. Leprince, unpublished data). determine whether the production of
Gas exchange rates and volatile produc- volatiles after desorption results from the
tion can also be measured by gas chro- heat treatment per se or not (Wilson and
matography (GC) (Kimmerer and McDonald, 1986).
Kozlowski, 1982; Gorecki et al., 1984; Klein
and Sachs, 1992; Leprince and Hoekstra, Laser photoacoustic spectroscopy
1998; Leprince et al., 1999), gas chromatog-
raphy–mass spectrometry (GC-MS) (Zhang
et al., 1994), high-pressure liquid chro- PA is an emerging technique that has over-
matography (HPLC) (Degoussée et al., 1995) come the problems associated with head-
or by using a Gilson differential respirome- space analysis. A PA set-up consists mainly
Dessication - Chap 04 18/3/02 1:55 pm Page 115

Methods for Quantifying Desiccation Phenomena 115

of two components: a line-tunable CO laser appears to be the most appropriate tech-

that excites gas molecules specifically nique for this purpose (Shachar-Hill and
according to their infrared fingerprint Pfeffer, 1996; Roberts, 2000). Several strate-
absorption, and parallel resonators. Each gies can be adopted using 13C-, 31P-, 14N- or
resonator is coupled to a sensitive micro- 15N-NMR, depending on the nature of the

phone in which the concentration of gases metabolite to be analysed. NMR studies

is sequentially measured based on an may or may not be destructive. The princi-
acoustic phenomenon (Harren and Reuss, ples of NMR spectroscopy and the advan-
1997; see Zuckerman et al., 1997, tages and disadvantages of in vivo applications as a non-invasive technique
for technical details of the experimental will be described below in Section 4.4 (see
set-up). This technique permits the probing also Shachar-Hill and Pfeffer, 1996).
of metabolic processes non-invasively that A first strategy is to monitor dynamic
result in the emission and/or absorption of changes of natural or enriched nuclei in the
ethylene, acetaldehyde, ethanol and CO2 samples over different intervals during dry-
from drying tissues without altering the ing. This approach may or may not be
water content (Leprince et al., 2000). destructive. In both cases, it yields dynamic
Furthermore, technical improvements are information about metabolic fluxes and fast
being made to allow the analysis of lipid responses of metabolism to physiological
peroxidation products such as ethene, perturbations. The natural abundance of 13C
ethane, pentane and hexane. PA techniques is only 1.1%. Thus 13C-NMR is not a sensi-
offer two important advantages over con- tive method and requires concentrations in
ventional headspace analysis: (i) biologi- the mM range. However, one can take
cally relevant gases can be detected with a advantage of this low sensitivity to trace
sensitivity limit of 100- to 1000-fold higher some metabolic changes by the detection of
than GC; and (ii) the time response is less compounds that accumulate to high levels.
than 1 min. The PA is set up as a flow- These metabolites include compatible
through system. The gas employed to dry solutes that accumulate in cyanobacteria
the tissues is also the carrier of the (Reed et al., 1985), sugars and oil in seeds
gas/volatile to be analysed. The disadvan- (Rutar, 1989; Ishida et al., 1990, 1996;
tages are: (i) to our knowledge, the access to Koizumi et al., 1995), and trehalose in fun-
PA is restricted to a handful of laboratories gal spores (Bécard et al., 1991). The non-
in The Netherlands ( invasive character of NMR may allow the
tracegasfac/), Germany, Italy and the US; (ii) time-course of metabolic events to be
the equipment is not commercially avail- directly followed and the subcellular local-
able and the current prototypes require the ization of some metabolites to be deter-
assistance of physicists and engineers to mined, for instance, in maturing or
take and process the measurements; (iii) a germinating seeds (Colnago and Seidl,
limited range of biologically relevant gases 1983; Ishida et al., 1990, 1996).
can be measured; and (iv) water vapour Alternatively, since the natural abun-
strongly interferes with the measurement dance of 13C is low, it is possible to label
and must be totally removed from the gas. specific metabolites and monitor their fate
through the cellular network of metabolic
pathways in vivo or in vitro with crude
4.3.2. NMR applications to measure steady- extracts (Dieuaide-Noubhani et al., 1995;
state concentrations and to assess metabolic Roberts, 2000; Roscher et al., 2000).
responses to drying (see also Section 4.4.2) Commercially n-13C-labelled sugars are
available for almost every carbon position
To determine the effects of desiccation on of sucrose, glucose and fructose molecules.
the dynamics of metabolic pathways, the Similarly 31P- and 14N-labelled compounds
flux of metabolites through the different can be used to monitor the dynamics of
paths must be known. NMR spectroscopy phosphorylated metabolites and amino
Dessication - Chap 04 18/3/02 1:55 pm Page 116

116 O. Leprince and E.A. Golovina

acids, respectively. Whether in vivo NMR 4.3.4. Oxidative stress and anhydrobiosis
is applicable to drying tissues remains to
be ascertained. A particular problem is to Whether oxidative stress is a cause or an
maintain reliably the specimen in the same effect of desiccation sensitivity has yet to
functional state and with the same water be resolved due to a large body of conflict-
content during the NMR measurements. ing evidence in the literature. The core of
A second strategy, which is not mutu- the problem is two fold. From a physiologi-
ally exclusive to that above, is to screen cal point of view, Hendry (1993) argued
chemical fingerprints of crude extracts that attempts to correlate free radical
obtained from different hydration levels. processes with desiccation tolerance must
This application will yield analytical infor- be done in relation to the characterization
mation about metabolites in a steady state. of other desiccation-induced damage. This
By comparing spectra of crude extracts must be done in order to assess whether
obtained during drying, it should be possi- free radicals generated during drying are a
ble to pinpoint the metabolites, the concen- cause or an effect of the loss of viability.
trations of which are mostly sensitive to Echoing the earlier review of Gutteridge
changes in water content during drying and Halliwell (1990) and Leprince et al.
(Fan, 1996; Noteborn et al., 2000). Roughly (1990), he pointed out that free-radical-
0.5–1 g of fresh material is often required mediated injury can occur before or after
to take an NMR spectrum, which could be the time of death during drying (Hendry,
a limiting factor if the amount of biological 1993). Thus, great caution should be exer-
material is restricted. cised in ascertaining whether oxidative
stress plays a role in desiccation-induced
injury and loss of desiccation tolerance.
4.3.3. Photosynthesis studies Oxidative injury in both animals and plants
generally results from stress-induced meta-
The recent methods to assess photosyn- bolic disturbances, particularly in the elec-
thetic activities have become non-destruc- tron transport chains. Considering the
tive. They exploit the interactions between technical difficulties in estimating meta-
light, gas exchange, operation of the photo- bolic activities during drying (see Section
synthetic electron transport and ambient 4.3, p. 114), even greater care should then
conditions. For these reasons, they are be taken in attempts to link oxidative dam-
widely used to study the photosynthetic age to desiccation-induced metabolic per-
responses to environmental stresses turbation. Various pathologies and
(Bukhov et al., 1989; Foyer et al., 1994). degenerative diseases have been linked to
They have also been applied to compare the mitochondrial dysfunction and generation
photosynthetic responses of anhydrobiotes of reactive O2 species (ROS) in mammals
with those of desiccation-sensitive plants (Yates and van Houten, 1997; Esposito et
(Schwab and Heber, 1984; Schwab et al., al., 1999; Wallace, 1999). Thus, it should
1989; see Chapter 7). The most applied be interesting to see whether parallels exist
technique is chlorophyll a fluorescence, between animal and plant anhydrobiotes.
which assesses the efficiency of electron The second problem regarding the puta-
transport through photosystem II and non- tive role of oxidative stress in desiccation
photochemical quenching processes associ-
tolerance and ageing is the methodology
ated with it. Light-induced electron
that has been employed so far. A survey of
transport in photosystem II can be studied
methods employed to detect oxidative stress
using fluorescence induction kinetics
in seeds can be found in Benson (1990).
(Vertucci and Leopold, 1986). Light-scatter-
ing measurements at 535 nm are also useful 1. The array of techniques that have been
to gain insights into the physical and chem- employed to assess the role of free-radical-
ical events associated with the formation of induced injury in anhydrobiotes is very
a membrane potential in thylakoids. small. A survey of the literature on molecu-
Dessication - Chap 04 18/3/02 1:55 pm Page 117

Methods for Quantifying Desiccation Phenomena 117

lar markers used to estimate free-radical- function and cell death (Yates and van
induced damage during drying or in the Houten, 1997). Oxidation of proteins by
dry state shows that the results are based ROS can induce protein fragmentation or
mostly on a thiobarbituric acid-reactive enzyme inactivation, leading to the disrup-
substances (TBARS) assay and a non-inva- tion of glycolysis (Hyslop et al., 1988) and
sive EPR spectroscopy technique (Table the Calvin cycle (Kaiser, 1979). Protein oxi-
4.1). The former measures malonyldialde- dation has been linked to various patholo-
hyde (MDA) as a breakdown product of gies in humans (Dean et al., 1993; Berlet
lipid peroxidation using a simple and and Stadtman, 1997) and to seed ageing
rapid assay (Heath and Packer, 1968) and (Zhang et al., 1997).
the latter estimates a carbon free radical of 3. Analytical procedures to estimate lipid
unknown origin (Hendry, 1993). As dis- hydroperoxides in crude extracts are
cussed below, doubts have been cast as to fraught with potential artefacts (Gutteridge
whether these two assays are reliable and Halliwell, 1990; Hageman et al., 1992;
enough for all anhydrobiotic material. Meagher and Fitzgerald, 2000). The prob-
2. Table 4.1 also shows that, in 98% of lems are numerous, ranging from the pres-
these studies, lipid peroxidation was mea- ence of contaminants (metal ions) that
sured as a marker of free-radical-induced initiate lipid peroxidation during tissue
injury. It must be recognized that proteins grinding to instability of peroxidized lipids
(Dean et al., 1993; Berlet and Stadtman, during the extraction and lack of sensitiv-
1997) and DNA (von Sonntag and ity. Furthermore, the nature of peroxidative
Schuchmann, 1987; Hageman et al., 1992; damage depends on the type of free-radical
Wiseman and Halliwell, 1996) are also sen- initiator and the membrane or lipid compo-
sitive to free radicals, albeit less than fatty sition (McKersie et al., 1990). Thus, the
acids. Interestingly, mitochondrial DNA is lipid peroxidation assays currently used in
more sensitive to ROS than nuclear DNA. seed science cannot be indiscriminately
Free-radical-induced DNA damage can sig- applied to all seeds. Unfortunately, the
nificantly contribute to mitochondrial dys- studies 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 vegetative
tissues. Examples of free-radical damage specific to DNA are oxidized
nucleotides such as thymine glycol, 8-hydroxy-2-deoxyguanine and
methylguanine (Hageman et al., 1992). Examples of free-radical damage to
protein are iminopeptides, carbonyl content and glutamyl-semialdehyde
residues of oxidatively modified proteins (Berlet and Stadtman, 1997).
Number of studies
over the past three
Methods decades
MDA or TBARS determinationa 17
Determination of an organic free radical by
in vivo EPR spectroscopy 11
Free fatty acid determination 6
Chemical modifications of lipids (e.g. conjugated
dienes, fatty acid composition) 5
Other techniques (determination of breakdown
products resulting from lipid peroxidation,
chemiluminescence, fluorescent probes) 9
Free-radical damage specific to DNA 0
Free-radical damage specific to protein 2
aMDA, malonyldialdehyde; TBARS, thiobarbituric acid-reactive substances.
Dessication - Chap 04 18/3/02 1:55 pm Page 118

118 O. Leprince and E.A. Golovina

assess whether the marker used as an index known to accumulate in desiccation-toler-

of oxidative injury was sensitive or appro- ant systems. Anthocyanins accumulate to
priate for the biological material or experi- large concentrations in leaves of resurrec-
mental conditions. For example, in oily tion plants. Hodges et al. (1999) have intro-
seeds, lipid peroxidation is dependent on duced several modifications to overcome
the triacylglycerol composition and struc- this interference in leaf extracts. However,
ture of oil reserves (Neff et al., 1992). the suggested improvements did not
Furthermore, in an assay of peroxidized appear to be reliable for the results shown
lipids of a crude extract obtained from oily in Table 4.2.
seeds, the triacylglycerol fraction may 5. An organic stable free radical has been
mask more important and significant linked to respiration, oxidative stress and
changes occurring in membrane lipids. desiccation tolerance (Hendry et al., 1992;
Another example further illustrates the Hendry, 1993; Leprince et al., 1995).
point. Table 4.2 compares two methods However, measurements of this organic
estimating the level of peroxidized lipids radical using non-invasive EPR have gener-
in germinating pea axes in relation to the ated conflicting evidence concerning its
loss of desiccation tolerance. Using the link to desiccation tolerance (Hendry,
assay developed by Jiang et al. (1991), 1993). Its chemical nature and localization
results suggest that there is a link between should be identified to ascertain whether
an increase in oxidative damage following this is a reliable method to estimate oxida-
drying and the loss of desiccation toler- tive stress in seeds. The fact that the EPR
ance. In contrast, the TBARS assay pro- signal is sensitive to liquid water makes it
vides inconclusive evidence. difficult to use in drying tissues. Studying
frozen specimens could lessen the problem
4. The limitations of the TBARS test have
but this approach will not be able to tell
been known for several years (Gutteridge
whether the radical is generated by drying
and Halliwell, 1990; Hodges et al., 1999)
or by freezing.
and include a lack of sensitivity and speci-
ficity and a tendency to overestimate MDA From these remarks, we conclude that
contents. Recently, it has been shown that positive results from a lipid peroxidation
several compounds commonly found in assay will provide evidence that a free-radi-
plant extracts (e.g. sugars, oligosaccharides, cal reaction has occurred either during
anthocyanins) also react with thiobarbi- drying or during drying and extraction. A
turic acid, thereby interfering strongly with negative result will not provide any evi-
the peroxidized products (Table 4.3). Non- dence one way or the other. Therefore, it is
reducing sugars and oligosaccharides are suggested that a range of assays should be

Table 4.2. Comparison of two methods measuring lipid peroxidation as a marker of oxidative damage in
crude extracts of germinating axes of pea before and after fast drying. TBARS were measured as in
Leprince et al. (1990) and calculated as in Du and Bramlage (1992) to take into account interference by
sugars. Lipid hydroperoxide levels were measured using the xylenol orange/ammonium sulphate reagent
according to the method of Jiang et al. (1991) and quantified using H2O2 as a standard. Data are the
average (± SE) of 3–5 replicates (O. Leprince, J. Fajerman and F.A. Hoekstra, unpublished observations).
TBARSa Lipid peroxide
Sensitivity to drying Treatment (mol mg1 dw) (mol equiv. H2O2 mg1 dw)
Tolerant Fresh 2.76 ± 1.93 15 ± 3
Dried 9.89 ± 1.79 16 ± 2
Intolerant Fresh 33.05 ± 15.01 22 ± 2
Dried 27.12 ± 9.00 71 ± 15
aTBARS, thiobarbituric acid-reactive substances.
dw, dry weight.
Dessication - Chap 04 18/3/02 1:55 pm Page 119

Methods for Quantifying Desiccation Phenomena 119

tested as a necessary prerequisite to 4.4. Spectroscopy Techniques

address the role of free-radical processes
in desiccation tolerance and seed As follows from a consideration of quan-
longevity. To be validated, these assays tum mechanics, an atom or molecule has
should be carried out on material that has discrete energy states. Spectroscopy is the
been treated with agents to generate oxida- measurement of the energy differences
tive stress such as ultraviolet (UV) radia- between these states. The energy differ-
tion and free-radical generators such as ences E can be measured by the absorp-
paraquat or H2O2. tion spectra of electromagnetic radiation.
An increasing number of methodologies In conventional spectroscopy, the fre-
are currently being developed to analyse quency is varied and the frequency at
free-radical-induced damage in vitro and in which maximal absorption occurs reflects
vivo. Most of these methods are derived the difference between the states. The fre-
from studies on animals, humans or food- quencies vary from the MHz range for NMR
stuffs. The challenge will be to adapt these to the GHz (microwave) range for EPR
techniques to seed science and anhydrobi- spectroscopy. The frequencies for absorp-
ology, keeping in mind the caveats tion spectroscopy range from 1012 Hz for IR
described in this chapter and the guide- to 1016 Hz for UV light. The frequencies of
X-rays and -irradiation are 1019 Hz and
lines provided by studies aimed at unravel-
1021 Hz, respectively.
ling the free-radical chemistry occurring in
living tissues. Among emerging methodolo-
gies, PA (see above), the use of specific flu-
4.4.1. Electron paramagnetic resonance (EPR)
orescent probes (LeBel et al., 1992) and
(see also Chapter 2)
fluorescence spectroscopy, spin-trapping
techniques and non-invasive EPR spec- General description
troscopy (see Section 4.4) and new spec-
troscopy assays (Jiang et al., 1991; LeBel et The energy differences that are studied
al., 1999; Junqua et al., 2000) warrant fur- with EPR spectroscopy are the result of the
ther investigation. interaction of unpaired electrons with a

Table 4.3. Interference of various compounds in the thiobarbituric acid-reactive

substances (TBARS) assay in crude extracts of plant and seed tissues. The TBARS
assay was performed using the procedure of Heath and Packer (1968) in the
presence of various amounts of sugars and anthocyanins. TBARS concentrations
were calculated from the difference between absorbance values at 532 and 600 nm
and expressed as a relative increase compared to extracts without interfering
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.
Dessication - Chap 04 18/3/02 1:55 pm Page 120

120 O. Leprince and E.A. Golovina

static magnetic field (Zeeman splitting). In Sample preparation is relatively easy. The
EPR spectroscopy the electromagnetic fre- sample is incubated in an aqueous solution
quency is kept constant and the magnetic of spin-probe or spin-labelled molecules
field is scanned (due to limitations of for a short time and then transferred into
microwave electronics). There are four fre- the resonator of an EPR spectrometer for
quencies available in EPR spectrometers: spectra recording. Usually, neutral spin-
1.1 GHz (L-band), 3.0 GHz (S-band), 9.5 probe molecules can readily pass through
GHz (X-band) and 35 GHz (Q-band). membranes and partition within cells over
Among them, the X-band is the most com- the polar and apolar phases according to
monly used. The interaction between their partition coefficients. The EPR signal
nuclei and the electron (hyperfine interac- of the spin-probe molecules outside cells
tions) causes the hyperfine splitting of the can be eliminated by broadening agents via
EPR spectrum. The spectral shape can give spin–spin interaction. Paramagnetic metal
information about the sample under study. ion complexes such as chromium oxalate
Only systems that contain non-paired or ferricyanide are often used as broaden-
electrons will give an EPR signal. Pairing ing agents. Thus, the EPR spectrum of spin
gives zero net electron magnetic moment. probes in the sample in the presence of
Since a paired spin system is energetically broadening agents is exclusively derived
favourable, chemical bonding normally from the inside of cells. Different aspects of
results in molecules that have no unpaired desiccation tolerance can be studied by the
electrons and, hence, no EPR signal occurs. analysis of EPR spectra of spin probes
Exceptions to this rule are transition-metal introduced into the cells.
ions, free radicals and free electron centres Spin labels are stable free radicals. The
such as those produced by high-energy unpaired electron belongs to the nitroxide
irradiation of macromolecules. Free radi- group, which is flanked by quaternary car-
cals produced in biological systems usually bon atoms of methyl groups, protecting
cannot be detected by EPR because of their the radical from recombination and
short half-life times, resulting in low accounting for the high stability of the
steady-state concentrations. label. The EPR spectra of nitroxide spin
The introduction of spin-label/probe labels have a three-line nitrogen hyperfine
methods has considerably increased the structure and are environmentally sensi-
possibilities for the application of EPR in tive. The variety of spin probes of differ-
biological systems. The spin-label group ent properties and the possibility of the
that is almost exclusively used is the attachment of nitroxides to biological
nitroxide moiety synthesized by Rozantzev molecules of interest have created exten-
(1970) and introduced by McConnell sive applications of this method in biol-
(McConnell and McFarland, 1970). ogy (Marsh, 1981; Morse, 1985).
There is a limitation to the hydrated sam- A problem that often arises in experi-
ple size that can be used for spectra record- ments with biological samples is chemical
ing because of dielectric losses caused by reduction of spin labels and spin probes.
water. However, the high sensitivity of the Despite the high stability in aqueous and
method allows the use of very small sample other media, nitroxides are susceptible to
sizes of less than 1 mg. The relative simplic- (reversible) reduction by some biological
ity of sample preparation and spectra metabolites (such as ascorbic acid and thi-
recording enables the operator to handle ols), electron transport chains and other
large numbers of samples per day. redox systems, resulting in the disappear-
ance of paramagnetism. Ferricyanide is Applications of EPR methods usually effective at limiting the rate of
reduction or reoxidizing reduced label
GENERAL REMARKS. The great variety of spin (Kaplan et al., 1973). Oxygenation, aeration
probes allows a multiplicity of information or the use of specific inhibitors can also be
about biological systems to be obtained. used to protect nitroxides against reduction
Dessication - Chap 04 18/3/02 1:55 pm Page 121

Methods for Quantifying Desiccation Phenomena 121

or to restore them from the reduced 4 daa wheat kernel

hydroxylamines (Marsh, 1981).
Another problem with the application of
the spin-label technique is the disturbance Dead
that might be caused by the guest molecules (a) tissue
themselves (e.g. membrane spin labels).
This is inherent in all methods using
reporter groups, in contrast to spectroscopic
methods that do not use guest molecules
(e.g. NMR, FTIR). Because of the high sensi-
tivity of the spin-label method, very low
concentrations of label can be used, which Difference (viable cells)
minimizes possible disturbance. However,
the possibility of obtaining information
about a specific environment of interest Oil
makes the EPR method attractive in the (b)
study of desiccation tolerance. In contrast,
NMR and FTIR give information that is
averaged along the sample, which can be
complementary to the information from
reporter group methods. Cytoplasm


The principle of the EPR spin-probe Fig. 4.1. (a) The electron paramagnetic resonance
method for the estimation of the relative (EPR) spectrum of 4-oxo-2,2,6,6-tetramethyl-1-
amount of viable cells is based on the fact piperidinyloxy (TEMPONE) in developing wheat
kernel that was harvested at 4 days after anthesis (4
that membranes of viable cells are imper-
daa) and dried on the ears. The thick line represents
meable to some broadening agents,
the broad component of the spectrum and
whereas the membranes of damaged cells originates from TEMPONE in dead tissue; the thin
are not (Keith and Snipes, 1974). Thus, the line represents the total spectrum. (b) Spectrum
EPR signal of spin-probe molecules inside showing the difference between the total spectrum
cells with disrupted membranes is and broad component, representing TEMPONE
quenched by a broadening agent, and the located in viable cells. Peaks originating from
total amplitude of the EPR signal from the TEMPONE in the aqueous cytoplasm of viable cells
sample will correlate with the amount of and from oil bodies are indicated. Total scan width
viable cells in a sample (Dobrucki et al., is 100 gauss. Spectra are reproduced from Golovina
et al. (2001).
1990). Because of the high sensitivity of the
method, it is possible to determine small
amounts of viable cells in mostly dead tis-
sue. This approach has been applied in the Many of the anhydrobiotic systems con-
study of desiccation tolerance acquisition tain oil bodies as storage material. In this
of proembryonic cells in wheat kernels, case, amphiphilic spin-probe molecules
which were slowly dried on the ear at an will partition into lipid bodies as well, and
early stage when proembryos could not be EPR spectra are composed of two compo-
detected morphologically (Fig. 4.1) nents originating from spin-probe mole-
(Golovina et al., 2001). Such an approach cules in aqueous (cytoplasm) and
allows the developmental death of wheat hydrophobic (oil) environments. These
endosperm cells during kernel develop- spectra differ in the distance between lines
ment (Golovina et al., 2000) and the (the isotropic splitting constant aiso is
progress of cell death after cold (Fig. 4.2) or around 16 G for an aqueous environment
imbibitional stress in neem seeds to be fol- and around 14 G for a lipid environment)
lowed (Sacandé et al., 2001). and in the position of the central line
Dessication - Chap 04 18/3/02 1:55 pm Page 122

122 O. Leprince and E.A. Golovina

plasmic (hcyt) and oil peaks (hoil) can be

Axis of
neem used for the quantitative assessment of the
proportion of viable cells in a sample
(Golovina and Tikhonov, 1994; Golovina et
al., 1997a,b; Leprince et al., 1999).
(a) Control
plasma membrane permeability can be esti-
mated by using the water-soluble nitroxide
radicals in the presence of a broadening
Cytoplasm agent (Miller and Barran, 1977; Golovina et
al., 1998; Hoekstra et al., 1999). The
method is based on the presence of tempo-
(b) 7 days rary defects in membranes that allow ferri-
cyanide ions to penetrate into the cell and
broaden the signal arising from spin probes
located in the cytoplasm. The line-height
(c) 28 days ratio of the lipid peak to the water peak
(L/W) will correlate with the number of fer-
ricyanide ions that have penetrated the cell
through the plasma membrane and can be
Fig. 4.2. Electron paramagnetic resonance (EPR) used to characterize plasma membrane
study of chilling damage of neem (Azadirachta permeability (Fig. 4.3).
indica) seeds using 4-oxo-2,2,6,6-tetramethyl-1-
piperidinyloxy (TEMPONE) as a spin probe. CELL VOLUME AND OSMOTIC EFFECT. The height
Spectrum (a), mature, fresh axes used as control; ratio of cytoplasmic to lipid peaks in EPR
spectra (b) and (c), whole embryos after,
spectra can be used to determine cell vol-
respectively, 7 and 28 days of storage under humid
ume changes under osmotic stress. This
conditions at 5°C. Spectra are plotted in the same
scale to allow comparison. The oil and cytoplasmic follows from the fact that spin-probe mol-
peaks are indicated in the high-field region (right ecules are equally distributed inside and
side) of spectrum (a). Total scan width is 100 gauss. outside the cells. The total cellular vol-
Spectra are reproduced from Sacandé et al. (2001). ume that is not accessible to broadening
agents will determine the amount of spin
probe that is separated from the broaden-
(g-factor), and can be resolved in the high ing agent and, hence, the line-height of
field (right-side) part of the spectrum the cytoplasmic component in the EPR
(Marsh, 1981). In the case of loss of mem- spectrum. This total volume is the prod-
brane integrity, ferricyanide ions that have uct of the number and volume of viable
penetrated the cell only broaden the signal cells. Cell division and enlargement of
of TEMPONE (4-oxo-2,2,6,6-tetramethyl-1- cells during imbibition and germination
piperidinyloxy) molecules localized in the (Golovina et al., 2001) and osmotically
aqueous cytoplasm. The signal of TEM- induced changes in cell volume (Miller,
PONE from oil bodies remains unbroad- 1978) can cause changes in the line-
ened, because ferricyanide cannot partition height of the cytoplasmic component.
into the lipid phase. The intensity of this The ratio between the line-heights of
hydrophobic signal can then be used as a cytoplasmic and lipid peaks can be used
measure of the total amount of cells in the to quantify the osmotic effect. However,
sample, whereas the intensity of the polar when the amount of oil changes during
cytoplasmic component represents the seed development (Golovina et al., 2001)
amount of cells with intact membranes or during germination (Sacandé et al.,
(Golovina et al., 1997a,b). The ratio R 2001), the height of the cytoplasmic line
between the heights of the aqueous cyto- can be used instead.
Dessication - Chap 04 18/3/02 1:55 pm Page 123

Methods for Quantifying Desiccation Phenomena 123

Typha latifolia 1998). Line-height differences arising from

differentially broadened lines due to
(a) 0s
slowed motion of spin-probe molecules
(Fig. 4.4, spectra a and b) are used to esti-
(b) 7s
mate viscosity (Keith and Snipes, 1974).
The line-height ratio between lines can be
converted to viscosity. Based on such an
approach, the changes in cytoplasmic vis-
cosity with drying of desiccation-tolerant
(c) 30 s and sensitive samples (Leprince et al.,
1999) and with the acquisition of desicca-
tion tolerance during seed development
(Golovina et al., 2001) have been estab-
When spin-probe motion slows further,
L not only is a progressive increase in differ-
(d) 60 s ential broadening observed, but also a dis-
tortion of the line shape (Fig. 4.4, spectrum
c). Rigidly immobilized, randomly oriented
radicals give a powder spectrum (Marsh,
1981), which can be used to characterize
biological glasses (Buitink et al., 1998, 1999,
2000b,c,d,f). In this case, the viscosity must
Fig. 4.3. Electron paramagnetic resonance (EPR)
be estimated using saturation transfer EPR
study of imbibitional leakage of Typha latifolia
pollen using 4-oxo-2,2,6,6-tetramethyl-1-
or pulsed EPR methods (see below).
piperidinyloxy (TEMPONE) as a spin probe. Pollen
was either directly incubated in a solution of PARTITIONING OF AMPHIPHILES INTO THE LIPID PHASE
TEMPONE/ferricyanide (spectrum a) or after a WITH DRYING. The shape of spin-probe spec-
previous rehydration in liquid germination medium tra depends on properties of the environ-
for 7 s (spectrum b), 30 s (spectrum c) and 60 s ment; therefore amphiphilic spin probes
(spectrum d). All the spectra exhibit contributions can be used to follow their partitioning
from the aqueous cytoplasm (W) and from lipid (L) with drying (Golovina et al., 1998; Buitink
(oil bodies) environments, which are resolved in the et al., 2000e; Hoekstra and Golovina, 2000;
high-field region (right side). The spectra were
Golovina and Hoekstra, 2002). The samples
normalized to the height of the lipid (L) peak. The
ratio W/L was taken as a measure of plasma
are preloaded with spin probes and allowed
membrane permeability (see explanation in the text, to dry. The EPR spectra recorded from the
Section 4.4.1). Total scan width is 80 gauss. Spectra samples at different moisture content will
are based on data from Hoekstra et al. (1999). be composed of spectra originating from
spin-probe molecules at different locations
(Fig. 4.5). They can be decomposed, and the
Because the shape of relative proportion of spin probes at the dif-
EPR spectra of spin probes is sensitive to ferent locations can be estimated (Hoekstra
molecular motion, cytoplasmic viscosity and Golovina, 2000; Golovina and
can be studied with spin-probe techniques Hoekstra, 2001).
(Keith and Snipes, 1974). To characterize
the shape of the spectrum originating from PHYSICAL PROPERTIES OF MEMBRANES. The small,
the cytoplasmic location of the spin probe, water-soluble spin probe TEMPO (2,2,6,6-
other components (lipid, starch-like) of the tetramethyl-1-piperidinyloxy) or spin-
spectrum have to be subtracted (Golovina labelled fatty acids, steroids or
et al., 2000, 2001). Alternatively, charged phospholipids are used to study the physi-
spin probes that do not partition into the cal properties of membranes (for refer-
lipid phase can be applied (Buitink et al., ences, see Berliner, 1976; Marsh, 1981;
Dessication - Chap 04 18/3/02 1:55 pm Page 124

124 O. Leprince and E.A. Golovina

(a) Cytoplasm
Root (a)

h0 h–1

(b) Oil bodies

h0/ h–1= 1.19

Axis (b)

(c) surface
glass h0/ h–1= 1.75

(c) (d) Combined

a:b:c = 5:10:85

Fig. 4.4. Electron paramagnetic resonance (EPR) Fig. 4.5. Typical electron paramagnetic resonance
spectra of 4-oxo-2,2,6,6-tetramethyl-1- (EPR) spectra of 4-oxo-2,2,6,6-tetramethyl-1-
piperidinyloxy (TEMPONE) in the cytoplasm of piperidinyloxy (TEMPONE) in aqueous cytoplasm
wheat seedling root (spectrum a) and hydrated (spectrum a), oil bodies (spectrum b), and of spin-
wheat axis (spectrum b). The ratio of the height of probe molecules immobilized at the membrane
the central line (h0) to the height of the high-field surface (spectrum c). Spectrum (d) is the sum of
line (h1) reflects cytoplasmic viscosity. The spectra (a), (b) and (c). Spectra were combined in
cytoplasmic viscosity is less in seedling root such a proportion that the relative amounts of spin
(h0/h1= 1.19) than that in hydrated wheat axis probe in spectra (a), (b) and (c) were 5%, 10% and
(h0/h1= 1.75). The EPR spectrum of TEMPONE in 85%, respectively. Spectrum (d) simulates the late
air-dried sucrose glass (spectrum c) is typical for stage of TEMPONE partitioning during drying. Total
rigidly immobilized, randomly oriented spin-probe scan width is 100 gauss. Spectra are based on data
molecules. The distance between the outer extremes from Golovina and Hoekstra (2001).
2Amax (in gauss) can be used to characterize the
slow motion of spin-probe molecules. Total scan
width is 100 gauss. Spectra are based on data from
Golovina and Hoekstra (2001). provides valuable information about mem-
brane dynamics, because the stable free-
radical doxyl group can be placed at
Morse, 1985). Isolated membranes can be different positions along the acyl chain.
labelled with TEMPO, which partitions Thus information can be obtained from dif-
between water and the membranes. The ferent depths in membranes, from the sur-
partitioning depends on membrane fluidity face to the core. Labelling model membranes
and aqueous-phase viscosity and can be poses no problem. The spin label is mixed at
used for the determination of the mem- 1 mol % with the lipids in organic solvent,
brane phase transition. However, the possi- which is subsequently removed by evapora-
ble partitioning of TEMPO into oil bodies tion. The anhydrous mixture is then dis-
complicates the in vivo membrane investi- persed in the appropriate amounts of
gations. The use of spin-labelled fatty acids aqueous phase. Labelling isolated biological
Dessication - Chap 04 18/3/02 1:55 pm Page 125

Methods for Quantifying Desiccation Phenomena 125

membranes poses more problems. Spin- teins and lipids in biological membranes
labelled fatty acids have first to be dis- (for references, see Marsh, 1981; Hemminga,
solved in ethanol and then added to the 1983) and glasses (Roozen et al., 1991). The
membranes in such a quantity that the final method has been successfully applied in
concentration of ethanol does not exceed 1 the study of biological glasses in anhydrobi-
mol %. Other ways of membrane labelling otic systems (Buitink et al., 1998, 1999;
have been reviewed by Marsh (1981). 2000b,c,d,f). This approach of measuring
Labelling membranes with doxyl stearates slow rotational motion has given stunning
in vivo following the same approach can be insight into the differences between biologi-
used but is more tricky than with isolated cal glasses and sugar or polymer glasses.
membranes. A small number of in vivo stud- For example, a remarkable observation orig-
ies have been published. Among them only inating from the ST-EPR measurements was
a few investigations have been conducted the occurrence of a second kinetic change
on desiccation-tolerant systems (Golovina in mobility at a definite temperature above
and Tikhonov, 1994; Vishnyakova et al., the glass transition temperature (Buitink et
2000; Golovina and Hoekstra, 2001). al., 2000f), which may have physiological
relevance for survival in the dry state (Fig.
10.3 in Chapter 10).
TOLERANT SYSTEMS. When conventional EPR Pulsed EPR. CW-EPR often cannot give
spectroscopy as described above yields the true values for the relaxation times of the
rigid-limit powder line shape (see Fig. 4.4, spin label, because of the inhomogeneous
spectrum c), it is insensitive to the rate of broadening of the lines. However, pulsed
molecular motion. The following two EPR EPR (electron spin echo technique) pro-
methods have been designed to overcome vides a direct method for the measurement
this problem and adapted to anhydrobiotic of relaxation times that give insight into
material. They are particularly suitable to the molecular dynamics of spin probes
characterize a glassy state. (Morse, 1985). This method has been used
to identify the glassy state in wheat seeds
Saturation-transfer EPR. Saturation-transfer
(Dzuba et al., 1993) and to characterize the
EPR (ST-EPR) allows the measurement of
motion of guest molecules in biological
very slow molecular motion with rotational
glasses of different moisture content
correlation times between 107 and 103
(Buitink et al., 2000a).
s. For comparison, conventional continuous
wave (CW) EPR enables rotational correla- EPR imaging (EPRI). The potential of using
tion times below 107 s to be resolved. In both NMR and EPR imaging was suggested
conventional EPR, motion averaging of the by Lauterbur (1973). However, while NMR
spectral anisotropy occurs within the time imaging (NMRI) has progressed into clinical
of spin–spin relaxation T2. In ST-EPR, this usage, the application of EPRI is restricted,
averaging occurs within the time of particularly in biological systems. This is
spin–lattice relaxation T1, which is 300 caused by the severe dielectric losses and
times longer than T2 for slow-moving consequent heating that occurs in aqueous
nitroxide molecules (Marsh, 1981). Thus, samples at conventional EPR frequencies (X-
ST-EPR extends the motional sensitivity of band). The dimension of the hydrated sam-
the spin-label technique to one that moni- ple that can be used for imaging is limited to
tors a 300-fold slower motion than with a few millimeters. This problem can be
conventional EPR. ST-EPR spectra of partly overcome by using low-frequency EPR
nitroxide spin probes can be analysed by (L-band) and a surface coil (Berliner and
independent line shape parameters. Using a Fujii, 1985). EPRI is used for visualizing
reference material of known viscosity, the paramagnetic centres in a sample. There are
molecular rotation can be calculated in an several biological applications in which
empirical way (Hemminga, 1983). ST-EPR EPRI has a significant advantage over NMRI:
has been used to study the motion of pro- the spatial distribution of O2 and redox
Dessication - Chap 04 18/3/02 1:55 pm Page 126

126 O. Leprince and E.A. Golovina

metabolism, mapping viable and non-viable this effect is proportional to the O2 concen-
cells, the diffusion of paramagnetically tration (Swartz, 1987).
labelled solutes, and mapping native free radi-
pH measurements. Spin-labelled amine
cals that are stable or trapped by incorporated
and carboxylic acids have been used to
spin traps at the sites of transient radical pro-
determine the pH in vesicles and cells
duction (Berliner and Fujii, 1986; Bacic et al.,
(Mehlhorn et al., 1982). The method is
1989; Dobrucki et al., 1990). In spite of the
based on the differential membrane perme-
potential advantage of EPRI, there are only a
ability for charged and neutral forms of
few cases in which the method has been
these spin probes. Because the equilibrium
applied to desiccation-tolerant systems. The
between charged and neutral amines and
pathways of bulk water penetration into wheat
acids depends on pH, the intracellular
kernels during imbibition have been studied
EPR signals of these spin probes can be
using the nitroxide radical TEMPOL (4-
used to calculate the intracellular pH.
Unfortunately, such an approach can only
(Smirnov et al., 1988; Golovina et al., 1991).
be applied in the presence of a solution of
To avoid the problem of dielectric losses, the
a broadening agent. To study the changes
kernels were placed in liquid nitrogen.
in pH in a sample during drying, specific
Using perdeuterated 15N nitroxides as imag-
pH-sensitive spin probes can be applied.
ing substances, tens of micrometre resolu-
The reversible effect of pH on EPR spectra
tion can be achieved. Such resolution was
is associated with proton exchange in the
sufficient to obtain EPRI of viable cells in
radicals. Protonated and non-protonated
hydrated lettuce seeds in the presence of fer-
forms have different EPR parameters. The
ricyanide (Walczak et al., 1987). The image
protonable group in the radical structure
enabled contrast between embryo and stor-
has to be close to the unpaired electron.
age tissue to be observed. The EPRI of the
Iminonitroxides are the most promising in
penetration and distribution of natural spin
this respect (Khramtsov and Weiner, 1988).
probes (humic substances) in wheat kernels
has also been demonstrated (Smirnov et al., Spin trapping. The free radicals that are
1991). produced in anhydrobiotic organisms dur-
ing water loss (Section 4.3.4) cannot be
detected by EPR because of their short half-
life resulting in low steady-state concentra-
BIOSIS.Additional EPR methods have been
tion, or the short relaxation times leading to
designed to study several biochemical and
very broad lines. These radicals can be
biophysical aspects of biology. However,
trapped specifically by spin traps and
these methods have not yet been applied
detected by EPR in organic extracts or in
specifically to seeds or other types of anhy-
vivo (Knecht and Mason, 1993). Four differ-
drobiotic tissues.
ent traps are commonly used in biological
Intracellular O2 concentration. Intracellular systems: 2-nitrosopropane (MNP), phenyl-
O2 affects the shape of EPR spectra of spin N-tert-butylnitrone (PBN), -(4-pyridyl-1-
probes because, as a paramagnetic mole- oxide)-N-tert-butylnitrone (POBN), and
cule, it causes line broadening. Broadening 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).
is proportional to O2 concentration, thereby The primary free radicals interact with the
allowing the intracellular concentration to double bond of diamagnetic spin-trap mole-
be calculated (Swartz, 1987). However, the cules and form radical adducts that are
application of this method may not be much more stable than the primary free
straightforward for drying biological mater- radicals. The radical adducts of these spin
ial because the rise of viscosity also intro- traps are nitroxide radicals. The primary
duces changes in line broadening. Another free radical can be identified either from the
approach is to use the effects of O2 on the spectra of radical adducts, or after purifica-
microwave power saturation: the presence tion of radical adducts and further identifi-
of O2 diminishes power saturation, and cation by mass spectroscopy. Various
Dessication - Chap 04 18/3/02 1:55 pm Page 127

Methods for Quantifying Desiccation Phenomena 127

shortcomings complicate the application of NMR signals can be characterized by

spin trapping in vivo in drying organisms: intensity, frequency, line shape and relax-
oxidation of the spin traps and reduction of ation times. All these characteristics are
the radical adducts, and amphiphilic affected by the physical and chemical envi-
behaviour which may relocate spin traps ronment of the magnetic nucleus and can
into the membranes (Knecht and Mason, be used to obtain information of biological
1993). interest such as the state of water, intracel-
lular pH and membrane dynamics. Signal
intensity is related to the number of mole-
4.4.2. Nuclear magnetic resonance cules that produce the signal. In relaxation
(see also Chapter 2) experiments, the intensity depends on the
time of signal registration and on the rate General description of magnetization decay. For quantitative
estimation of peaks in NMR spectra, inte-
Analogous to EPR spectroscopy, NMR spec- gration of the lines should be used because
troscopy is based on the resonance absorp- of the different relaxation times of the sig-
tion of electromagnetic radiation by the nals. The limits of integration are deter-
system during the transition between two mined by the signal-to-noise ratio of the
discrete energy states. The energy differ- signal and the overlapping with other sig-
ences studied in NMR spectroscopy are due nals in a spectrum.
to the interaction of nuclear magnetic Local fields originating from the local
moments with the magnetic field (Zeeman electron density modify the external field
splitting for nuclei). The energy differences imposed on magnetic nuclei. As a result, the
are smaller than those in EPR because of resonance frequency of a nucleus depends
the smaller magnetic moment of nuclei. on its chemical environment, which is
This explains why electromagnetic radia- called chemical shift. Magnetization relaxes
tion in the radiofrequency range is required exponentially, and the faster the decay, the
to excite the transitions that produce the broader the line in the spectrum. Broad
NMR signal, whereas that in the microwave lines have lower amplitudes and overlap
range is used in EPR spectroscopy. with other lines, which leads to poorly
Because the energy differences in NMR resolved spectra. In living systems, the vari-
are small, the differences in number of ations of magnetic susceptibility across the
nuclei at different energy levels are also sample cause line broadening, which makes
small. As a consequence, the signal it difficult to record high-resolution spectra
strength is weak, which makes NMR an from dense heterogeneous tissue, such as
inherently insensitive technique. Only seeds, and from tissues containing air-
those nuclei that have a non-zero spin spaces (leaves and roots).
quantum number resulting in non-zero The T1 (spin–lattice or longitudinal) and
magnetic moment can be used. The split- T2 (spin–spin or transverse) relaxation
ting between energy levels depends on the times characterize the magnetization decay
strength of the magnetic field and the mag- because of the interaction of the nuclear
netogyric ratio of the nucleus. The highest magnetic moments with the environment
magnetogyric ratio and the almost 100% (T1) and with each other (T2). Relaxation
natural abundance make proton (1H) NMR times are mostly determined by the
the most sensitive. The reasonably high motional properties of the nucleus.
magnetogyric ratio and 100% natural abun- Measurements of relaxation are particu-
dance of 31P nuclei give moderately good larly important in NMR studies of tissue
receptivity for in vivo phosphorus NMR. In water when information about the exis-
contrast, the 13C nucleus has very low tence of different water fractions in the tis-
receptivity because of its low natural abun- sue is required. In practice, the
dance, but, as a label, this isotope could be measurements are easier to conduct than to
useful (Schneider, 1997; Roberts, 2000). interpret (Ratcliffe, 1994).
Dessication - Chap 04 18/3/02 1:55 pm Page 128

128 O. Leprince and E.A. Golovina

In basic NMR experiments, the sample is cal systems because of the generally high
placed in a magnetic field, and the NMR sig- water content, the high natural abundance
nal is generated by irradiation of the sample and the high magnetogyric ratio of 1H. This
with a radio-frequency field, given as pulses allows the use of low-field NMR instead of
of different sequences. A single pulse cre- expensive high-field NMR magnets.
ates a net magnetization, which is regis-
tered. The magnetization decays to zero, MEASUREMENTS OF WATER CONTENT. 1H low-field
and the time-dependence of the decay (free NMR allows the non-destructive measure-
induction decay) is recorded. In low-field ment of the water content in biological sys-
studies, this decay is analysed directly. In tems with high precision. There are two
high-field NMR, the decay is converted into types of analytical NMR commonly used in
spectra by Fourier transformation. The NMR this respect – continuous wave (CW) NMR
spectrum is the plot of intensity against fre- (wide-line) (Pohle and Gregory, 1968) and
quency of the radio-frequency field. All pulsed NMR (Martin et al., 1980), the latter
NMR applications developed for studying now being generally adopted. In CW-NMR
living systems can be divided into four the amount of liquid water is estimated from
groups: (i) detection of water signal; (ii) the area under the absorption peak. The sig-
NMR imaging; (iii) high-resolution multinu- nal from water strongly ‘bound’ to biopoly-
clear NMR spectroscopy; and (iv) solid-state mers is not visible because of broadening.
NMR spectroscopy (Ratcliffe, 1994). The signals from liquid water and oil are not
To exploit the advantage of a non-inva- resolved, but the contribution of oil to the
sive technique, NMR experiments need to signal can be estimated by drying.
minimize the physiological perturbation Pulsed NMR can be used to analyse the
and maintain the tissue in a physiologi- different water fractions. In pulsed NMR
cally controllable state. In this respect, the all protons are excited by a short intense
whole plant, cell suspensions and intact radio frequency (RF) pulse resulting in a
seeds are the easiest tissues, and excised free induction signal, which decays when
tissues the most demanding (Ratcliffe, the pulse is switched off. The initial
1994). Often, it is necessary to submerge a amplitude of free induction decay (FID) is
sample in water to avoid differences in proportional to the total number of pro-
magnetic susceptibility between air and tons in a sample. The signals due to nuclei
cellular material, a practice that is incom- in different physical states decay at differ-
patible with drying organisms. Proper O2 ent rates: signals due to protons in solid
supply and illumination have to be main- state decay faster (microseconds) than
tained, especially in densely packed sam- those in liquid phase (from milliseconds
ples. In solid-state NMR, when magic angle to seconds). This signal decay can be
spinning is applied, it is impossible to con- analysed to reveal the contribution of dif-
trol the physiological state because of the ferent proton fractions. To avoid the influ-
extreme conditions (more than 1000 rota- ence of inhomogeneity of magnetic field
tions per minute) imposed on the sample. and water diffusion on the rate of decay,
special sequences of pulses such as spin-
echo (SE) or Carr–Purcell–Meiboom–Gill The NMR study of water in living (CPMG) are used (Farrar and Becker,
systems 1971). In air-dry samples, the signal decay
from water associated with polymers
GENERAL REMARKS. The study of water in (mainly starch) can be distinguished easily
anhydrobiotes is of particular interest from that of oil protons on the basis of the
because with drying and rehydration both considerable differences in spin–spin
water content and water properties change. relaxation time T2. Such an approach is
NMR is a powerful tool to study water in widely used for rapid and non-destructive
vivo. There is no problem with the sensitiv- determination of moisture and oil content
ity of detecting the water signal in biologi- in air-dry seeds (e.g. Tiwari et al., 1974;
Dessication - Chap 04 18/3/02 1:55 pm Page 129

Methods for Quantifying Desiccation Phenomena 129

Gambhir and Agarwala, 1985; Brusewitz Compartmentation is the reason why

and Stone, 1987; Gambhir, 1992; Rubel, more than one fraction of water is generally
1994; Warmsley, 1998). In hydrated seeds, observed in hydrated living systems. A the-
drying or D2O exchange can be used to ory of transverse relaxation in compart-
separate the NMR signal of free water from mented systems has been developed, based
that of oil (Ratkovic et al., 1982a). on the chemical exchange and diffusion
Because the different water fractions properties of the water (Belton and
have the same chemical shift, only pulsed Ratcliffe, 1985). Two to three water frac-
NMR can be used to characterize them in tions have been shown in hydrated tissue
living tissues. The changes in water frac- originating from different plant cell com-
tions with different relaxation characteris- partments (Bacic and Ratkovic, 1984;
tics can be followed during the Belton and Ratcliffe, 1985; Snaar and van
dehydration or rehydration of anhydrobi- As, 1992). However, it appears that there is
otic systems. This gives insight into the no simple relationship between the multi-
role of the different water fractions in bio- exponential character of T2 and the com-
logical systems (Seewaldt et al., 1981; partmentation of the water (Ratcliffe, 1994).
Ratkovic et al., 1982a; Aksyonov and The heterogeneity in cellular size and com-
Golovina, 1986a,b; Ishida et al., 1987, position, subcellular compartmentation,
1988b; Bacic et al., 1992; Golovina and and plasmalemma and tonoplast permeabil-
Aksyonov, 1993; Marconi et al., 1993). ity could have influenced the multi-expo-
However, data on different water fractions nential decay curves (Snaar and van As,
must be interpreted with extreme caution 1992). The detection of the simultaneous
(Ratcliffe, 1994). Different water fractions presence of water of different relaxation
with specific relaxation times can be dis- behaviour in anhydrobiotes with reduced
criminated only if there is no fast exchange MC may have been caused by the inhomo-
of protons between the fractions in the geneous water distribution within the
NMR time window. In the case of fast organisms. Thus, the water with long T2 (or
exchange between protons, only one relax- slow-relaxing water) observed in wheat ker-
ation time is observed. The number of pro- nels during the first hours of imbibition is
tons of different mobility and their thought to be localized around the embryo
relaxation times will determine the and in the vascular bundle, whereas the
observed effective relaxation time. When fast-relaxing water is thought to be associ-
associated with macromolecules, water ated with starchy endosperm (Golovina and
protons have shorter relaxation times, Aksyonov, 1993).
which will influence the overall relaxation
time. This is the reason why T1 (spin–lattice WATER SELF-DIFFUSION COEFFICIENT. The behav-
relaxation time) and T2 (spin–spin relax- iour of water in living systems can also be
ation time) values are lower in cellular characterized by the water self-diffusion
water than in bulk water and decrease fur- coefficient. The diffusion coefficient is
ther with water loss. Thus, T2 values can measured by the pulsed (spin-echo) NMR
also be used to measure moisture content technique in the presence of a (pulsed)
(MC) (Ratkovic, 1987). Below 0.2 g H2O g1 field gradient (Fukushima and Roeder,
dry weight, the relationship between relax- 1981). In addition to nuclear magnetic
ation times (T1 and T2) and moisture con- relaxation, the spin-echo amplitude
tent is reversed (Clegg et al., 1982; Ratkovic decreases in the presence of a field gradient
et al., 1982b; Wolk et al., 1989). Because if water changes its position during the
the increase in T1 and T2 at low water con- measurement. Diffusion coefficients as a
tents has also been observed in measure of water mobility can be calcu-
starch/water systems besides anhydrobiotic lated from the signal decay in the presence
organisms, the increase might be attributed of a field gradient. As in the case of relax-
to water molecules jumping from one sorp- ation times, self-diffusion coefficients of
tion site to another. cellular water are lower than those of bulk
Dessication - Chap 04 18/3/02 1:55 pm Page 130

130 O. Leprince and E.A. Golovina

water and decrease with drying (Clegg et NMR imaging

al., 1982). This can be caused by the pres-
ence of diffusion barriers (membranes or GENERAL REMARKS. NMRI is mainly based on
cell walls) or macromolecules. These the detection of the water signal. 1H reso-
macromolecules can cause either obstruc- nance frequency is independent of the
tion of the diffusion or water binding (Seitz location of the water in a tissue, so that tis-
et al., 1981; Back et al., 1991). As a result, sue water signal is averaged across the
the diffusion coefficient in hydrated anhy- whole sample. The spatial distribution of
drobiotes has been shown to be 2–5 times the water signal can be obtained if a mag-
smaller than that of bulk water (Clegg et netic field gradient is applied, which arises
al., 1982; Fleischer and Werner, 1992). In from the dependence of the resonance fre-
Artemia cysts the diffusion coefficient has quency of NMR signals on the magnetic
been measured from 0.02 to 1.49 g water field strength. In spite of the simplicity of
g1 dry weight, the minimum value being the principle of NMRI, its practical appli-
almost 50 times lower in the dry cysts than cation is rather complicated. Information
in the hydrated cysts (Seitz et al., 1981). on the spatial distribution of water or water
properties (relaxation times or diffusion
MEMBRANE PERMEABILITY. Paramagnetic ions coefficients) can be obtained. Dynamic
(Mn2+) cause a decrease in relaxation times information can be obtained from time-
due to their interaction with nuclei. Conlon dependent properties of the image. There
and Outhred (1972) proposed a method of are two different experimental approaches
measuring membrane permeability to water, in NMRI: imaging large objects (roots,
based on the change in relaxation time of stems or whole plants) with low spatial
intracellular water that is in diffusional resolution, and imaging small samples
exchange with an extracellular MnCl2 solu- (seeds, excised tissues) with high spatial
tion. From the estimated water-exchange resolution (NMR microscopy) (Ratcliffe,
time and the cell dimension, the diffusion 1994; Ishida et al., 2000).
permeability coefficient Pd can be calcu- Spatial resolution is mainly determined
lated (Stout et al., 1977, 1978; Bacic and by the signal/noise ratio, but other factors
Ratkovic, 1984). Unfortunately, this such as short relaxation times and the pres-
approach cannot be applied to the systems ence of air space cause intensity loss and a
that are subjected to drying, because the tis- decrease in spatial resolution. The develop-
sue has to be in Mn2+ solution. ment of NMRI has led to a resolution that
The pulsed-gradient spin-echo method approaches the dimension of single cells in
proposed by Stejskal and Tanner (1965) plant tissues (Connelly et al., 1987). The
can be used to study the in situ membrane theoretical limit is considered as 10  10 
permeability for water during drying. The 10 µm (Ratcliffe, 1994). While NMR is not
method allows the water diffusion to be yet able to compete with optical microscopy
measured over the time between two in its resolution of cellular structures, it has
pulses of field gradient. The presence of the great advantage of being non-invasive
partly permeable barriers causes the and, thus, can be used to monitor function-
decrease in the apparent diffusion coeffi- ing plant tissue. The ability to resolve struc-
cient for water, so that the permeability of tures depends not only on resolution but
membranes for water and the size of water also on the image contrast, which is deter-
compartments can be calculated (Tanner, mined by the differences in signal intensity
1978; von Meerwall and Ferguson, 1981). between different regions of the sample.
This approach has been applied to follow Knowledge of relaxation properties of the
the changes in membrane properties in tissue water is central to the understanding
developing barley seeds (Ishida et al., of image contrast. Nitroxide radicals
1995) and to calculate the size of oil bodies (Magin et al., 1986; Swartz et al., 1986) and
in rape seeds (Fleischer et al., 1990; paramagnetic ions (Ishida et al., 2000) can
Fleischer and Werner, 1992). be used as contrasting agents.
Dessication - Chap 04 18/3/02 1:55 pm Page 131

Methods for Quantifying Desiccation Phenomena 131

WATER DISTRIBUTION IN SEEDS DURING MATURATION signal of the externally supplied water
AND GERMINATION. It is possible to map sta- (Connelly et al., 1987). The changes in relax-
tionary, diffusing and flowing water in ation times of tissue water during seed matu-
plant tissue (Ratcliffe, 1994). NMRI enables ration or germination cause changes in the
the water distribution inside seeds to be image contrast. Relaxation times of water
determined. The brightness of the image is depend on the interaction of water with
proportional to the proton density. macromolecules. The synthesis of storage
Experiments with seeds of different species substances during maturation and their
have shown that the signal/noise ratio in hydrolysis during germination result in an
the image is sometimes limited by the short apparent decrease or increase in brightness
relaxation time for tissue water (T2 < 10 of the NMR image (Ishida et al., 1990, 1995;
ms) (Connelly et al., 1987). The sensitivity McIntyre et al., 1995), so that solubilized
problem can be overcome to some extent parts of the storage tissue can become visi-
by signal averaging, since the time scale for ble. The changes in image contrast during
detectable structural changes in germinat- precocious germination of Phaseolus vul-
ing seeds is long in comparison with the garis seeds after ethylene treatment have
time required to obtain an image. In NMR been attributed to changes in the water sta-
images of seeds, a clear distinction tus and water redistribution from the cotyle-
between axis and storage tissue can be don to the axis (Fountain et al., 1998).
obtained (Connelly et al., 1987; Kano et al.,
1990; Hou et al., 1996; Fountain et al., THE DISTRIBUTION OF OIL AND SUCROSE IN SEEDS.
1998; Carrier et al., 1999). The changes in The spatial image of other compounds,
water distribution during drying and rehy- mainly lipids and carbohydrates that accu-
dration have shown the transfer routes for mulate in storage tissue, can be mapped in
water (Ruan and Litchfield, 1992; Ruan et vivo using the chemical-shift imaging (CSI)
al., 1992; Song et al., 1992; Kovacs and technique (Bottomley et al., 1984). The 1H
Nemenyi, 1999). NMR spectra of water, oil and sugars have
The water content may be more uni- different chemical shifts, but the peaks are
formly distributed in seeds than proton not resolved unless the water peak is sup-
NMRI indicates. This discrepancy arises pressed. The CSI technique applied to 1
from the inhomogeneity of the susceptibil- day germinating mung bean seeds has
ity of the sample associated with the pres- shown uniformly distributed oil, which
ence of cell walls and storage substances allowed the changes in the image with ger-
(Back et al., 1991). Eccles et al. (1988) mination to be attributed to the bulk water
applied pulsed gradient spin-echo and fraction (Connelly et al., 1987). Oil and
steady gradient NMRI to maturing wheat sucrose have been mapped in fresh maize
kernels and found the spatial distribution kernels (Koizumi et al., 1995), germinating
of the self-diffusion coefficient of water. barley seeds (Ishida et al., 1990) and in
The diffusion was slowest in endosperm developing pea seeds (Tse et al., 1996).
and highest in the vascular bundle. Back et
al. (1991) used the dependence between
the self-diffusion coefficient of water and High-resolution multinuclear NMR
the relative water content obtained by spectroscopy
Callaghan et al. (1979) to correct the proton
map for wheat grain and showed the more GENERAL REMARKS. High-resolution multi-
uniform distribution of water in the cor- nuclear NMR is used to detect ions and
rected image. metabolites of low molecular weight, the
For experiments in which germination of intracellular pH, the subcellular compart-
seeds has to be followed over many hours in mentation of compounds and the flux
the magnet, it is necessary to maintain a con- through metabolic pathways (Ratcliffe,
tinuous water supply to the seeds, while at 1994; Schneider, 1997; Roberts, 2000). Low
the same time minimizing the spectroscopic concentration of the molecules of interest
Dessication - Chap 04 18/3/02 1:55 pm Page 132

132 O. Leprince and E.A. Golovina

and low receptivity of nuclei other than 1H than that of the 1H nucleus, which reduces
make this approach rather insensitive. The overlapping in the spectra. Secondly, the
sensitivity increases with increasing field low natural abundance of 13C opens possi-
strength. High-resolution NMR spectrome- bilities for labelling the tissue and monitor-
ters are usually equipped with high-field ing metabolic pathways. The biological use
superconducting magnets in the range of NMR to study metabolism is described in
4.7–14.1 T, corresponding to a 1H fre- Section 4.3.2. 13C NMR has also been used
quency of 200–600 MHz. The sensitivity to establish changes in soybean seeds dur-
can be increased by multiple scanning and ing maturation and germination. The mois-
usually permits the detection of millimolar ture content-dependent disappearance or
concentrations of metabolites (Ratcliffe, appearance of narrow peaks associated with
1994). To increase the sensitivity further, sugars in in vivo NMR spectra is indicative
the tissue volume within the detector has of the presence of free water in these seeds
to be maximized. Cell suspensions and (Ishida et al., 1987, 1988a). The sensitivity
excised tissues are more suitable for such of natural abundance 13C NMR can be
experiments than whole plants or seeds. enhanced, by applying low-speed magic-
angle spinning (Ni and Eads, 1992) or by
1H NMR. Different nuclei can be used for dif- the detection of 13C by protons coupled to
ferent purposes. The high sensitivity makes the 13C nucleus (Heidenreich et al., 1998).
1H attractive for metabolite detection. 13C labelling gives opportunities for probing

Nevertheless, the need to suppress the water different metabolic pathways, such as lipid
signal and the complexity of spectra limit synthesis in soybean ovules (Schaeffer et
the possibilities for in vivo 1H NMR. The al., 1975) and the metabolism of dormancy-
small differences in chemical shift and con- breaking chemicals in red rice (Footitt et
siderable overlapping of broad signals in tis- al., 1995).
sues make 1H spectra poorly resolved. For
example, in germinating seeds only peaks 31P NMR. In vivo 31P NMR has many applica-

from sugars and oil under conditions of par- tions because of the convenient magnetic
tial water signal suppression can be properties of the 31P nucleus and the physi-
resolved (Koizumi et al., 1995; Ishida et al., ological importance of the information that
1996). 1H NMR spectra of oil in dry seeds can be deduced from the spectra. The mea-
can be obtained because the signals from surement of cytoplasmic and vacuolar pH is
other nuclei are broadened due to immobi- one of the most important applications of in
lization. However, the resolution of lines is vivo 31P NMR, which is based on the depen-
not good because of differences in magnetic dence on pH of the chemical shift of Pi.
susceptibility. The magic-angle sample This, together with the slow exchange of Pi
spinning (MASS) technique eliminates line across the tonoplast, allows the origin of the
broadening arising from differences in mag- Pi signal – either cytoplasmic or vacuolar –
netic susceptibility due to fast mechanical to be determined and, consequently, the
rotation about an axis, making a magic angle cytoplasmic and vacuolar pH. A number of
(54°55), and resulting in 1H spectra from important phosphorylated metabolites can
dry seeds with a good resolution (Rutar, be resolved in 31P spectra. For some of them
1989). 1H NMR is widely used to analyse (Pi, polyphosphates), information on the
tissue extracts for the presence of specific subcellular distribution can also be obtained
compounds such as, for example, betaine in because of the pH-dependent chemical shift.
wild-type and transformed Arabidopsis 31P NMR has been applied to study the pH

thaliana seeds (Alia et al., 1998). of intracellular compartments in germinat-

ing seeds of Phacelia tanacetifolia (Espen et
13C NMR. 13C NMR is more attractive for al., 1995). Changes in chemical shifts of the
application in vivo for two reasons. First, pH-dependent 31P signal from cytoplasmic
the chemical shift scale of the 13C nucleus and vacuolar inorganic phosphate correlate
is more than an order of magnitude greater with seed germination. 31P can also be used
Dessication - Chap 04 4/4/02 2:19 pm Page 133

Methods for Quantifying Desiccation Phenomena 133

to monitor phosphorus compounds and choline (DPPC). It was shown that the head
their changes during maturation and germi- groups are in a rigid state above and below
nation of seeds, both in extracts and in vivo. the phase transition for both dry DPPC and
Because of line broadening in in vivo exper- a mixture of dry DPPC and trehalose.
iments, a lower number of phosphorus com- Tsvetkova et al. (1998) used 31P NMR in a
pounds can be resolved (Ishida et al., 1987, comparative study of the interaction of glu-
1988a) in comparison with extracts (Ricardo cose, trehalose and hydroxyethyl starch
and Santos, 1990). 31P spectra can be used with dry DPPC. The differential effect of car-
for the identification of the appearance or bohydrates on the behaviour of head groups
disappearance of vacuoles in seeds during has been related to the role of trehalose in
germination and maturation (Ishida et al., membrane protection upon drying.
1990). Phospholipids arranged in bilayers or in
an inverted hexagonal phase have different
line shapes (31P pattern) (Cullis and de Structure and dynamics of cellular Kruijff, 1979). These differences between
membranes bilayer and hexagonal phase spectra arise
from the fact that the lipids are restricted in
GENERAL REMARKS. NMR provides a rapid, motion to the plane of the membrane in the
non-invasive method for investigating the lamellar state. In the case of the hexagonal
state of membranes in isolated cellular phase, a rapid motion about the cylinder
fractions and in living tissues. The axis averages the chemical shift anisotropy.
approach in the study of membrane struc- These differences in 31P pattern can be used
ture and dynamics is solid-state NMR, to detect the presence of either phase.
because of the anisotropic nature of the For many years researchers have been
membranes. The main nuclei used for this interested in the membrane transition upon
study are 31P and 2H. Sometimes, labelling drying from the bilayer into the hexagonal
with 13C has been used, although the line phase (Simon, 1974). In an attempt to
shape is difficult to analyse. detect this membrane transition, Priestley
and de Kruijff (1982) applied 31P NMR to
31P NMR The chemical shift of the phospho- several dry biological systems. The in vivo
lipids depends on the orientation of the spectra were complicated by the superposi-
phosphate groups with respect to the mag- tion of the signals from phospholipids and
netic field. In the case of unrestricted phosphorus-containing compounds. Pollen
motion, all directions are averaged and the of Typha latifolia was the most suitable for
spectrum is isotropic and contains the nar- spectra analysis. At 5.2% MC, the line
row symmetrical 31P NMR line (Cullis and shape of the spectrum was broad and not
de Kruijff, 1979). In some cases, peaks from suitable for analysis. At MC  8.8%, only
different phospholipids can be resolved isotropic signals from phosphorus low-
(Smith, 1985). In the case of restricted weight molecules could be identified, but,
mobility of phospholipids in membranes, at 10.9% MC, a clear peak from phospho-
the spectrum is anisotropic. The shape of lipids organized in bilayers became evi-
the anisotropic 31P NMR spectrum depends dent. Thus, no evidence was obtained for
on the type and rate of motion of the phos- the presence of a hexagonal phase in the
pholipids. Thus, 31P NMR spectra are sensi- pollen on drying to 10.9% MC.
tive to the physical state of the
phospholipids. From the spectra, the order 2H NMR. The relatively small quadrupole

parameter can be calculated (Smith, 1985). moment of deuterium makes it an ideal

There are a few examples of the successful probe of membrane lipids (Smith, 1985).
application of 31P NMR in the field of desic- Fatty acids labelled with 2H at different
cation tolerance. Lee et al. (1986, 1989) positions must be synthesized. The 2H
studied the interaction of trehalose with the NMR spectrum of membranes contains
phospholipid, dipalmitoylphosphatidyl- three clearly separated lines (‘rabbit ears’),
Dessication - Chap 04 18/3/02 1:55 pm Page 134

134 O. Leprince and E.A. Golovina

and the separation relates to the ordering of netic field. IR spectroscopy is sensitive to
the 2H-labelled segment. Quadrupole split- vibrations that modulate a molecule’s
ting, overall pattern and relaxation times dipole moment. The range of frequencies
are usually used to characterize 2H spectra. is around 1012–1014 Hz or 400–4000 cm1.
Spin–lattice relaxation is sensitive to rela- The main problem of IR spectroscopy is
tively rapid motions, whereas spin–spin high water absorption in the IR region.
relaxation is sensitive to slow motions D2O substitution or dry films are often
(Smith, 1985). This technique can be used used. In plotting IR spectra, the intensity
to study membrane phase transitions, the of absorption (A) against wave number
influence of acyl chain saturation on mem- (1/
) is used. The main characteristics of
brane fluidity and changes in membrane the absorption band are wave number of
fluidity. the maximum absorption (Amax), the width
2H NMR was applied by Lee et al. (1986, of the band determined at half of the
1989) in a study on the effect of interaction height of Amax, the optical density at Amax
of trehalose with dry DPPC on the behav- and the shape of the band. Every band can
iour of acyl chains. 2H quadrupole spectra be assigned to a certain chemical group
of dry DPPC labelled at the 7th position and a certain type of vibration. In the case
showed that the disorder of lipid acyl of simple molecules, IR spectra consist of
chains is much greater in the case of inter- narrow lines. In the case of macromole-
action of DPPC with trehalose above the cules, the spectrum is characterized by rel-
phase transition than in hydrated or dry atively broad bands because of the
DPPC without trehalose. The new type of overlapping of a great number of individ-
liquid-crystalline phase observed in the dry ual lines corresponding to different types
mixture of trehalose and DPPC is believed of bonds and different conformations.
to play a main role in maintaining mem-
brane stability in dehydrating organisms. Biological applications
13C NMR. 13C-labelledphospholipids can be With the introduction of FTIR spectrome-
used to study the particular dynamics of ters in the 1970s, in vivo studies became
membranes in the interfacial region. Lee et possible, which was not the case with the
al. (1989) used 13C-labelled sn-2-carbonyl grating IR spectrometers because of their
of DPPC to study the influence of the inter- low energy throughput. FTIR spectroscopy
action of dry DPPC with trehalose on inter- can be used for the analysis of certain com-
facial behaviour. No changes in 13C NMR pounds, or to study the interaction between
powder spectra were observed during the molecules. In dry organisms, the technique
phase transition of a dry mixture of is particularly useful because of the
DPPC/trehalose, whereas hydrated DPPC ‘absence’ of water. The absorption of water
exhibited pronounced changes during the usually obscures other absorption bands
phase transition. and thus complicates the interpretation of
spectra. A considerable advantage of in vivo
FTIR spectroscopy is that it permits the
4.4.3. Fourier transform infrared (FTIR)
analysis of macromolecules in their natural
environment as opposed to in a solvent. A
disadvantage is that information is obtained General description of infrared
on the average vibrational absorption of all
molecular groups contributing to the IR-
Infrared (IR) spectroscopy deals with the absorption band under study.
transition between vibrational energy lev- For analysis of small samples or the loca-
els that permanently exist in a system, in tion of certain compounds in specific tis-
contrast to NMR and EPR where the transi- sues, an IR microscope fixed to the optical
tion occurs between energy levels that bench can be used. Improvement in sensi-
arise in a system only in an external mag- tivity has been reached by the application of
Dessication - Chap 04 18/3/02 1:55 pm Page 135

Methods for Quantifying Desiccation Phenomena 135

liquid nitrogen-cooled MCT (mercury/cad- tein secondary structure with dehydration

mium/telluride) detectors, which allow (Wolkers and Hoekstra, 1995, 1997;
pollen, microorganisms or slices of seeds to Golovina et al., 1997c; Wolkers et al.,
be studied. Peak positions or presence of 1998a,b). Conformational changes of pro-
shoulders in the spectra can be analysed by teins can be derived from peak positions
computer-assisted derivative analysis (Susi in the amide I (1600–1700 cm1) and II
and Byler, 1983) and deconvolution (Byler (around 1550 cm1) regions (Byler and
and Susi, 1986) procedures, respectively. Susi, 1986; Surewicz and Mantsch, 1988).
Depending on transmittance and scattering The amide I band mainly arises from the
characteristics of a sample, a transmission, C O stretching vibration of the peptide
reflection or attenuated total reflection groups, and the amide II band from the
(ATR) mode can be used. N–H bending vibration of the protein
An example of the in vivo analysis of cer- backbone (Susi et al., 1967). The C O
tain compounds in seeds is scanning in the stretching frequency is very sensitive to
transmission mode along a slice of tissue. changes in the nature of the hydrogen
Thus, it has been confirmed that the aleu- bonds arising from the different types of
rone layer is enriched in proteins and the secondary structure. This causes a charac-
endosperm in starch. In the case of dehy- teristic set of IR-absorption bands for each
drating organisms, the change in molecular type of secondary structure (Susi et al.,
interactions or conformation is of interest. 1967). Curve fitting of the different bands
The occurrence of an absorption band allows, to a certain extent, the amounts of
around 2850 cm1 originating from the sym- -helix, random coil, turn and -sheet
metric stretching vibration of CH2 can safely structures to be established (Surewicz et
be attributed to acyl chains, either from oil al., 1993). In some model enzyme sys-
or from membranes. If the organism is low tems, a highly characteristic low wave
in oil, it is possible to follow, in vivo, the number band (around 1625 cm1 in the
decrease in C–H vibrational freedom in the amide I region (the intermolecular
acyl chains of membranes with dehydration extended -sheets) is indicative of the for-
(Cameron et al., 1983; Crowe et al., 1989; mation of large protein aggregates with
Hoekstra et al., 1992). Restriction of vibra- drying (Prestrelski et al., 1993). These
tional freedom by molecular interaction aggregates have also been found in vivo on
(van der Waals interactions in the case of gel heat denaturation. The stability of pro-
phase formation) leads to shifts of the teins against heat denaturation can be fol-
absorption peaks to lower wave number and lowed by scanning over a range of
sharpening of these peaks. If the sample temperatures (Wolkers and Hoekstra,
holder is temperature-controlled, it is possi- 1997; Wolkers et al., 1998a). In the situa-
ble to determine the gel-to-liquid crystal tion where the absorption band of water
transition temperature of these membranes (HOH scissoring vibration band at
from shifts in the absorption maxima with 1650 cm1) interferes with the proper esti-
temperature. The same information can be mation of the different protein secondary
obtained from shifts in other absorption structures, H2O can be replaced by D2O,
bands, e.g. the asymmetric CH2 stretch which causes a downward shift in wave
around 2920 cm1 and the C O stretch of number. The accessibility of the proteins
the ester bond of the acyl chains around for D2O can help identify the protein sec-
1740 cm1 (Sowa et al., 1991). Although the ondary structure.
general melting behaviour of oil in seeds Recently, it was established that the
can also be analysed by other techniques glassy state can be studied in vivo by
(e.g. differential scanning calorimetry), that inspection of the OH-stretch at around
of membranes is difficult with other meth- 3300 cm1 (Wolkers et al., 1998c, 1999).
ods because of the small amount involved. The interaction of sugars with proteins or
In vivo FTIR spectroscopy has been with polar head groups has been verified
successfully applied in the study of pro- in dry model systems in the 3300 cm1
Dessication - Chap 04 18/3/02 1:55 pm Page 136

136 O. Leprince and E.A. Golovina

(Wolkers et al., 1998d) and 1240 cm1 thereby limiting the range of moisture
regions (Crowe et al., 1996), respectively. content that can be studied (Buitink et al.,
Such interaction upon desiccation has not 1996; Sacandé et al., 2000). However, the
been established with certainty in vivo due future of DSC in studying anhydrobiosis
to the possible absorption of other molecu- is questionable since no major difference
lar groups in these regions. Although in in the calorimetric properties of water was
vivo FTIR spectroscopy has disadvantages found between desiccation-tolerant and
in that it is an averaging technique and sensitive organisms (Sun et al., 1994;
that it is difficult to establish with cer- Buitink et al., 1996; Fig. 10.2, Chapter 10).
tainty from which molecules the spectra
originate, it has the considerable advan-
tage that molecules are studied in their 4.5.2. Electron microscopy
native environment. The disadvantages
can be partly alleviated by parallel in vitro Owing to technical difficulties in studying
experiments, also employing other meth- ultrastructural characteristics of organ-
ods of analysis. elles in the dry state and upon rehydration,
two promising microscopic techniques are
worth mentioning because they can be con-
4.5. Additional Techniques to Study sidered as non-invasive techniques: atomic
Biochemical and Biophysical Aspects of force microscopy (AFM) and low-tempera-
Desiccation Tolerance ture scanning electron microscopy
(LTSEM). AFM is particularly suitable for
4.5.1. Differential scanning calorimetry imaging, non-invasively, the surface topog-
(DSC) raphy of membranes at a nanometer scale.
Furthermore, AFM can be used to obtain
DSC is applied to the study of thermal information on the mechanical properties
events associated with lipid and water of surfaces (Heinz and Hoh, 1999;
phase/state transition. In plant anhydro- Claessens et al., 2000). LTSEM overcomes
biotes, it is used for two main purposes: problems linked to aqueous fixation. It
(i) to determine the calorimetric proper- allows a fast and direct observation of
ties of water present in the system; and freeze–fractured specimens with great reso-
(ii) to construct a state–phase diagram in lution without altering the sample water
which the glass transition temperature content. Application of LTSEM was found
(Tg) and the ice formation/melting temper- to be powerful for studying ultrastructural
ature are plotted as a function of moisture damage resulting from imbibitional injury
content (Vertucci, 1990; Leprince and in seeds (Leprince et al., 1998; Nijsse et
Vertucci, 1995; Buitink et al., 1996). The al., 1998; Sacandé et al., 2001) and cellu-
calorimetric behaviour of the glass transi- lar collapse in lichens (Scheidegger et al.,
tion can be characterized although DSC 1995). In the near future, new technologi-
does not give direct access to the physical cal developments (so-called semi-in-lens)
and biological properties of glasses. will improve the resolution, which is cur-
Sometimes, the heat released during the rently limited to 100 nm in most commer-
glass transition is below the sensitivity of cially available equipment. Non-invasive
the equipment. For example, in seed fixation (freeze-substitution) and a new
species such as rice and tobacco, Tg can- non-aqueous fixative for immunocyto-
not be detected by DSC (O. Leprince and J. chemistry (acrolein) are becoming avail-
Buitink, unpublished data). Furthermore, able for transmission electron microscopy
in oily seeds such as neem and Impatiens, studies (Grote et al., 1999), allowing
the lipid melting transitions often mask microscope observation without disturb-
the thermal events associated with water, ing the sample water content.
Dessication - Chap 04 18/3/02 1:55 pm Page 137

Methods for Quantifying Desiccation Phenomena 137

4.6. Acknowledgements l’Agriculture et de la Pêche, the Contrat

de Plan Etat-Région and INRA; E.A.G.
The authors thank Dr F.A. Hoekstra for gratefully acknowledges the financial
his contribution to the section on spec- support by a grant from the Wageningen
troscopy methods and for critically read- Centre for Food Sciences and by NATO
ing the manuscript. O.L. acknowledges collaborative linkage grant # LST.CLG
the financial support of the Ministère de 975082.

4.7. References

Aksyonov, S.I. and Golovina, E.A. (1986a) Admission and distribution of water in wheat seeds dur-
ing swelling. Soviet Plant Physiology 33, 124–130.
Aksyonov, S.I. and Golovina, E.A. (1986b) Specific features of water relations of plant seeds during
imbibition and maturation. Studia Biophysica 111, 169–172.
Alia, Hayashi, H., Chen, T.H.H. and Murata, N. (1998) Transformation with a gene for choline oxi-
dase enhances the cold tolerance of Arabidopsis during germination and early growth. Plant,
Cell and Environment 21, 232–239.
Bacic, G. and Ratkovic, S. (1984) Water exchange in plant tissue studied by proton NMR in the pres-
ence of paramagnetic centers. Biophysical Journal 45, 767–776.
Bacic, G., Nilges, M.J., Magin, R.L., Walczak, T. and Swartz, H.M. (1989) In vivo localized ESR spec-
troscopy reflecting metabolism. Magnetic Resonance in Medicine 10, 266–272.
Bacic, G., Srejic, R., Lahajnar, G., Zupancic, I. and Ratkovic, S. (1992) Water and lipids in maize seed
embryos: a proton NMR relaxation and diffusion study. Seed Science and Technology 20, 233–240.
Back, P.J., Coy, A., Xia, Y., Callaghan, P.T., Diamante, L.M. and Umbach, S.L. (1991) Some biological
applications of motional contrast in NMR microscopy. International Journal of Biological
Macromolecules 13, 181–189.
Bécard, G., Doner, L.W., Rolin, D.B., Douds, D.D. and Pfeffer, P.E. (1991) Identification and quantifica-
tion of trehalose in vesicular-arbuscular mycorrhizal fungi by in vivo carbon-13 NMR and HPLC
analyses. New Phytologist 118, 547–552.
Belton, P.S. and Ratcliffe, R.G. (1985) NMR and compartmentation in biological tissues. Progress in
NMR Spectroscopy 17, 241–279.
Benson, E.E. (1990) Free Radical Damage in Stored Germplasm. International Board of Plant Genetic
Resources, Rome, 128 pp.
Berlet, B.S. and Stadtman, E.R. (1997) Protein oxidation in aging disease, and oxidative stress.
Journal of Biological Chemistry 272, 20313–20316.
Berliner, L.J. (1976) Spin Labeling: Theory and Applications. Academic Press, New York, 592 pp.
Berliner, L.J. and Fujii, H. (1985) Magnetic resonance imaging of biological specimens by electron
paramagnetic resonance of nitroxide spin labels. Science 227, 517–519.
Berliner, L.J. and Fujii, H. (1986) EPR imaging of diffusion processes in biologically relevant poly-
mers. Journal of Magnetic Resonance 69, 68–72.
Bottomley, P.A., Foster, T.H. and Leue, W.M. (1984) In vivo nuclear magnetic resonance chemical
shift imaging by selective irradiation. Proceedings of the National Academy of Sciences USA 81,
Brusewitz, G.H. and Stone, M.L. (1987) Wheat moisture by NMR. Transactions of the American
Society of Agricultural Engineers 30, 858–862.
Buitink, J., Walters-Vertucci, C., Hoekstra, F.A. and Leprince, O. (1996) Calorimetric properties of
dehydrating pollen. Analysis of a desiccation-tolerant and an intolerant species. Plant
Physiology 111, 235–242.
Buitink, J., Claessens, M.M.A.E., Hemminga, M.A. and Hoekstra, F.A. (1998) Influence of water con-
tent and temperature on molecular mobility and intracellular glasses in seeds and pollen. Plant
Physiology 118, 531–541.
Buitink, J., Hemminga, M.A. and Hoekstra, F.A. (1999) Characterization of molecular mobility in seed
tissues: an electron paramagnetic resonance spin probe study. Biophysical Journal 76,
Dessication - Chap 04 18/3/02 1:55 pm Page 138

138 O. Leprince and E.A. Golovina

Buitink, J., Dzuba, S.A., Hoekstra, F.A. and Tsvetkov, Y.D. (2000a) Pulsed EPR spin-probe study of
intracellular glasses in seed and pollen. Journal of Magnetic Resonance 142, 364–368.
Buitink, J., Hemminga, M.A. and Hoekstra, F.A. (2000b) Is there a role for oligosaccharides in seed
longevity? An assessment of intracellular glass stability. Plant Physiology 122, 1217–1224.
Buitink, J., Hoekstra, F.A. and Hemminga, M.A. (2000c) Molecular mobility in the cytoplasm of let-
tuce radicles correlates with longevity. Seed Science Research 10, 285–292.
Buitink, J., Leprince, O., Hemminga, M.A. and Hoekstra, F.A. (2000d) Molecular mobility in the cyto-
plasm: an approach to describe and predict lifespan of dry germplasm. Proceedings of the
National Academy of Sciences USA 97, 2385–2390.
Buitink, J., Leprince, O. and Hoekstra, F.A. (2000e) Dehydration-induced redistribution of
amphiphilic molecules between cytoplasm and lipids is associated with desiccation tolerance in
seeds. Plant Physiology 124, 1413–1425.
Buitink. J., van den Dries, I.J., Hoekstra, F.A., Alberda, M. and Hemminga, M.A. (2000f) High critical
temperature above Tg may contribute to the stability of biological systems. Biophysical Journal
79, 1119–1128.
Buitink, J., Leprince, O., Hemminga, M.A. and Hoekstra, F.A. (2000g) The effects of moisture and
temperature on the ageing kinetics of pollen: interpretation based on cytoplasmic mobility.
Plant, Cell and Environment 23, 967–974.
Bukhov, N.G., Sabat, S.C. and Mohany, P. (1989) Sequential loss of photosynthetic functions during
leaf desiccation as monitored by chlorophyll fluorescence transient. Plant Cell Physiology 30,
Byler, D.M. and Susi, H. (1986) Examination of the secondary structure of proteins by deconvolved
FTIR spectra. Biopolymers 25, 469–487.
Callaghan, P.T., Jolley, K.W. and Lelievre, J. (1979) Diffusion of water in the endosperm tissue of
wheat grains as studied by pulsed field gradient nuclear magnetic resonance. Biophysical
Journal 28, 133–141.
Cameron, D.G., Martin, A. and Mantsch, H.H. (1983) Membrane isolation alters the gel to liquid crys-
tal transition of Acholeplasma laidlawii B. Science 219, 180–182.
Carrier, D., Kendall, E., Bock, C.A., Cunningham, J.E. and Dunstan, D. (1999) Water content, lipid
deposition and (+)-abscisic content in developing white spruce seeds. Journal of Experimental
Botany 50, 1359–1364.
Claessens, M.M.A.E., Leprince, O., van Aelst, A.C., Hoekstra, F.A. and Leermakers, F.A.M. (2000)
Atomic force microscopy topography of plasma membranes of anhydrobiotes in relation to imbi-
bitional injury. In: Erdey, D., Berjak, P., Farrant, J.M. and Pammenter, N.W. (eds) 3rd
International Workshop on Desiccation Tolerance and Sensitivity of Seeds and Vegetative Plant
Tissues. Itala, p. 12.
Clegg, J.S., Seitz, P., Seitz, W. and Hazlewood, C.F. (1982) Cellular responses to extreme water loss:
the water-replacement hypothesis. Cryobiology 19, 306–316.
Colnago, L.A. and Seidl, P.R. (1983) Application of carbon-13 nuclear magnetic resonance to the ger-
mination of soybean seeds in vivo. Journal of Agricultural and Food Chemistry 31, 459–461.
Conlon, T. and Outhred, R. (1972) Water diffusion permeability of erythrocytes using an NMR tech-
nique. Biochimica et Biophysica Acta 288, 354–364.
Connelly, A., Lohman, J.A.B., Loughman, B.C., Quiquampoix, H. and Ratcliffe, R.G. (1987) High reso-
lution imaging of plant tissues by NMR. Journal of Experimental Botany 38, 1713–1723.
Crowe, J.H., Hoekstra, F.A. and Crowe, L.M. (1989) Membrane phase transitions are responsible for
imbibitional damage in dry pollen. Proceedings of the National Academy of Sciences USA 86,
Crowe, J.H., Hoekstra, F.A., Nguyen, K.H.N. and Crowe, L.M. (1996) Is vitrification involved in
depression of the phase transition temperature in dry phospholipids? Biochimica et Biophysica
Acta 1280, 187–196.
Cullis, P.R. and de Kruijff, B. (1979) Lipid polymorphism and the functional roles of lipids in biologi-
cal membranes. Biochimica et Biophysica Acta 559, 399–420.
Dean, R.T., Gieseg, S. and Davies, M.J. (1993) Reactive species and their accumulation on radical-
damaged proteins. Trends in Biochemical Sciences 18, 437–441.
Degoussée, N. Triantaphylidès, C., Starek, S., Iacazio, G., Martini, D., Bladien, C., Voisine, R. and
Montillet, J.-L. (1995) Measurement of thermally produced volatile alkanes: an assay for plant
hydroperoxy fatty acid evaluation. Analytical Biochemistry 224, 524–531.
Dessication - Chap 04 18/3/02 1:55 pm Page 139

Methods for Quantifying Desiccation Phenomena 139

Dieuaide-Noubhani, M., Raffard, G., Canioni, P., Pradet, A. and Raymond, P. (1995) Quantification of
compartmented metabolic fluxes in maize root tips using isotope distribution from 13C- and 14C-
labeled glucose. Journal of Biological Chemistry 270, 13147–13159.
Dobrucki, J.W., Demsar, F., Walczak, T., Woods, R.K., Bacic, G. and Swartz, H.M. (1990) Electron spin
resonance microscopy of an in vitro tumour model. British Journal of Cancer 61, 221–224.
Du, Z. and Bramlage, W.J. (1992) Modified thiobarbituric acid assay for measuring lipid oxidation in
sugar-rich tissue extracts. Journal of Agricultural and Food Chemistry 40, 1566–1570.
Dzuba, S.A., Golovina, E.A. and Tsvetkov, Yu.D. (1993) Echo-induced EPR spectra of spin probes as a
method for identification of glassy state in biological objects. Journal of Magnetic Resonance
101, 134–138.
Eccles, C.D., Callaghan, P.T. and Jenner, C.F. (1988) Measurement of the self-diffusion coefficient of
water as a function of position in wheat grain using nuclear magnetic resonance imaging.
Biophysical Journal 53, 77–82.
Espen, L., Morgutti, S., Alisi, C., Pirovano, L., Ragg, E. and Cocucci, S.M. (1995) Germination and pH
of intracellular compartments in seeds of Phacelia tanacetifolia. Physiologia Plantarum 93,
Esposito, L.A., Melov, S., Panov, A., Cottrell, B.A. and Wallace, D.C. (1999) Mitochondrial disease in
mouse results in increased oxidative stress. Proceedings of the National Academy of Sciences
USA 96, 4820–4825.
Fan, T.W.-M. (1996) Recent advances in profiling plant metabolites by multinuclear and multidimen-
sional NMR. In: Shachar-Hill, Y. and Pfeffer, P.E. (eds) Nuclear Magnetic Resonance in Plant
Biology. Current Topics in Plant Physiology, Vol. 16, American Society of Plant Physiologists,
Rockville, Maryland, pp. 181–256.
Farrar, T.C. and Becker, E.D. (1971) Pulse and Fourier Transform NMR: Introduction to Theory and
Methods. Academic Press, New York, 115 pp.
Fleischer, G. and Werner, A. (1992) Study of water and oil diffusion in rape seeds with sorption–
desorption and NMR techniques. Biochimica et Biophysica Acta 1116, 305–308.
Fleischer, G., Skirda, V.D. and Werner, A. (1990) NMR-investigation of restricted self-diffusion of oil
in rape seeds. European Biophysical Journal 19, 25–30.
Footitt, S., Vargas, D. and Cohn, M.A. (1995) Seed dormancy in red rice. X. A 13C-NMR study of the
metabolism of dormancy-breaking chemicals. Physiologia Plantarum 94, 667–671.
Fountain, D.W., Forde, L.C., Amith, E.E., Owens, K.R., Bailey, D.G. and Callaghan, P.T. (1998) Seed
development in Phaseolus vulgaris L cv Seminole. 3. NMR imaging of embryos during ethylene-
induced precocious germination. Seed Science Research 8, 357–365.
Foyer, C.H., Lelandais, M. and Kunert, K.J. (1994) Photooxidative stress in plants. Physiologia
Plantarum 92, 696–717.
Fukushima, E. and Roeder, S.B.W. (1981) Experimental Pulsed NMR: a Nuts and Bolts Approach.
Addison-Wesley, Reading, Massachusetts, 539 pp.
Gambhir, P.N. (1992) Application of low-resolution pulsed NMR to the determination of oil and
moisture in oilseeds. Trends in Food Science and Technology 3, 191–196.
Gambhir, P.N. and Agarwala, A.K. (1985) Simultaneous determination of moisture and oil content in
oilseeds by pulsed nuclear magnetic resonance. Journal of the American Oil Chemists Society
62, 103–108.
Golovina, E.A. and Hoekstra, F.A. (2002) Membrane behavior as influenced by partitioning of
amphiphiles during drying: a comparative study in anhydrobiotic plant systems. Comparative
Biochemistry and Physiology (in press).
Golovina, E.A. and Tikhonov, A.N. (1994) The structural differences between the embryos of viable
and nonviable wheat seeds as studied with the EPR spectroscopy of lipid-soluble spin labels.
Biochimica et Biophysica Acta 1190, 385–392.
Golovina, E.A., Smirnov, A.I., Yakimchenko, O.E. and Aksyonov, S.I. (1991) EPR-tomography of
paths by which an aqueous solution of nitroxyl radical enters the wheat caryopsis. Soviet Plant
Physiology 38, 90–94.
Golovina, E.A., Tikhonov, A.N. and Hoekstra, F.A. (1997a) An electron paramagnetic resonance spin-
probe study of membrane-permeability changes with seed aging. Plant Physiology 114, 383–389.
Golovina, E.A., Wolkers, W.F. and Hoekstra, F.A. (1997b) Behaviour of membranes and proteins during
natural seed ageing. In: Ellis, R.H., Black, M., Murdoch, A.J. and Hong, T.D. (eds) Basic and Applied
Aspects of Seed Biology. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 787–796.
Dessication - Chap 04 18/3/02 1:55 pm Page 140

140 O. Leprince and E.A. Golovina

Golovina, E.A., Wolkers, W.F. and Hoekstra, F.A. (1997c) Long-term stability of protein secondary
structure in dry seeds. Comparative Biochemistry and Physiology 117A, 343–348.
Golovina, E.A., Hoekstra, F.A. and Hemminga, M.A. (1998) Drying increases intracellular partitioning
of amphiphilic substances into the lipid phase: impact on membrane permeability and signifi-
cance for desiccation tolerance. Plant Physiology 118, 975–986.
Golovina, E.A., Hoekstra, F.A. and van Aelst, A.C. (2000) Programmed cell death or desiccation tol-
erance: two possible routes for wheat endosperm cells. Seed Science Research 10, 365–379.
Golovina, E.A., 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 Experimental
Botany 52, 1015–1027.
Golovina, Y.A. and Aksyonov, S.I. (1993) Role of water state in metabolism activation in seeds during
first steps of imbibition. In: Côme, D. and Corbineau, F. (eds) Basic and Applied Aspects of Seed
Biology, Vol 2. ASFIS, Paris, pp. 375–380.
Gorecki, R.J., Harman, G.E. and Mattick, L.R. (1984) The volatile exudates from germinating pea
seeds of different viability and vigor. Canadian Journal of Botany 63, 1035–1039.
Gros, J.B., Achard, C. and Dussap, C.G. (1992) Solubilité de l’oxygène dans les milieux alimentaires
liquides. Mesures et prédiction. Sciences des Aliments 12, 47–61.
Grote, M., Reichelt, R. and Wiermann, R. (1999) A new protocol to prepare dry plant specimens for
electron microscopy and immunocytochemistry. Micron 30, 65–70.
Gutteridge, J.M.C. and Halliwell, B. (1990) The measurement and mechanism of lipid peroxidation in
biological systems. Trends in Biochemical Sciences 15, 129–135.
Hageman, J.J., Bast, A. and Vermeulen, N.P.E. (1992) Monitoring of oxidative free radical damage in
vivo: analytical aspects. Chemico-Biological Interactions 82, 243–293.
Hailstones, M.D. and Smith, M.T. (1989) Thermally-derived volatile aldehydes in relation to seed
viability in soybean seeds. Seed Science and Technology 17, 649–658.
Halliwell, B. and Gutteridge, J.M.C. (1984) Lipid peroxidation, oxygen radicals and antioxidant ther-
apy. Lancet June 23rd, 1396–1397.
Hamilton, S., Hamilton, R.J. and Sewell, P.A. (1992) Extraction of lipids and derivative formation. In:
Hamilton, R.S. and Hamilton, S. (eds) Lipid Analysis. A Practical Approach. IRL Press, Oxford,
pp. 13–64.
Harren, F.J.M. and Reuss, J. (1997) Spectroscopy. Photoacoustics. In: Trigg, G.I. (ed.) Encyclopedia of
Applied Physics, Vol. 19. VCH Publisher, New York, pp. 413–435.
Heath, R.L. and Packer, L. (1968) Photoperoxidation in isolated chloroplasts. I. Kinetics and stoi-
chiometry of fatty acid peroxidation. Archives of Biochemistry and Biophysics 125, 189–198.
Heidenreich, M., Spyros, A., Köckenberger, N., Chandrakumar, N., Bowtell, R. and Kimmich, R.
(1998) CYCLOCROP mapping of 13C labelled compounds: perspectives in polymer science and
plant physiology. In: Blümler, P., Blümich, B., Botto, R. and Fukishima, E. (eds) Spatially
Resolved Magnetic Resonance: Methods, Materials, Medicine, Biology, Rheology, Geology,
Ecology, Hardware. Wiley-VCH, New York, pp. 21–52.
Heinz, W.F. and Hoh, J.H. (1999) Spatially resolved force spectroscopy of biological surfaces using
the atomic force microscope. Trends in Biotechnology 17, 143–171.
Hemminga, M.A. (1983) Interpretation of ESR and saturation transfer ESR spectra of spin labeled
lipids and membranes. Chemistry and Physics of Lipids 32, 323–383.
Hendry, G.A.F. (1993) Oxygen, free radical processes and seed longevity. Seed Science Research 3,
Hendry, G.A.F., Finch-Savage, W.E., Thorpe, P.C., Atherton, N.M., Buckland, S.M., Nilsson, K.A. and
Seel, W.E. (1992) Free radical processes and loss of seed viability during desiccation in the
recalcitrant species Quercus robur L. New Phytologist 122, 273–279.
Hodges, D.M., DeLong, J.M., Forney, C.F. and Prange, R.K. (1999) Improving the thiobarbituric acid-
reactive-substances assay for estimating lipid peroxidation in plant tissues containing antho-
cyanin and other interfering compounds. Planta 207, 604–611.
Hoekstra, F.A. and Golovina, E.A. (2000) Impact of amphiphile partitioning on desiccation tolerance.
In: Black, M., Bradford, K.J. and Vasques-Ramos, J. (eds) Seed Biology: Advances and
Applications. CAB International, Wallingford, UK, pp. 43–55.
Hoekstra, F.A. and van Roekel, T. (1983) Isolation-inflicting injury to mitochondria from fresh
pollen gradually overcome by an active strengthening during germination. Plant Physiology
73, 995–1001.
Dessication - Chap 04 18/3/02 1:55 pm Page 141

Methods for Quantifying Desiccation Phenomena 141

Hoekstra, F.A., Crowe, J.H. and Crowe, L.M. (1992) Germination and ion leakage are linked with
phase transitions of membrane lipids during imbibition of Typha latifolia pollen. Physiologia
Plantarum 84, 29–34.
Hoekstra, F.A., Golovina, E.A., van Aelst, A.C. and Hemminga, M.A. (1999) Imbibitional leakage from
anhydrobiotes revisited. Plant, Cell and Environment 22, 1121–1131.
Hou, J., Kendall, E. and Simpson, G. (1996) Nuclear magnetic resonance microimaging of water uptake
and distribution in caryopses of wild oat (Avena fatua). Journal of Experimental Botany 48, 683–692.
Hyslop, P.A., Hinshaw, D.B., Halsey, W.A., Schraufstätter, I.U., Sauerheber, R.D., Spragg, R.G.,
Jackson, J.H. and Cochrane, C.G. (1988) Mechanisms of oxidant-mediated cell injury. The gly-
colytic and mitochondrial pathways of ADP phosphorylation are major intracellular targets inac-
tivated by hydrogen peroxide. Journal of Biological Chemistry 263, 1665–1675.
Ishida, N., Kano, H., Kobayashi, T., Hamaguchi, H. and Yoshida, T. (1987) Estimation of biological
activities by NMR in soybean seeds during maturation. Agricultural and Biological Chemistry
51, 301–307.
Ishida, N., Kano, H., Kobayashi, T., Hamaguchi, H. and Yoshida, T. (1988a) The relationship between
imbibitional damage and initial water content of soybeans. Agricultural and Biological
Chemistry 52, 2771–2775.
Ishida, N., Kano, H., Kobayashi, T. and Yoshida, T. (1988b) Analysis of physical states of water in
soybean seeds by NMR. Agricultural and Biological Chemistry 52, 2777–2781.
Ishida, N., Kobayashi, T., Masuda, R., Kano, H., Yoshida, T. and Ogawa, H. (1990) Tracing metabolic
changes in soybean cotyledons during germination by NMR. Agricultural and Biological
Chemistry 54, 1359–1365.
Ishida, N., Ogawa, H. and Kano, H. (1995) Diffusion of cell-associated water in ripening barley seeds.
Magnetic Resonance Imaging 13, 745–751.
Ishida, N., Koizumi, M. and Kano, H. (1996) Location of sugars in barley seeds during germination by
NMR microscopy. Plant, Cell and Environment 19, 1415–1422.
Ishida, N., Koizumi, M. and Kano, H. (2000) The NMR microscope: a unique and promising tool for
plant science. Annals of Botany 86, 259–278.
Jiang Z.-Y., Woollard, A.C.S. and Wolff, S.P. (1991) Lipid hydroperoxide measurement by oxidation
of Fe2+ in the presence of xylenol orange. Comparison with the TBA assay and an iodometric
method. Lipids 26, 853–856.
Junqua, M., Biolley, J.-P., Pie, S., Kanoum, M., Duran, R. and Goulas, P. (2000) In vivo occurrence of
carbonyl residues in Phaseolus vulgaris proteins as a direct consequence of a chronic ozone
stress. Plant Physiology and Biochemistry 38, 853–861.
Kaiser, W.M. (1979) Reversible inhibition of the Calvin cycle and activation of oxidative pentose
phosphate cycle in isolated intact chloroplasts by hydrogen peroxide. Planta 145, 377–382.
Kano, H., Ishida, T., Kobayashi, T. and Koisumi, M. (1990) 1H-NMR imaging analysis of changes in
free water distribution in barley and soybean seeds during maturation. Japanese Journal of Crop
Science 59, 503–509.
Kaplan, J., Canonico, P.G. and Caspary, W.J. (1973) Electron spin resonance studies of spin-labeled
mammalian cells by detection of surface membrane signals. Proceedings of the National
Academy of Sciences USA 70, 66–70.
Keith, A.D. and Snipes, W. (1974) Viscosity of cellular protoplasm. Science 183, 666–668.
Khramtsov, V.V. and Weiner, L.M. (1988) Proton exchange in stable nitroxyl radicals: pH-sensitive
spin probes. In: Volodarsky, L.B. (ed.) Imidazoline Nitroxides, Vol II, Applications. CRC Press,
Boca Raton, Florida, pp. 37–80.
Kimmerer, T.W. and Kozlowski, T.T. (1982) Ethylene, ethane, acetaldehyde, and ethanol production
by plants under stress. Plant Physiology 69, 840–847.
Klein, J.D. and Sachs, M. (1992) Measurement of water uptake and volatile production by coated
wheat seeds and subsequent seedling growth. Seed Science and Technology 20, 299–305.
Knecht, K.T. and Mason, R.P. (1993) In vivo spin trapping of xenobiotic free radical metabolites.
Archives of Biochemistry and Biophysics 303, 185–194.
Koizumi, M., Ishida, N. and Kano, H. (1995) Location of sucrose and oils in a maize seed by NMR
microscopy. Bioscience, Biotechnology and Biochemistry 59, 2321–2323.
Kovacs, A.J. and Nemenyi, M. (1999) Moisture gradient vector calculation as a new method for evalu-
ating NMR images of maize (Zea mays L) kernels during drying. Magnetic Resonance Imaging
17, 1077–1082.
Dessication - Chap 04 18/3/02 1:55 pm Page 142

142 O. Leprince and E.A. Golovina

Lauterbur, P.C. (1973) Image formation by induced local interactions: examples employing Nuclear
Magnetic Resonance. Nature 242, 190–191.
LeBel, C.G., Collins, J. and Gebicki, J.M. (1999) Hydroperoxide assay with the ferric–xylenol orange
complex. Analytical Biochemistry 273, 149–155.
LeBel, C.P., Ischiropoulos, H. and Bondy, S.C. (1992) Evaluation of the probe 2,7-dichlorofluorescin
as an indicator of reactive oxygen species formation and oxidative stress. Chemical Research in
Toxicology 6, 227–231.
Lee, C.W.B., Waugh, J.S. and Griffin, R.G. (1986) Solid-state NMR study of trehalose/1,2-dipalmitoyl-
sn-phosphatidylcholine interactions. Biochemistry 25, 3737–3742.
Lee, C.W.B., Das Gupta, S.K., Mattai, J., Shipley, G.G., Abdel-Mageed, O.H., Makriyannis, A. and
Griffin, R.G. (1989) Characterization of the lambda phase in trehalose-stabilized dry membranes
by solid-state NMR and X-ray diffraction. Biochemistry 28, 5000–5009.
Leprince, O. and Hoekstra, F.A. (1998) The responses of cytochrome redox state and energy metabo-
lism to dehydration support a role for cytoplasmic viscosity in desiccation tolerance. Plant
Physiology 118, 1253–1264.
Leprince, O. and Vertucci, C.W. (1995) Characterization of calorimetric behaviour of intracellular
glasses in bean axes in relevance with storage stability. Plant Physiology 109, 1471–1481.
Leprince, O., Deltour R., Thorpe, P.C., Atherton, N.M. and Hendry, G.A.F. (1990) The role of free rad-
icals and radical processing systems in loss of desiccation tolerance in germinating maize (Zea
mays L.). New Phytologist 116, 573–580.
Leprince, O., Vertucci, C.W., Hendry, G.A.F. and Atherton, N.M. (1995) The expression of desicca-
tion-induced damage in orthodox seeds is a function of oxygen and temperature. Physiologia
Plantarum 94, 233–240.
Leprince, O., van Aelst, A.C., Pritchard, H.W. and Murphy, D.J. (1998) Oleosins prevent oil-body coa-
lescence during seed imbibition as suggested by a low-temperature scanning electron micro-
scope study of desiccation-tolerant and -sensitive oilseeds. Planta 204, 109–119.
Leprince, O., Buitink, J. and Hoekstra, F.A. (1999) Axes and cotyledons of recalcitrant seeds of
Castanea sativa Mill. exhibit contrasting responses of respiration to drying in relation to desic-
cation sensitivity. Journal of Experimental Botany 50, 1515–1524.
Leprince, O., Harren, F.J.M., Buitink, J., Alberda, M. and Hoekstra, F.A. (2000) Metabolic dysfunction
and unabated respiration precede the loss of membrane integrity during dehydration of germi-
nating radicles. Plant Physiology 122, 597–608.
Lievonen, S.M., Laaksonen, T.J. and Roos, Y.H. (1998) Glass transition and reaction rates: nonenzy-
matic browning in glassy and liquid systems. Journal of Agricultural and Food Chemistry 46,
Lynch, R.M. and Clegg, J.S. (1986) A study of metabolism in dry seeds of Avena fatua L. evaluated by
incubation with ethanol-1-C14. In: Leopold, A.C. (ed.) Membranes, Metabolism and Dry
Organisms. Cornell University Press, Ithaca, New York, pp. 50–59.
Magin, R.L., Wright, S.M., Niesman, M.R., Chan, H.C. and Swartz, H.M. (1986) Liposome delivery of
NMR contrast agents for improved tissue imaging. Magnetic Resonance in Medicine 3, 440–447.
Marconi, E., Carnovale, E., Di Nola, A. and Brosio, E. (1993) NMR assessment of water uptake in dif-
ferent Vigna spp. seeds. International Journal of Food Science and Technology 28, 25–33.
Marsh, D. (1981) Electron spin resonance: spin labels. In: Grell, E. (ed.) Membrane Spectroscopy.
Molecular Biology, Biochemistry and Biophysics, Vol. 31. Springer-Verlag, Berlin, pp. 51–142.
Martin, M.L., Martin, G.J. and Delpeuch, J.J. (1980) Practical NMR Spectroscopy. Heyden, London,
460 pp.
McConnell, H.M. and McFarland, B.G. (1970) Physics and chemistry of spin labels. Quarterly
Reviews of Biophysics 3, 91–136.
McIntyre, D.J.O., Peters, A.M., Bowtell, R.W., Bingham, K., Mahsfield, P. and Morris, P.G. (1995)
Mapping the utilization of protein stores during the germination of the castor bean using the
NMR microscope. Plant Physiology Suppl. 108, 43.
McKersie, B.D., Hoekstra, F.A. and Krieg, L.C. (1990) Differences in the susceptibility of plant mem-
brane lipids to peroxidation. Biochimica et Biophysica Acta 1030, 11–126.
Meagher, E.A. and Fitzgerald, G.A. (2000) Indices of lipid peroxidation in vivo: strengths and limita-
tions. Free Radical Biology and Medicine 28, 1745–1750.
Mehlhorn, R.J., Candau, P. and Packer, L. (1982) Measurements of volume and electrochemical gradi-
ents with spin probes in membrane vesicles. Methods in Enzymology 88, 751–762.
Dessication - Chap 04 18/3/02 1:55 pm Page 143

Methods for Quantifying Desiccation Phenomena 143

Miller, R.W. (1978) Osmotically induced removal of water from fungal cells as determined by a spin
probe technique. Plant Physiology 62, 741–745.
Miller, R.W. and Barran, L.R. (1977) The effect of ionic surface active agents on macroconidial
plasma membranes of Fusarium sulphureum. Canadian Journal of Microbiology 23, 1373–1383.
Morse, P.D. (1985) Structure–function relationships in cell membranes as revealed by spin labeling
EPR. In: Benga G. (ed.) Structure and Properties of Cell Membranes, Vol. 3. CRC Press, Boca
Raton, Florida, pp. 195–236.
Nash, T.S. III, Reiner, A., Demmig-Adams B., Kilian, E., Kaiser, W.M. and Lange, O.L. (1990) The
effect of atmospheric desiccation and osmotic water stress on photosynthesis and dark respira-
tion of lichens. New Phytologist 116, 269–276.
Neff, W.E., Mounts, S.T.L., Rinsch, W., Franket, E.N. and Weltoum, M.A.M. (1992) Effect of triacyl-
glycerol composition and structure on oxidative stability of oils from selected soybean
germplasm. Journal of the American Oil Chemists Society 69, 111–118.
Ni, Q.W. and Eads, T.M. (1992) Low-speed magic-angle-spinning carbon-13 NMR of fruit tissue.
Journal of Agricultural and Food Chemistry 40, 781–787.
Nijsse, J., Erbe, E., Brantjes, N.B.M., Schel, J.H.N. and Wergin, W.P. (1998) Low-temperature scanning
electron microscopic observations on endosperm in imbibed and germinated lettuce seeds.
Canadian Journal of Botany 76, 509–516.
Noteborn, H.P.J.M., Lommen, A., van der Jagt, R.C. and Weseman, J.M. (2000) Chemical fingerprint-
ing for the evaluation of unintended secondary metabolic changes in transgenic food crops.
Journal of Biotechnology 77, 103–114.
Pohle, W.D. and Gregory, R.L. (1968) Application of wide-line NMR to analysis of cereal products
and fats and oils. Journal of the American Oil Chemists Society 45, 775–777.
Prestrelski, S.J., Tedeschi, N., Arakawa, T. and Carpenter, J.F. (1993) Dehydration-induced conforma-
tional transitions in protein and their inhibition by stabilizers. Biophysical Journal 65, 661–671.
Priestley, D.A. and de Kruijff, B. (1982) Phospholipid motional characteristics in a dry biological sys-
tem. A 31P nuclear magnetic resonance study of hydrating Typha latifolia pollen. Plant
Physiology 70, 1075–1078.
Ratcliffe, R.G. (1994) In vivo NMR studies of higher plants and algae. In: Callow, J.A. (ed.) Advances
in Botanical Research, Vol. 20. Academic Press, London, pp. 43–123.
Ratkovic, S. (1987) Proton NMR of maize seed water: the relationships between spin-lattice relax-
ation time and water content. Seed Science and Technology 15, 147–154.
Ratkovic, S., Bacic, G., Radenovic, G. and Vucinic, Z. (1982a) Water in plants: a review of some
recent NMR studies concerning the state and transport of water in leaf, root, and seed. Studia
Biophysica 91, 9–18.
Ratkovic, S., Denic, M., Lahajnar, G. and Zupancic, I. (1982b) Biological systems with low water con-
tent: NMR approach to the state of water in plant seed. In: Franks, F. and Mathias, S.F. (eds)
Biophysics of Water. John Wiley & Sons, New York, pp. 312–314.
Reed, R.H., Warr, S.R.C., Richardson, D.I., Moore, D.J. and Stewart, W.D.P. (1985) Multiphasic
osmotic adjustment in a euryhaline cyanobacterium. FEMS Microbiology Letters 28, 225–229.
Ricardo, C.P.P. and Santos, H. (1990) Application of 31P NMR to monitor phosphorus compounds and
their changes during germination of legume seeds. Journal of Experimental Botany 41, 79–87.
Roberts, J.K.M. (2000) NMR adventures in the metabolic labyrinth within plants. Trends in Plant
Sciences 5, 30–34.
Rogerson, N.E. and Matthews, S. (1977) Respiratory and carbohydrates changes in developing pea
(Pisum sativum L.) seeds in relation to their ability to withstand desiccation. Journal of
Experimental Botany 28, 304–313.
Roozen, M.J.G.W., Hemminga, M.A. and Walstra, P. (1991) Molecular motion in glassy water–malto-
oligosaccharide (maltodextrin) mixtures as studied by conventional and saturation-transfer spin-
probe ESR spectroscopy. Carbohydrate Research 215, 229–237.
Roscher, A., Kruger, N.J. and Ratcliffe, R.G. (2000) Strategies for metabolic flux analysis in plants
using isotope labelling. Journal of Biotechnology 77, 81–102.
Rozantsev, E.G. (1970) Free Nitroxide Radicals. Plenum Press, New York.
Ruan, R. and Litchfield, J.B. (1992) Determination of water distribution and mobility inside maize
kernels during steeping using magnetic resonance imaging. Cereal Chemistry 69, 13–17.
Ruan, R., Litchfield, J.B. and Eschoff, S.R. (1992) Simultaneous and nondestructive measurements of
transient moisture profiles and structural changes in maize kernels during steeping using micro-
scopic nuclear magnetic resonance. Cereal Chemistry 69, 600–606.
Dessication - Chap 04 18/3/02 1:55 pm Page 144

144 O. Leprince and E.A. Golovina

Rubel, G. (1994) Simultaneous determination of oil and water contents in different oilseeds by pulsed
nuclear magnetic resonance. Journal of the American Oil Chemists Society 71, 1057–1062.
Rutar, V. (1989) Magic angle sample spinning NMR spectroscopy of liquids as a nondestructive
method for studies of plant seeds. Journal of Agricultural and Food Chemistry 37, 67–70.
Sacandé, M., Buitink, J. and Hoekstra, F.A. (2000) A study of water relations in neem (Azadirachta
indica) seed that is characterized by complex storage behaviour. Journal of Experimental Botany
51, 635–642.
Sacandé, M., Golovina, E.A., van Aelst, A.C. and Hoekstra, F.A. (2001) Viability loss of neem
(Azadirachta indica) seeds associated with membrane phase behaviour. Journal of Experimental
Botany 52, 919–931.
Schaeffer, J., Stejskal, E.O. and Beard, C.F. (1975) Carbon-13 nuclear magnetic resonance analysis of
metabolism in soybeans labelled by 13CO2. Plant Physiology 55, 1048–1053.
Scheidegger, C., Schroeter, B. and Frey, B. (1995) Structural and functional processes during water
vapour uptake and desiccation in selected lichens with green algal photosymbionts. Planta 197,
Schneider, B. (1997) In vivo nuclear magnetic resonance spectroscopy of low-molecular-weight com-
pounds in plant cells. Planta 203, 1–8.
Schwab, K.B. and Heber, U. (1984) Thylakoid membrane stability in drought-tolerant and drought-
sensitive plants. Planta 161, 37–45.
Schwab, K.B., Schreiber, U. and Heber, U. (1989) Response of photosynthesis and respiration of res-
urrection plants to desiccation and rehydration. Planta 177, 217–227.
Seewaldt, V., Priestley, D.A., Leopold, A.C., Feigenson, G.W. and Goodsaid-Zalduondo, F. (1981)
Membrane organization in soybean seeds during hydration. Planta 152, 19–23.
Seitz, P.K., Chang, D.C., Hazlewood, C.F., Rorschach, H.E. and Clegg, J.S. (1981) The self-diffusion of
water in Artemia cysts. Archives of Biochemistry and Biophysics 210, 517–524.
Shachar-Hill, Y. and Pfeffer, P.E. (1996) Nuclear Magnetic Resonance in Plant Biology. Current Topics
in Plant Physiology, Vol. 16, American Society of Plant Physiologists, Rockville, USA, 260 pp.
Shiota, H. and Kamada, H. (2000) Acquisition of desiccation tolerance by cultured carrot cells upon
ectopic expression of C-ABI3, a carrot homolog of ABI3. Journal of Plant Physiology 156,
Simon, E.W. (1974) Phospholipids and plant membrane permeability. New Phytologist 73, 377–420.
Smirnov, A.I., Yakimchenko, O.E., Aksyonov, S.I., Golovina, E.A., Lichtenstein, G.I. and Lebedev,
Y.S. (1988) Distribution of an aqueous solution of a probe during wheat grain imbibition as
investigated by EPR tomography. Soviet Plant Physiology 35, 516–520.
Smirnov, A.I., Yakimchenko, O.E., Golovina, E.A., Bekova, S.K. and Lebedev, Y.S. (1991) EPR imag-
ing with natural spin probes. Journal of Magnetic Resonance 91, 386–391.
Smith, I.C. (1985) Structure and dynamics of cell membranes as revealed by NMR techniques. In:
Benga, G. (ed.) Structure and Properties of Cell Membranes, Vol 3. CRC Press, Boca Raton,
Florida, pp. 237–260.
Snaar, J.E.M. and van As, H. (1992) Probing water compartments and membrane permeability in
plant cells by 1H NMR relaxation measurements. Biophysical Journal 62, 1654–1658.
Song, H., Litchfield, J.B. and Morris, H.D. (1992) Three-dimential microscopic MRI of maize kernels
during drying. Journal of Agricultural Engineering Research 53, 51–69.
Sowa, S., Connor, K.F. and Towill, L.E. (1991) Temperature changes in lipid and protein structure
measured by Fourier transform infrared spectrophotometry in intact pollen grains. Plant Science
78, 1–9.
Stejskal, E.O. and Tanner, J.E. (1965) Spin diffusion measurements: spin-echoes in the presence of a
time-dependent field gradient. Journal of Chemical Physics 42, 288–292.
Stout, D.G., Cotts, R.M. and Steponkus, P.L. (1977) The diffusional water permeability of Elodea leaf
cells as measured by nuclear magnetic resonance. Canadian Journal of Botany 55, 1623–1631.
Stout, D.G., Steponkus, P.L. and Cotts, R.M. (1978) Nuclear magnetic resonance relaxation times and
plasmalemma water exchange in ivy bark. Plant Physiology 62, 636–641.
Sun, W.Q., Irving, T.C. and Leopold, A.C. (1994) The role of sugar, vitrification and membrane phase
transition in seed desiccation tolerance. Physiologia Plantarum 90, 621–628.
Surewicz, W.K. and Mantsch, H.H. (1988) New insight into protein secondary structure from resolu-
tion-enhanced infrared spectra. Biochimica et Biophysica Acta 952, 115–130.
Surewicz, W.K., Mantsch, H.H. and Chapman, D. (1993) Determination of protein secondary structure
by Fourier transform infrared spectroscopy: a critical assessment. Biochemistry 32, 389–394.
Dessication - Chap 04 18/3/02 1:55 pm Page 145

Methods for Quantifying Desiccation Phenomena 145

Susi, H. and Byler, D.M. (1983) Protein structure by Fourier transform infrared spectroscopy: second
derivative spectra. Biochemical and Biophysical Research Communications 115, 391–397.
Susi, H., Timasheff, S.N. and Stevens, L. (1967) Infrared spectra and protein conformations in aque-
ous solutions. I. The amide I band in H2O and D2O solutions. Journal of Biological Chemistry
242, 5460–5466.
Swartz, H.M. (1987) Measurement of pertinent oxygen concentrations in biological systems. Acta
Biochimica et Biophysica Hungaricae 22, 277–293.
Swartz, H.M., Chen, K., Pals, M., Sentjurc, M. and Morse, P.D. II (1986) Hypoxia-sensitive NMR con-
trast agents. Magnetic Resonance in Medicine 3, 169–174.
Tanner, J.E. (1978) Transient diffusion in a system partitioned by permeable barriers. Application to
NMR measurements with a pulsed field gradient. Journal of Chemical Physics 69, 1748–1754.
Tiwari, P.N., Gambhir, P.N. and Rajan, T.S. (1974) Rapid and nondestructive determination of seed oil
by pulsed nuclear magnetic resonance technique. Journal of the American Oil Chemists Society
51, 104–109.
Tse, T.Y., Spanswick, R.M. and Jelinski, L.W. (1996) Quantitative evaluation of NMR and MRI meth-
ods to measure sucrose concentration in plants. Protoplasma 194, 54–62.
Tsvetkova, N.M., Phillips, B.L., Crowe, L.M., Crowe, J.H. and Risbud, S.H. (1998) Effect of sugars on
headgroup mobility in freeze-dried dipalmitoylphosphatidylcholine bilayers: solid-state 31P
NMR and FTIR studies. Biophysical Journal 75, 2947–2955.
Vertucci, C.W. (1990) Calorimetric studies of the state of water in seed tissues. Biophysical Journal
58, 1463–1471.
Vertucci, C.W. and Leopold, A.C. (1986) Physiological activities associated with hydration level in
seeds. In: Leopold, A.C. (ed.) Membranes, Metabolism and Dry Organisms. Cornell University
Press, Ithaca, New York, pp. 35–49.
Vertucci, C.W. and Leopold, A.C. (1987) Oxidative processes in soybean and pea seeds. Effect of
light, temperature and water content. Plant Physiology 84, 1038–1043.
Vishnyakova, E.A., Ruuge, A.E., Golovina, E.A., Hoekstra, F.A. and Tikhonov, A.N. (2000) Spin-label-
ing study of membranes in wheat embryo axes. I. Partitioning of doxyl stearates into the lipid
domains. Biochimica et Biophysica Acta 1467, 380–394.
von Meerwall, E. and Ferguson, R.D. (1981) Interpreting pulsed-gradient spin-echo diffusion experi-
ments with permeable membranes. Journal of Chemical Physics 74, 6956–6959.
von Sonntag, C. and Schuchmann, H.-P. (1987) Radical-mediated DNA damage in presence of oxy-
gen. Methods in Enzymology 186, 511–520.
Walczak, T., Demsar, F., Gabrys, H. and Swartz, H.M. (1987) Seed germination: EPR imaging study.
In: Abstracts of 29th Rocky Mountain Conference, Denver, Colorado, 2–6 August, p. 150.
Wallace, D.C. (1999) Mitochondrial diseases in man and mouse. Science 283, 1482–1493.
Warmsley, J. (1998) Simultaneous determination of oil and moisture in seeds by low-resolution
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 decreases
starch 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 responds
to 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: role
in 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 Phaseolus
vulgaris 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.
Wolkers, W.F. and Hoekstra, F.A. (1997) Heat stability of proteins in desiccation tolerant cattail
pollen (Typha latifolia): a Fourier transform infrared spectroscopic study. Comparative
Biochemistry and Physiology 117A, 349–355.
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 Arabidopsis
thaliana. The Plant Journal 16, 133–143.
Dessication - Chap 04 18/3/02 1:55 pm Page 146

146 O. Leprince and E.A. Golovina

Wolkers, W.F., Bochicchio, A., Selvaggi, G. and Hoekstra, F.A. (1998b) Fourier transform infrared
microspectroscopy detects changes in protein secondary structure associated with desiccation
tolerance in developing maize embryos. Plant Physiology 116, 1169–1177.
Wolkers, W.F., Oldenhof, H., Alberda, M. and Hoekstra, F.A. (1998c) A Fourier transform infrared
microspectroscopy 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 conformational
changes of poly-L-lysine as influenced by drying rate and carbohydrates. Biochimica et
Biophysica Acta 1425, 127–136.
Wolkers, W.F., Tetteroo, F.A.A., Alberda, M. and Hoekstra, F.A. (1999) Changed properties of the
cytoplasmic matrix associated with desiccation tolerance of dried carrot somatic embryos. An in
situ Fourier transform infrared study. Plant Physiology 120, 153–163.
Yates, F.M. and van Houten, B. (1997) Mitochondrial DNA damage is more extensive and persists
longer than nuclear DNA damage in human cells following oxidative stress. Proceedings of the
National Academy of Sciences USA 94, 514–519.
Zhang, M., Maeda, Y., Furihata, Y., Nakamuru, Y. and Esashi, Y. (1994) A mechanism of seed deterio-
ration in relation to the volatile compounds evolved in dry seeds themselves. Seed Science
Research 4, 49–56.
Zhang, M., Nagata, S., Miyazawa, K., Kikuchi, H. and Esashi, Y. (1997) A competitive enzyme-linked
immuno assay to quantify acetaldehyde-protein adducts that accumulate in dry seeds during
aging. Plant Physiology 113, 397–402.
Zuckermann, H., Harren, F.J.M., Reuss, J. and Parker, D.H. (1997) Dynamics of acetaldehyde produc-
tion during anoxia and post-anoxia in red bell pepper studied by photoacoustic techniques.
Plant Physiology 113, 925–932.
Dessication - Chap 05 18/3/02 2:07 pm Page 147

Part III

Biology of Dehydration
Dessication - Chap 05 18/3/02 2:07 pm Page 148
Dessication - Chap 05 18/3/02 2:07 pm Page 149

5 Desiccation Sensitivity in Orthodox and

Recalcitrant Seeds in Relation to Development

Allison R. Kermode1 and Bill E. Finch-Savage2

1Department of Biological Sciences, Simon Fraser University, Burnaby, BC,
V5A 1S6, Canada; 2Horticulture Research International,Wellesbourne,
Warwick CV35 9EF, UK

5.1. Introduction 150

5.2. 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 155 Seed development in recalcitrant species 157 Time-dependent effects of storage and drying rate 159 Desiccation tolerance differs between seed tissues 160
5.2.6. Mechanisms underlying the acquisition of desiccation
tolerance: recent findings and speculations 161 The effects of premature desiccation during the
tolerant and intolerant stages of orthodox seed
development 161 Cellular and metabolic changes during the
transition to a desiccation-tolerant state 161 Desiccation-tolerance mechanisms in sensitive
seeds 170
5.3. Conclusions 174
5.4. References 175

© CAB International 2002. Desiccation and Survival in Plants: Drying Without Dying
(eds M. Black and H.W. Pritchard) 149
Dessication - Chap 05 19/3/02 3:56 pm Page 150

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

5.1. Introduction the embryo passes into a metabolically

inactive or quiescent state.
The development of most seeds can be The majority of seeds are referred to as
divided conveniently into three confluent ‘orthodox’, in which desiccation occurs as
stages (Fig. 5.1). During histodifferentiation, a pre-programmed and final stage in their
the single-celled zygote undergoes exten- development (Fig. 5.1). Seeds of the ortho-
sive mitotic division, and the resultant cells dox type and other desiccation-tolerant
differentiate to form the basic body plan of structures such as spores and pollen are
the embryo (the axis and cotyledons); con- unique in the degree of water loss toler-
currently, there is the formation of the ated; as much as 90–95% of the original
triploid endosperm or haploid megagameto- water is removed during their develop-
phyte. Thereafter, cell division ceases dur- ment. In this dehydrated state, the seed can
ing the seed expansion stage and there is survive the vagaries of the environment
cell expansion and the deposition of and, unless dormant, will resume full meta-
reserves (normally proteins along with bolic activity, growth and development
lipids or carbohydrates), primarily in the when conditions conducive to germination
storage tissues (i.e. cotyledons, endosperm are provided (Fig. 5.1). This chapter dis-
or megagametophyte). Finally, the develop- cusses some of the mechanisms underlying
ment of most seeds is terminated by some desiccation tolerance of seeds and recent
degree of drying (maturation drying), which approaches to elucidate the precise roles of
results in a gradual reduction in metabo- protective molecules and repair processes
lism as water is lost from seed tissues and at the cellular and subcellular levels.

Development Germination growth

Histodifferentiation Maturation Desiccation Dry


Cell division Reduced Quiescence Renewed Reserve

Cell expansion metabolism (Mature dry metabolism breakdown
seed) (respiration,
Reserve deposition nucleic acid and
protein synthesis)

Dormancy Cell elongation

(sometimes) Cell division

Desiccation-intolerant Desiccation-tolerant Desiccation-intolerant

Histodifferentiation Cell expansion Maturation drying Germination and growth

Fig. 5.1. Some events associated with seed development, germination and growth. (From Kermode, 1995.)
Dessication - Chap 05 18/3/02 2:07 pm Page 151

Desiccation Sensitivity in Relation to Seed Development 151

An important approach to elucidating age upon subsequent rehydration and an

the basis of desiccation tolerance in seeds inappropriate proportion or distribution of
is comparative analyses between seeds that freezable and non-freezable (bound) water
differ in their capacity to withstand water within the seed (Berjak et al., 1992;
loss, i.e. seeds of orthodox and recalcitrant reviewed in Bewley and Oliver, 1992;
plant species. ‘Orthodox’ seeds can be Vertucci and Farrant, 1995). Recent research
stored for long periods under conventional in this area will be discussed briefly but see
conditions, i.e. in the dry state and at low also Chapters 6–8 of this volume.
temperature. Recalcitrant seeds, on the
other hand, do not undergo maturation
drying, nor are they capable of withstand- 5.2. Development and Acquisition of
ing water loss of the magnitude of that Desiccation Tolerance
experienced by orthodox seeds. The seeds
are shed at relatively high moisture con- 5.2.1. Changes in water status during
tents and are highly susceptible to desicca- development of orthodox seeds
tion injury; in order to remain viable, they
must not undergo any substantial change The three major phases of seed develop-
in moisture. They are not storable under ment characteristic of orthodox seeds
conditions suitable for orthodox seeds and, (namely histodifferentiation, expansion
even when stored under moist conditions, and maturation drying; Fig. 5.1) are marked
their viability is frequently brief and only by distinctive changes in fresh weight, dry
rarely exceeds a few months (reviewed in weight and water content (Fig. 5.2). During
Chin and Roberts, 1980; Bewley and Black, histodifferentiation and early cell expan-
1994; Smith and Berjak, 1995; Vertucci and sion, there is a rapid increase in whole
Farrant, 1995; Berjak and Pammenter, seed fresh weight and water content.
1997; Pammenter and Berjak, 1999). Thus Generally, a period of rapid dry-weight
the terms ‘orthodox’ and ‘recalcitrant’ have gain follows (when whole seed fresh
been used to describe the storage behaviour weight is relatively stable); this takes place
of seeds. A category intermediate between during the later part of the seed expansion
orthodox and recalcitrant is now recog- phase of development. Most seeds lose
nized (e.g. coffee) in which seeds survive water during this phase as reserves are
desiccation but become damaged during deposited primarily within storage tissues,
dry storage at low temperatures (0°C and displacing water from the cells. This
20°C) (Ellis et al., 1990, 1991a). It is decline in water content slows as the seed
important to note, however, that the situa- approaches its maximum dry weight. Then,
tion is more complex and there is a gradual as the seed undergoes maturation drying
continuum of desiccation tolerance across and approaches quiescence, there is a
orthodox and recalcitrant species. period of fresh weight loss accompanied by
The question arises as to whether the a rapid decline in whole seed water con-
desiccation sensitivity of recalcitrant seeds tent (Kermode, 1990; Fig. 5.2).
is at least partially the result of an insuffi- Little is known about the mechanism and
cient accumulation of protective proteins, route of water loss from seeds. Some studies
or whether other factors (including a lack suggest the existence of a passive mecha-
of protective sugars) are more important. nism whereby water is lost primarily by
Since desiccation tolerance is arguably a evaporation from the surface of surrounding
quantitative feature (Vertucci and Farrant, seed structures (Nechiporenko and
1995), the amount of protective proteins, or Rybalova, 1983; Lee and Atkey, 1984;
the rate at which the proteins accumulate, Goncharova et al., 1985). Another suggestion
may determine the level of tolerance. is that water moves from the seed to the par-
Other features that may be part of the ent plant by a metabolically active process,
basis of desiccation sensitivity include an i.e. the plant actually ‘pumps’ the water from
inability to repair desiccation-induced dam- the seed (Meredith and Jenkins, 1975).
Dessication - Chap 05 18/3/02 2:07 pm Page 152

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

Expansion Maturation
Histodifferentiation (reserve deposition) drying





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 orthodox
seeds. Three major periods are noted: I, rapid fresh-weight gain; II, rapid dry-weight gain; III, fresh-weight
loss. (From Kermode and Bewley, 1986.)

In soybean and castor bean, the desicca- drying of seeds at a desiccation-tolerant

tion period is most probably initiated by stage of their development promotes germi-
the severing of the vascular supply to the nation upon subsequent rehydration. Air-
seed (funiculus detachment) and senes- dried grains of wheat not only germinate at
cence of the pod or capsule (Greenwood an earlier stage of development than non-
and Bewley, 1982; Murray and Nooden, dried grains, but at later stages may also
1986). This would suggest that relocation germinate at a faster rate than their non-
of water from the seed to the parent plant dried counterparts (Mitchell et al., 1980;
is not the means by which water loss Symons et al., 1983). Seeds of Phaseolus
occurs. Similarly, pectic substances in the vulgaris (French bean) undergo a transition
lumina of xylem elements of the rachis of to a desiccation-tolerant state around 26
wheat and barley (laid down during the DAP (days after pollination) approximately
final stages of grain maturation) may lead halfway through development (Dasgupta et
to the progressive dehydration of the ear by al., 1982). Seeds at 26–32 DAP can be
cutting off its water supply (Cochrane, induced to germinate to increasing extents
1985). if first dried over silica gel, whereas those
dried at 22 DAP fail to germinate when
subsequently rehydrated and they eventu-
5.2.2. Acquisition of desiccation tolerance ally deteriorate. The 22 DAP seeds do not
during development of orthodox seeds recover their full cellular and metabolic
integrity following the premature drying
Seeds of orthodox plant species cannot tol- treatment. At later times of development,
erate drying at all stages of their develop- however, the seeds acquire a tolerance of
ment. During very early development, seeds desiccation and also germinability is
are generally intolerant of drying, but they induced.
later undergo a transition to a desiccation- A similar situation exists for the castor
tolerant state at a particular time (reviewed bean seed (Ricinus communis) (Kermode
in Kermode, 1990, 1995). In many cases, and Bewley, 1985). Here, germinability is
Dessication - Chap 05 18/3/02 2:07 pm Page 153

Desiccation Sensitivity in Relation to Seed Development 153

not achieved until 50–55 DAP, whereas pre- (brought about by air-drying to 10% water
mature drying will promote the germina- content) during the course of germination,
tion ability of seeds as young as 25 DAP. At while the cotyledons remain tolerant for a
earlier stages of development, drying not considerably longer period (Senaratna and
only fails to induce germination ability, but McKersie, 1983).
also kills the seed. For both P. vulgaris and As will be discussed below, changes that
R. communis, the seeds acquire a tolerance occur on dehydration of the most
of desiccation and an ability to be potenti- desiccation-sensitive seeds (e.g. those of the
ated to germinate by this treatment around mangrove, Avicennia marina) can be very
25 days after development commences. similar to changes brought about by desic-
The transition to a desiccation-tolerant cation of orthodox seeds during the intoler-
state approximately midway through ant stage following germination (Farrant et
development is also characteristic of other al., 1986). Some recalcitrant seeds initiate
seeds, e.g. soybean, maize, barley, germination-related metabolism shortly
Agrostemma githago (Adams and Rinne, after shedding (reviewed in Vertucci and
1981; deKlerk, 1984; Bartels et al., 1988; Farrant, 1995) and, in A. marina, 10–15
Bochicchio et al., 1988). Tolerance of desic- days before shedding (Farrant et al., 1993b).
cation is gained over only a few days of As germination events progress, the seeds
development (e.g. between 20 and 25 DAP become increasingly sensitive to drying and
in R. communis); it is achieved well before attempting to store these seeds is akin to
the completion of major developmental storage of germinated, orthodox seeds
events such as reserve deposition and the (Farrant et al., 1986, 1988). There is no
commencement of normal maturation dry- clear-cut event delineating the end of seed
ing (Kermode and Bewley, 1985; Kermode development and the start of germination;
et al., 1986) (Figs 5.1 and 5.3). during both phases, recalcitrant seeds
appear to remain metabolically active,
although the axes may undergo a very brief
5.2.3. Loss of desiccation tolerance following period of relative quiescence.
germination of orthodox seeds

During germination, seeds initially remain 5.2.4. Effects of the rate and extent of
tolerant of reimposed desiccation, but at desiccation on the acquisition of tolerance of
some stage after axis elongation this ability orthodox seeds
is lost (Fig. 5.1). Germinating soybean
seeds are tolerant of drying during the The rate at which drying is imposed during
early stages, up to 6 h after commencing early development is critical for the subse-
imbibition, but they become increasingly quent expression of germinability, and
intolerant after this time. Thus, desiccation thus, when it is stated that a seed acquires
at 36 h after the start of imbibition kills the a tolerance of desiccation at a particular
seed (Senaratna and McKersie, 1983). The stage during its development, it is neces-
plasma membrane appears to be a major sary to define the rate of water loss to
site of damage in seeds during the desicca- which it is subjected (see Chapters 2 and
tion intolerant stage of germination, as 3). Whole seeds of several legumes (Adams
indicated by ultrastructural studies et al., 1983; Ellis et al., 1987) and R. com-
(Crevecoeur et al., 1976) and by the munis (Kermode and Bewley, 1985) are
increased solute and electrolyte leakage unable to withstand rapidly imposed dry-
upon subsequent rehydration (Senaratna ing (over silica gel or under regimes similar
and McKersie, 1983). There appears to be a to ambient laboratory conditions) at early
differential sensitivity of different seed tis- stages (i.e. during most of development,
sues with respect to the loss of desiccation prior to maturation drying) and exhibit no
tolerance. For example, axes of soybean germinability upon subsequent rehydra-
rapidly lose their tolerance to desiccation tion. This contrasts with seeds at the same
Histodifferentiation Cell expansion Maturation drying Germination/growth

Dessication - Chap 05

Initiation of High rate of Cessation of

storage protein storage protein storage protein
synthesis synthesis synthesis Germination Induction of
and post- LEA protein
Inhibition of Initiation of Termination of germinative synthesis

germination LEA protein development growth

synthesis Inhibition of
Adjustment to reserve
Inhibition of drying breakdown
germination +
2:07 pm

Preparation for
+ germination

+ +
+ +
Page 154

Desiccation ABA Water

‘Vascular stress
ABA High
factors’ osmolarity
A.R. Kermode and B.E. Finch-Savage

plant Water
stress –
– Precocious

Fig. 5.3. Events during the development and germination/growth of seeds that are affected by desiccation, high osmolarity or abscisic acid (ABA). Desiccation
tolerance is generally acquired by seeds around mid-maturation, when late embryogenesis abundant (LEA) proteins and other protective substances are synthesized.
Induction of a subset of LEA proteins also occurs following the transition to germination and growth, i.e. in seedlings and plant vegetative tissues when they are
subjected to water-deficit-related stresses. (From Kermode, 1995.)
Dessication - Chap 05 18/3/02 2:07 pm Page 155

Desiccation Sensitivity in Relation to Seed Development 155

stage of development dried slowly over sat- occurs during the first 55 days of develop-
urated salt solutions or air-dried while ment of R. communis seeds, although these
enclosed in the pod, where full germinabil- seeds can tolerate slow drying at stages as
ity is evident. Tolerance of rapid drying early as 25 DAP (Kermode and Bewley,
generally occurs only at or near the com- 1985). Seeds (and isolated embryos) of some
pletion of reserve deposition (as indicated members of the Gramineae, on the other
by the attainment of maximum dry weight) hand, can survive and germinate following a
just after the onset of natural drying drastic drying treatment (which reduces
(Rogerson and Matthews, 1977; Kermode their water content to around 5%) at rela-
and Bewley, 1985; Ellis et al., 1987), tively early stages of development (Bartels et
although there are exceptions (see subse- al., 1988; Bochicchio et al., 1988). The rea-
quent discussion). sons for the variation between species in the
Gradual water loss may allow protective rate of water loss tolerated during their
changes to occur and hence increase the development are not known.
seed’s resistance to disruption by dehydra-
tion. Rapid drying presumably would not
allow such protective changes to take place 5.2.5. Variation in desiccation tolerance
and may cause considerable disruption to across species
cellular membranes and internal structures (see also Chapter 8)
(see Section 5.2.6). As a result, the seed
requires time for metabolic readjustment A wide range of species growing in differ-
(i.e. repair) following rapid drying. This ent habitats produce seeds that cannot sur-
cannot take place during drying itself vive drying to the low moisture content
because the seed reaches a critical dry (and that enables prolonged storage of orthodox
quiescent) state before the repair processes seeds. These species are spread widely
can be initiated. Such repair is also through the plant kingdom and updated
impeded upon imbibition because of a too- lists continue to be produced (Chin and
rapid influx of water, which cannot be Roberts, 1980; Hofmann and Steiner, 1989;
accommodated by the weakened or dam- Hong et al., 1996). Since Roberts (1973)
aged structural components of the cell. In introduced the terms orthodox and recalci-
fact, rapid rates of drying may predispose trant, it has become clear that the degree to
seeds to imbibitional injury, as indicated which seeds can survive desiccation varies
by increased rates of solute leakage, a greatly both within and between non-
symptom of cellular membrane disruption. orthodox species. The moisture contents to
However, slowing the rate of hydration which seeds of different species can sur-
may prevent a loss of germinability of vive dehydration ranges from just less than
rapidly dried seeds by allowing time for that of vegetative tissues to almost com-
repair to occur. Germinability of soybean plete tolerance. A recent study has shown a
seeds (following an accelerated ageing continuous scale of desiccation tolerance
treatment) is increased from 10% to 90% across 64 orthodox and recalcitrant
by controlling the rate of imbibition species, with critical water contents for
(Tilden and West, 1985). Since seeds survival ranging from 0.1 to > 1.2 g g1 dry
become capable of surviving rapid water weight (Sun, 1999). Even in genetically
loss during later development, they must related species there is wide variation in
acquire a greater cellular and metabolic desiccation tolerance at shedding, for
resistance to this event or have an example, within the genera Shorea, Hopea
enhanced ability to effect repair during the and Dipterocarpus (Tompsett, 1987), Citrus
early stages of imbibition. and Quercus (Sun, 1999) and Coffea
The capacity to withstand rapid or slow (Dussert et al., 1999). Species within the
desiccation during the tolerant phase of genus Acer (Hong and Ellis, 1990; Dickie et
orthodox seed development varies between al., 1991; Fig. 5.4) produce orthodox and
species. An intolerance of rapid desiccation recalcitrant seeds.
Dessication - Chap 05 18/3/02 2:07 pm Page 156

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

The restrictive categorization of seeds Consideration of variation in the desicca-

introduced by Roberts (1973), particularly tion tolerance of seeds across species as a
with the introduction of an intermediate continuum of behaviour in response to dry-
category (Ellis et al., 1990, 1991a,b), ing is arguably more realistic (Farrant et
remains useful in so far as it fulfils its origi- al., 1988; Berjak and Pammenter, 1997;
nal purpose to describe storage behaviour. Pammenter and Berjak, 1999). Such varia-
However, it does not accurately reflect tion, while tedious to categorize, provides
knowledge of seed response to desiccation. an opportunity to increase our understand-
Seed dry weight

Norway maple
Seed moisture content (%)





Seed development


Germination (%)


40 Germination Germination
before drying after drying


August September October November

Seed development

Fig. 5.4. Seed development on adjacent trees of orthodox Acer platanoides (Norway maple) and recalcitrant
Acer pseudoplatanus (sycamore). Adapted from data in Hong and Ellis, 1990 and Dickie et al., 1991.
Dessication - Chap 05 18/3/02 2:07 pm Page 157

Desiccation Sensitivity in Relation to Seed Development 157

ing of the basis of desiccation tolerance, known what proportion of this variation is
which has only just begun to be exploited. genetic in origin or due to the environ-
A number of studies have shown that ments in which seed development, storage
desiccation-sensitive seeds do not pass or drying occurred. Temperature of drying
through a fully desiccation-tolerant phase can alter tolerance to desiccation (Ellis et
during their development, but tolerance al., 1990, 1991a; Berjak et al., 1994). Rate
tends to increase to a maximum near the of drying and the extent of storage before
time of shedding as moisture content drying are also important time-dependent
declines (Figs 5.4 and 5.5; reviewed by factors that could alter the extent of metab-
Finch-Savage, 1996; Berjak and olism-induced damage accumulated dur-
Pammenter, 1997). Thus, the extent of ing desiccation (Pammenter and Berjak,
seed maturity at harvest is one of a num- 1999). Such damage would alter the seed’s
ber of factors, discussed below, that deter- inherent capacity for desiccation tolerance
mines the degree of desiccation tolerance and may obscure discrete critical water
observed in sensitive seeds (e.g. as deter- potentials for survival. A further compli-
mined by critical water content experi- cating factor is that the onset of metabo-
ments). The increase in desiccation lism leading to the completion of
tolerance as water content declines during germination is often observed in recalci-
development (Fig. 5.5) and the apparent trant seeds following harvest or natural
continuous range of critical moisture con- shedding (Farrant et al., 1988; Pammenter
tents observed across species suggests that and Berjak, 1999), and this is known to
desiccation tolerance is a quantitative fea- progressively increase sensitivity in toler-
ture. However, it is more accurate to ant species (Section 5.2.3).
express the degree of desiccation tolerance
in terms of water potential as this reflects Seed development in recalcitrant
the amount of water available to the cyto-
plasm (see Chapter 2). When data are pre-
sented in this way, there is convincing With the exception of A. marina, the phys-
evidence that tolerance appears to be iology of seed development following ini-
acquired in discrete water potential steps tial histodifferentiation has strong
during development (Farrant and Walters, similarities across the recalcitrant species
1998), and the tolerance of species may be so far studied in detail (reviewed by Finch-
grouped according to these steps (Walters, Savage, 1996; Berjak and Pammenter,
1999; see Chapter 9), though in other stud- 1997). This general pattern of recalcitrant
ies this was thought unlikely (Sun, 1999). seed development is also similar to that of
It is argued that these critical water poten- orthodox seeds before they reach maxi-
tials, which have different water activities mum dry weight (mass maturity) and
and associated metabolic processes rapidly lose water following vascular sepa-
(reviewed by Vertucci and Farrant, 1995; ration (Finch-Savage, 1996; Farrant et al.,
see also Chapters 2 and 9), may be related 1997). For example, when adjacent trees of
to specific desiccation stresses and dis- the sympatric species Acer pseudoplatanus
crete patterns of gene expression (Walters, (recalcitrant) and Acer platanoides (ortho-
1999). Thus, critical water potentials may dox) are compared, there is a strong tempo-
be determined by specific tolerance mech- ral correlation in developmental events
anisms or by sufficient accumulation of such as the accumulation of seed reserves
desiccation protectants. and the development of germinability (Fig.
Within a species, seed tolerance of des- 5.4). However, there are a number of com-
iccation can vary according to provenance mon characteristics of recalcitrant seed
even to the extent that one species, neem development that contrast with those of
(Azadirachta indica), has been described orthodox seeds and which result in seeds
as both orthodox and recalcitrant (Berjak adapted for rapid germination and estab-
and Pammenter, 1997). However, it is not lishment. In general, recalcitrant seeds at
Dessication - Chap 05 18/3/02 2:07 pm Page 158

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

Seed development

70 Reserve accumulation




Moisture content at 50% viability (%)


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 content
at 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) is
fitted to 1989 data (r 2 = 0.937, d.f. 5). (From Finch-Savage and Blake, 1994.)

shedding have had almost no net loss of 1993; A. pseudoplatanus, Hong and Ellis,
water; they can still be accumulating dry 1990). In contrast, viviparous germination
weight, they retain active metabolism, is a common event in other tropical recal-
remain desiccation-sensitive and have no citrant species such as Telfairia occiden-
requirement for desiccation to stimulate talis (Akoroda, 1986) and A. marina
subsequent germination. Despite these (Farrant et al., 1993b).
apparent adaptations for rapid germina- During development, tolerance to desic-
tion, a few temperate recalcitrant species cation increases throughout reserve accu-
are dormant at shedding (Aesculus hip- mulation as percentage moisture content
pocastanum, Tompsett and Pritchard, decreases in most recalcitrant seeds as it
Dessication - Chap 05 18/3/02 2:07 pm Page 159

Desiccation Sensitivity in Relation to Seed Development 159

does in orthodox species (Hong and Ellis, opment in recalcitrant species have not sig-
1990; Finch-Savage, 1996; Berjak and nificantly improved their desiccation toler-
Pammenter, 1997; Farrant et al., 1997). ance, suggesting inherent limitations to the
These changes are concomitant with a development of full tolerance.
reduction in vacuolar volume, the appear- A. marina will tolerate little drying and
ance of a semi-viscous state and other can be considered to be at the extreme sen-
changes associated with desiccation toler- sitive end of the tolerance continuum
ance (Farrant and Walters, 1998). However, across species; developmental age has lit-
in recalcitrant seeds there appears to be no tle influence on the desiccation sensitivity
clear end point to development. For exam- of seeds (Farrant et al., 1993b). In some
ple, seeds of Quercus robur are shed at dif- other species, maximum desiccation toler-
ferent moisture contents in different years ance is reached at a point before shedding
on the same tree and those shed with the (e.g. A. pseudoplatanus, Hong and Ellis,
lowest moisture content are most tolerant 1990) and tolerance may then subse-
to desiccation (Fig. 5.5; Finch-Savage and quently decline (e.g. Litchi chinensis,
Blake, 1994). There is a linear relationship Clausena lansium and Coffea arabica)
between moisture content at harvest (pre- (Ellis et al., 1991a; Fu et al., 1994). This
mature and at shedding) and the moisture decrease in tolerance may be due to the
content at which 50% of seeds remain initiation of germination (Farrant et al.,
viable during drying. This contrasts with 1988; Hong and Ellis, 1992). A gradual
orthodox species, where desiccation toler- decrease in tolerance is then shown as ger-
ance continues to increase after the acqui- mination proceeds in desiccation-sensitive
sition of maximum seed dry weight, during seeds (Farrant et al., 1988) as it does in
maturation drying, which results in a qui- orthodox seeds (Hong and Ellis, 1992).
escent seed (Sanhewe et al., 1996). In Q.
robur, development is indeterminate, but Time-dependent effects of storage
consistent in several respects with that of
and drying rate
orthodox seeds shed early before mass
maturity and therefore before full desicca- Many recalcitrant seeds are characteristi-
tion tolerance is acquired (Finch-Savage cally large and consequently dry slowly.
and Blake, 1994). It is therefore tempting to But even when the seeds are of similar size
suggest that the level of desiccation toler- to comparable orthodox seeds, such as in
ance may depend upon how far seeds of a A. pseudoplatanus (recalcitrant) and A.
species progress through development, platanoides (orthodox), the recalcitrant
possibly an evolutionary consequence of Acer takes 12 times as long to reach 20%
the environmental and selection pressures moisture content under the same drying
that were exerted on them in the past. conditions (Greggains et al., 2000a). A fur-
These ideas are taken further in a compari- ther characteristic of recalcitrant seeds is
son of development in orthodox P. vulgaris that even in the absence of desiccation they
seeds, and the recalcitrant seeds of A. hip- deteriorate rapidly and are therefore short-
pocastanum and A. marina (Farrant et al., lived. Differences in the reported critical
1997). One possibility in temperate cli- water contents for recalcitrant seeds can be
mates is that the onset of winter truncates greatly influenced by these factors, and the
seed development so that full tolerance threshold water potentials for a number of
does not develop. However, seeds of Q. other physiological processes can be highly
robur produced on trees grown outside the dependent on the postharvest history of the
temperate climate zone in the apparently seed (Tompsett and Pritchard, 1998). A
non-limiting environment and season common observation is that if seeds are
length of South Africa also failed to pro- stored before drying they become more
duce desiccation-tolerant seeds (Finch- desiccation-sensitive (Finch-Savage et al.,
Savage and Blake, 1994). In addition, 1996). This may be because of damage
experimental manipulations of seed devel- accumulated during storage (Greggains et
Dessication - Chap 05 18/3/02 2:07 pm Page 160

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

al., 2000b) or because storage has allowed (Vertucci et al., 1991). It appears from this
the initiation of germination (Farrant et al., and similar published work that desicca-
1985; Berjak et al., 1989). However, in tion sensitivity in recalcitrant seeds and
some other species, particularly in seeds excised embryonic axes can occur at a min-
shed early, development may continue so imum of two levels, which are influenced
there is a considerable delay before the by the rate of drying:
initiation of germination (Berjak and
1. The removal of freezable water is toler-
Pammenter, 1997).
ated and minimum survivable moisture
Drying rate can affect the apparent toler-
content coincides with the quantity of non-
ance of whole seeds (Farrant et al., 1985;
freezable (matrix-bound) water in the tis-
Pritchard, 1991; Pammenter et al., 1998,
sue. Farrant et al. (1988) suggested that
1999; Chapter 3), although this is not
recalcitrant seeds, unlike orthodox seeds,
always the case (Finch-Savage, 1992). In
require this bound water for the mainte-
embryonic axes, as discussed below, rapid
nance of membrane integrity.
drying consistently improves their survival
2. Seed viability is lost as freezable (free)
to lower moisture contents, perhaps
water is removed.
because there is less time for damage to
accumulate during drying. However, The former situation usually occurs in
Pammenter and Berjak (1999) pointed out rapidly dried embryonic axes (Berjak et
that rapid drying does not confer improved al., 1992), but has also been reported in
desiccation tolerance because axes that relatively desiccation-tolerant recalcitrant
have been rapidly dried to lower moisture seeds (Finch-Savage, 1992; Finch-Savage
contents do not survive long under ambi- et al., 1993). The second situation occurs
ent conditions. in more sensitive recalcitrant species. A
third level of sensitivity, which is unaf-
fected by drying rate, is thought to occur Desiccation tolerance differs
in the most sensitive seed: mechanical
between seed tissues
damage resulting from a reduction in cell
In general, when isolated, the embryonic volume in the early stages of drying
axis of desiccation-sensitive species is (Pammenter and Berjak, 1999). In all
more tolerant than when it is dried in the cases, solute leakage precedes viability
whole seed (Berjak et al., 1990; Pammenter loss during desiccation, suggesting that
et al., 1991; Leprince et al., 1999), which significant membrane damage has
may result from its more rapid drying occurred. These differences in critical
when excised than when in situ (Berjak et moisture levels are consistent with the
al., 1990). It may also relate to a signifi- concept of discrete water potential steps
cantly smaller proportion of ‘matrix-bound’ associated with desiccation tolerance
(Finch-Savage, 1992; Finch-Savage et al., (Farrant and Walters, 1998; Walters, 1999)
1993) or non-freezable water (water that is and may be related to the different desic-
bound or structure-associated; Berjak et al., cation stresses encountered. In species
1992) in the axis compared to storage tis- where loss of viability appears to coincide
sues. Desiccation tolerance of Landolphia with removal of non-freezable water
kirkii axes is also affected by developmen- (Berjak et al., 1992; Finch-Savage, 1992),
tal status and, in contrast to the whole it may be that these seeds lack mecha-
seed, immature axes are more tolerant than nisms required to stabilize membranes as
mature ones (Berjak et al., 1992) as a result water is removed. Where viability is lost
of a lower content of non-freezable water in as freezable water is removed, other
the immature axes. Despite their greater mechanisms may be limiting, such as the
desiccation tolerance, the immature axes, provision of adequate protection against
unlike mature axes, do not survive expo- free-radical attack. The provision of puta-
sure to very low temperatures and are tive protective mechanisms in seeds is
therefore unsuitable for cryopreservation reviewed in the following section.
Dessication - Chap 05 18/3/02 2:07 pm Page 161

Desiccation Sensitivity in Relation to Seed Development 161

5.2.6. Mechanisms underlying the ment), but the physical nature of such
acquisition of desiccation tolerance: recent changes is enigmatic. While drastic
findings and speculations changes to membranes have been observed
upon their drying in vitro (Crowe et al., The effects of premature desiccation 1992), the evidence suggests that mem-
during the tolerant and intolerant stages of branes (in the desiccation-tolerant state)
orthodox seed development appear to be protected against certain
major alterations (e.g. transformation of the
Premature desiccation during the early
bilayer arrangement to hexagonal-type
developmental stages of P. vulgaris (up to
arrangements). The importance of a preser-
22 DAP, i.e. during the desiccation-sensi-
vation of basic membrane composition dur-
tive stage) drastically reduces the metabolic
ing drying is obvious, for cells would
and cellular integrity of the axis upon sub- surely perish without the prompt re-estab-
sequent rehydration. Particularly evident is lishment of functioning membranes upon
a loss in the capacity to recover polyribo- rehydration when they are challenged by a
some levels and to resume protein synthe- swiftly changing hydration environment.
sis (Dasgupta et al., 1982). Considerable Fourier transform infrared microspec-
damage is inflicted upon cellular organelles troscopy has been useful for elucidating
(including protein bodies and mitochon- some of the changes to membranes and
dria) and upon the nuclear membrane. In other cellular constituents (e.g. proteins) fol-
contrast, such severe perturbations do not lowing desiccation at the tolerant and sensi-
occur following desiccation at a tolerant tive stages of seed development (Wolkers et
stage, e.g. at 32 DAP. Moreover, the limited al., 1998b, 1999; see Chapter 4). Isolated
damage that is sustained during drying at immature maize embryos acquire a toler-
this stage is rapidly reversed following ance to rapid drying between 22 and 25
rehydration; cells regain their normal DAP, but can tolerate slow drying from 18
appearance within a very short time. DAP onwards. Rapid drying at the tolerant
Studies on the effects of desiccation stages is associated with lower membrane
during the sensitive stages of seed develop- permeability upon rehydration in contrast
ment (or germination) suffer the limitation to embryos rapidly dried at a sensitive stage,
of not distinguishing between the causes of in which there is an almost complete loss of
desiccation intolerance and changes during membrane integrity. In addition, there is a
the death of cells as a consequence of greater proportion of -helical protein struc-
undergoing desiccation. Nevertheless, a tures in embryos rapidly dried at a tolerant
few of these studies (particularly those that versus an intolerant stage (Wolkers et al.,
have compared the effects of drying at the 1998b). The proportion of -helical protein
sensitive and tolerant stages) have pro- structures increases in the axes of embryos
vided some useful information on the cel- during slow drying of 20 and 25 DAP seeds
lular sites and/or metabolic processes that (as compared with that within fresh devel-
are most susceptible to damage during des- oping seeds at these stages), and this factor
iccation/rehydration, and hence require coincides with the acquisition of additional
protection for retention of viability (see tolerance of desiccation.
Chapters 9 and 12). As implied earlier, the
integrity of membranes in seeds is of cru-
cial importance to the maintenance of via- Cellular and metabolic changes
bility; any undue disruption of the during the transition to a desiccation-tolerant
membrane systems during drying is likely state
to be of immediate consequence once the
seed imbibes. It is probable that some DEHYDRINS AND LATE EMBRYOGENESIS ABUNDANT
changes in membrane structure are pro- PROTEINS ARE PRODUCED AS PART OF THE DEVELOP-
voked as a consequence of desicccation MENTAL PROGRAMME. As noted earlier,
(even during the tolerant stages of develop- orthodox seeds are not capable of with-
Dessication - Chap 05 18/3/02 2:07 pm Page 162

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

standing desiccation at all stages during glycine content and a high hydrophilicity
their development, but their potential index) also accumulate in Escherichia coli
acquisition of tolerance is usually sub- and in the yeast Saccharomyces cerevisiae
stantially earlier than the onset of the nat- as an adaptive response to hyperosmotic
ural drying event itself. A highly conditions; the authors suggest that most
abundant set of hydrophilic proteins LEA proteins are part of a more wide-
exhibiting temporal regulation during spread group that they term ‘hydrophilins’
seed development (i.e. the late embryoge- (Garay-Arroyo et al., 2000).
nesis abundant (LEA) proteins first A subset of the LEA proteins (including
described in cotton) has been implicated the LEA D-11 family and some denoted
in desiccation tolerance (Dure, 1993; see RAB (responsive to abscisic acid (ABA)) in
also Chapters 1, 10 and 11). The genes rice) have been termed dehydrins; they
encoding these proteins arise as highly exhibit some common features in their
coordinately regulated sets, which on this structure that may be important for their
basis comprise two distinct classes in cot- putative protective function (Close, 1996).
ton (Hughes and Galau, 1989); the mRNAs Dehydrin genes exhibit a flexible expres-
that correspond to the two classes peak sion repertoire, being responsive to both
just prior to, or during, desiccation developmental and environmental cues
(Hughes and Galau, 1989). LEA protein (reviewed by Thomas et al., 1991).
synthesis constitutes a large proportion of Transcription of these genes is also
the translational activity of the cotton induced in virtually all seedling tissues
embryo during late maturation (up to subjected to water stress (i.e. non-lethal
25%), regulated at the level of transcrip- desiccation). Thus, the protective role of
tion, i.e. by the abundance of lea mRNAs dehydrins in the survival of water loss is
(Hughes and Galau, 1987). In mature cot- purported to be dual: during maturation
ton embryos they comprise about 2% of drying of the developing seed and follow-
the total soluble protein (Dure, 1993) or ing germination/growth of the mature seed
about 30% of the non-storage protein moi- (i.e. in seedlings or plant vegetative tissues
ety (Hughes and Galau, 1987). undergoing mild water stress) (Fig. 5.3).
Since their description in cotton, mes- Precocious appearance of the proteins and
sages homologous to the lea cDNAs of cot- their mRNAs can be induced in cultured
ton (representing at least five conserved immature embryos by exogenous ABA
families of corresponding proteins) have treatment. It has been hypothesized that,
been found in abundance in mature dry during normal development, high levels of
embryos and storage organs of many ABA induce the accumulation of these
diverse plant species including polypeptides and hence prepare the
Arabidopsis thaliana, several crop species embryo for desiccation or possible cellular
and gymnosperms. Protein families related disruption upon subsequent rehydration
(reviewed by Kermode, 1990, 1995; Bray,
to some of the LEA proteins are induced
1991, 1993; Bewley and Oliver, 1992;
during drying of xerophytic species, e.g.
Chandler and Robertson, 1994; Ingram and
the desiccation-tolerant resurrection plant
Bartels, 1996).
(Craterostigma plantagineum), which is
capable of surviving in the desiccated state REGULATION OF DEHYDRIN AND LEA GENE EXPRESSION
for long periods and resumes full physio- BY ABA. Since ABA has been implicated as a
logical activity within several hours of mediator of stress responses, especially
rehydration (Bartels et al., 1990; where water stress is concerned, its poten-
Piatkowski et al., 1990; reviewed in tial role as the primary regulator of lea
Ingram and Bartels, 1996; see Chapter 11). genes in vegetative tissues has been inves-
The desiccation-related proteins accumu- tigated (reviewed by Bray, 1991; Chandler
late in leaves; some are also present within and Robertson, 1994; Kermode, 1995;
roots and in seeds. Proteins sharing fea- Ingram and Bartels, 1996; Plant and Bray,
tures with plant LEA proteins (e.g. a high 1999). In some cases (but not all) ABA
Dessication - Chap 05 18/3/02 2:07 pm Page 163

Desiccation Sensitivity in Relation to Seed Development 163

application stimulates the accumulation of of maize has been undertaken in order to

the mRNAs in the absence of water stress, elucidate the possible regulatory role of
and there is some evidence that endoge- ABA and to address whether discrete paral-
nous ABA plays a regulatory role in their lel ABA and stress response pathways exist
expression in seedling tissues (Fig. 5.3). in developing maize embryos (Finkelstein,
For example, there is excellent correlation 1993). However, substantially different
(e.g. in barley and maize) between the results have been obtained depending on
amounts of mRNA and ABA in shoots, the type of LEA protein under study and
roots and aleurone layers from either well- more systematic and detailed investigation
watered, dehydrated or dehydrated/rehy- is needed. Several 23- to 25-kDa proteins
drated seedlings (Chandler et al., 1988; corresponding to RAB17 are expressed nor-
Gomez et al., 1988). Other stresses that mally in the ABA-deficient mutants of
lead to increased endogenous ABA (e.g. maize (vp-2 and vp-5) in contrast to the
salt, cold and wounding) are often also dependency on applied ABA for their
capable of eliciting expression of these expression in vegetative tissues of the
genes. The most convincing evidence for mutant seedlings (Pla et al., 1989). Likewise,
the role of ABA in dehydrin gene expres- the regulation of the Rab28 gene (a homo-
sion comes from studies of ABA-deficient logue of the cotton lea D34 gene) in excised
mutants of maize (Pla et al., 1989, 1991). young embryos of the ABA-deficient vp-2
When exposed to dehydration stress, mutant closely resembles that found in non-
seedlings homozygous for mutations lead- mutant excised young embryos (Pla et al.,
ing to vivipary (e.g. vp2 and vp5) fail to 1991). In contrast, embryos of the ABA-
elevate ABA levels and show a correspond- insensitive mutant of maize (vp-1) do not
ing inability to produce dehydrins. Similar accumulate Rab28 transcripts to significant
results have been found in an ABA-defi- amounts during development; surprisingly,
cient mutant of tomato (Cohen and Bray, induction of Rab28 mRNA can be achieved
1990; reviewed by Bray, 1991). in these young vp-1 embryos by ABA treat-
What is the evidence that ABA plays a ment (Pla et al., 1991). Expression of the
central regulatory role in the expression of maize Em gene (a group 1 lea gene) may be
lea genes within the developing seed? dependent on both the presence of ABA
Generally, the mRNAs encoding LEA pro- within embryos and its perception via a
teins are detected in embryos around mid- functional Vp-1 gene product; it is barely
development; highest levels of expression detectable in the ABA-deficient mutant
occur either at incipient desiccation or embryos and is undetectable in the ABA-
during maturation drying itself (Gomez et insensitive vp-1 embryos. Nevertheless, vp-1
al., 1988; Mundy and Chua, 1988; Close et embryos do exhibit a response to both ABA
al., 1989). The mRNAs are preserved in and osmotica at the molecular level, since
the mature dry seed but are rapidly they accumulate specific gene products (22-
degraded upon imbibition, although, in and 30-kDa polypeptides) differentially
some cases, certain proteins persist follow- upon imposition of osmotic stress or exoge-
ing imbibition (Han et al., 1996). nous ABA (Butler and Cumming, 1993).
Precocious appearance of the proteins and The Vp-1 gene is thought to encode a
their mRNAs can be induced in cultured novel type of transcription activator, which
immature embryos by exogenous ABA. plays a role in the expression of ABA-
Thus, high levels of ABA during mid- responsive genes during seed development
development are thought to induce the (e.g. maize globulin and Em genes), similar
accumulation of these polypeptides and to the Abi-3 gene product in Arabidopsis
hence prepare the embryo for desiccation and other species (reviewed by Giraudat et
or possible cellular disruption upon subse- al., 1994; Hattori et al., 1995; McCarty,
quent rehydration (Fig. 5.3). 1995). The promoter elements of the rice
A comparative analysis of wild-type, Osem gene (an Em-type gene) required for
ABA-deficient and ABA-insensitive mutants regulation by VP-1 have been identified
Dessication - Chap 05 18/3/02 2:07 pm Page 164

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

(Hattori et al., 1995). These include 1 plantlets subjected to drought stress.

TACGTGTC (an ABA-responsive element or Thus, there may be two regulation path-
ABRE), a small sequence located just down- ways that mediate dehydrin transcript
stream of the ABRE and a quantitiative ele- accumulation in seeds and stressed vegeta-
ment (the sph box/RY repeat), conserved in tive tissues – an ABA-dependent pathway
many seed-specific gene promoters. and an ABA-independent pathway;
Accumulation of group 3 LEA proteins together, these pathways may have cumula-
in maturing maize embryos may be depen- tive effects (Giordani et al., 1999).
dent upon ABA but appears to have no Ectopic expression of the Abi-3 gene
specific requirement for the Vp-1 gene product (Giraudat et al., 1992) allows the
product (Thomann et al., 1992). ABA-mediated activation of lea genes in
Interestingly, when an ABA-deficient, vegetative tissues of A. thaliana (Parcy et
viviparous mutant of maize (vp-5) is al., 1994). Seed viability is not altered in
manipulated either genetically or via ABA-deficient (aba) and ABA-insensitive
biosynthesis inhibitors to induce gib- (abi-3) mutants of A. thaliana, yet seeds of
berellin (GA) deficiency during early seed double mutants exhibiting these two traits
development, vivipary is suppressed in do not undergo desiccation on the parent
developing kernels and the seeds acquire plant, are intolerant of artificial desiccation
desiccation tolerance and storage longevity and fail to produce some of the late abun-
(White et al., 2000). Major accumulation of dant proteins (Koornneef et al., 1989;
GA1 and GA3 occurs in wild-type maize Meurs et al., 1992). These double-mutant
kernels, just prior to a peak in ABA content seeds accumulate only low amounts of the
during development. It is speculated that major storage proteins and are deficient in
these GAs induce a developmental pro- several low-molecular-weight polypep-
gramme that leads to vivipary in the tides, both soluble and bound, some of
absence of normal amounts of ABA, and which are heat-soluble. During develop-
that a reduction of GAs re-establishes an ment (14–20 DAP), the low amounts of var-
ABA/GA ratio appropriate for suppression ious maturation-specific proteins are
of germination and induction of matura- degraded and proteins characteristic of ger-
tion. Induction of GA deficiency does not mination are induced, in the absence of
suppress vivipary in vp-1 mutant kernels, germination. Here, the seed developmental
suggesting that VP-1 acts downstream of programme is not completed, and there is a
both GA and ABA in programming seed premature (yet incomplete) switching to a
development (White et al., 2000). germination programme in the absence of
Two ABA-deficient mutants of sun- substances presumed to be protective
flower have been isolated – nd-1, an albino, against desiccation. Seeds become desicca-
non-dormant and lethal mutant exhibiting tion-tolerant when the plants are watered
a very low ABA content and no accumula- with an ABA analogue (LAB 173711) or by
tion of ABA in response to stress, and w-1, incubating isolated immature seeds (11–15
a wilty mutant, with reduced ABA accu- DAP) with ABA and sucrose. Whereas
mulation during embryo and plantlet sucrose may protect desiccation-sensitive
development and drought stress (Giordani structures from damage, ABA inhibits pre-
et al., 1999). The w-1 mutant exhibits a cocious germination and may be required
reduction of dehydrin transcripts in the for, or is accompanied by, completion of
early stages of embryo development as the seed developmental programme and
compared with wild-type embryos, indicat- associated acquisition of desiccation toler-
ing that ABA affects dehydrin accumula- ance (Meurs et al., 1992). In another study,
tion; however, the amount of dehydrin expression of a specific lea gene (a group 1,
transcripts appears to be independent of D19/Em homologue) was found to be
ABA content during late embryogenesis. slightly reduced in seeds of the
Accumulation of dehydrin transcripts Arabidopsis aba mutant, but was reduced
occurs in the leaflets and cotyledons of nd- by approximately tenfold in the abi-3
Dessication - Chap 05 18/3/02 2:07 pm Page 165

Desiccation Sensitivity in Relation to Seed Development 165

mutant; expression in the double mutant OTHER PROTECTIVE PROTEINS IMPLICATED IN DESICCA-
was not studied (Finkelstein, 1993). A dif- TION TOLERANCE. Specific small heat-shock
ferent Arabidopsis abi-3 mutant (abi-3-3, proteins (HSPs) of the cytosolic classes (I
isolated by screening for mutants that ger- and II) accumulate in seeds of several plant
minate in the presence of the GA biosyn- species. These proteins appear to be homo-
thetic inhibitor, Uniconazol) showed geneously distributed in all tissues of the
abnormal seed development, remaining seed and a role in the acquisition of desic-
green until maturity, had dramatically cation tolerance has been suggested (Coca
reduced amounts of storage proteins, was et al., 1994; Wehmeyer et al., 1996; see
desiccation-sensitive, and lacked dor- Chapters 1 and 10). In Arabidopsis and
mancy, indicative of a possible role for the other species, class I small HSPs are first
Abi-3-3 gene in the control of the synthesis detected during mid-maturation and
of seed storage proteins and desiccation become most abundant in dry seeds
protectants (Nambara et al., 1992). ABI5, a (Wehmeyer et al., 1996; Carranco et al.,
member of the family of basic leucine zip- 1999). In some seeds (e.g. Arabidopsis), the
per transcription factors, regulates a subset proteins decline rapidly during germination
of lea genes during seed development and (Wehmeyer et al., 1996); in others (e.g. sun-
in vegetative tissues in the presence of flower), they persist (Coca et al., 1994). The
ABA (Finkelstein and Lynch, 2000). Abi-3 gene product may activate expression
Further studies to clarify the role of of genes encoding specific small HSPs dur-
ABA and other components of the signal ing seed development. Transcriptional acti-
transduction pathway leading to lea gene vator mutants of Arabidopsis (abi-3-6,
expression and other late maturation fus3-3 and lec1-2) that are desiccation-sen-
events in developing seeds will be awaited sitive have undetectable amounts of HSP17.4
with interest. Recessive mutants of (abi-3-6) or highly reduced amounts of the
Arabidopis with lesions at the Fusca3 protein (fus3-3 and lec1-2), i.e. less than 2%
(fus3) and Leafy Cotyledon (lec1) gene loci of that in wild-type seeds (Wehmeyer and
lead to various abnomalities during mid- Vierling, 2000). Interestingly, a chimeric
embryogenesis and late embryogenesis, gene consisting of the small HSP gene pro-
including loss of dormancy and failure to moter linked to -glucuronidase (GUS)
acquire desiccation tolerance (Kirik et al., shows strong expression in mutant seeds
1998). FUS3 and LEC1 modulate the abun- that are heat-stressed, indicating that the
dance of ABI3 protein in seeds and syner- genes are under distinct developmental
gistic interactions between the three and stress regulation.
proteins (ABI3, FUS3 and LEC1) are Polypeptides produced in sunflower
thought to control various key events, seeds (e.g. HSP17.6 and HSP17.9, belong-
including accumulation of chlorophyll and ing to different families of cytoplasmic
anthocyanins, sensitivity to ABA and small HSPs) are indistinguishable from
expression of individual members of the low-molecular-weight HSPs expressed in
12S storage protein gene family (Parcy et vegetative tissues in response to water
al., 1997). Interestingly, part of FUS3 (a deficit, but they are different from homolo-
continuous stretch of 100 amino acids) gous proteins expressed in response to
shows significant similarity to the B3 thermal stress (Coca et al., 1994; Carranco
domain of the ABI3 and VP-1 proteins et al., 1997, 1999). Proteins immunologi-
(Luerssen et al., 1998), a domain which cally related to two sunflower small HSPs
interacts with the RY cis promoter motif of are detected in unstressed vegetative tis-
several seed proteins. Thus, both FUS3 and sues of the desiccation-tolerant resurrec-
ABI3 may be essential components of a reg- tion plant C. plantagineum and are
ulatory network acting in concert through induced to higher levels in these tissues
the RY-promoter element to control gene by water stress and heat shock. In desicca-
expression during late embryogenesis and tion-sensitive Craterostigma callus tissue,
seed development (Reidt et al., 2000). there are no detectable small HSP-related
Dessication - Chap 05 18/3/02 2:07 pm Page 166

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

polypeptides, but their expression, and the However, it is possible that the attainment
concurrent acquisition of desiccation tol- of a critical level of reserves is required
erance is induced by exogenous ABA before the seed can withstand desiccation
(Alamillo et al., 1995). (Kermode, 1997). Highly vacuolated cells
Small HSPs, immunologically related to (hence containing little reserve material)
a 20-kDa HSP from desiccation-sensitive may undergo severe mechanical disrup-
chestnut (Castanea sativa) seeds have been tion during water loss, and tearing or
detected in orthodox and recalcitrant seeds shearing of membranes (or other cellular
of 13 woody species; hence additional pro- components) could lead to irreversible
teins or mechanisms are likely to be changes in their internal morphology. The
involved in desiccation tolerance (Collada presence of a critical level of cellular
et al., 1997) (see later discussion). reserves would limit such changes (Table
Major intrinsic proteins (MIPs) are a 5.1). The quantity of reserves may also
family of channel proteins that are mainly merit consideration in relation to the loss
represented by aquaporins in plants. They of tolerance during seed germination. As
are generally divided into TIPs (tonoplast noted earlier, while soybean seed axes
intrinsic proteins) and PIPs (plasma mem- rapidly lose their tolerance to desiccation
brane intrinsic proteins) according to their during the course of germination, the
subcellular localization (reviewed by cotyledons remain tolerant for a consider-
Maurel et al., 1997). The vacuolar mem- ably longer period (Senaratna and
brane protein, -TIP (a water-channel pro- McKersie, 1983). The major breakdown of
tein), accumulates during seed maturation reserves within the cotyledons is a post-
in the parenchyma cells of seed storage germinative event; however, catabolism of
organs. Synthesis of this integral membrane reserves within the axes occurs relatively
protein does not appear to be related (in a early (i.e. during germination) to provide a
quantitative manner) to storage protein source of nutrients. The decline of
deposition and a role in seed desiccation, reserves below a critical level within the
cytoplasmic osmoregulation and/or seed axes may contribute to a loss of desicca-
rehydration has been suggested (Johnson et tion tolerance within this germinating tis-
al., 1989). The water-channel activity of the sue. Interestingly, certain seed storage
protein can be regulated by phosphoryla- proteins are suggested to play a more
tion and the protein assembles as a 60 Å  direct role in desiccation tolerance. One
60 Å square in which each subunit is member of the vicilin superfamily in pea
formed by a heart-shaped ring comprised of (psp54) is expressed during seed desicca-
-helices. This structure is remarkably sim- tion and is not detected prior to this stage;
ilar to that of mammalian PIPs, suggesting the mRNA encoding the protein declines
that the molecular design of functionally soon after imbibition, but can be detected
analogous and genetically homologous in vegetative tissues in response to water-
aquaporins is maintained between the plant deficit-related stresses and ABA (Castillo
and animal kingdoms (Daniels et al., 1999). et al., 2000). A lower-molecular-weight
In the desiccation-tolerant resurrection protein (p1), which corresponds to the C-
plant C. plantagineum, homologues to PIPs terminal third of p54, shares some proper-
and TIPs are regulated by dehydration and ties with dehydrins and is suggested to
ABA, with members of a subset of PIPs protect chromatin structure during desic-
(PIPa) being regulated by ABA-dependent cation (Castillo et al., 2000). Seed storage
and ABA-independent pathways (Mariaux globulins of spermatophytes are thought to
et al., 1998). have evolved from a group of ancient
In many seeds, the acquisition of desic- single-domain proteins of prokaryotes
cation tolerance during the seed expansion and fungi functional in cellular desicca-
stage of development occurs well before tion/hydration processes (Baumlein et al.,
the completion of reserve deposition. 1995; Shutov et al., 1998).
Table 5.1. Possible components of desication tolerance in seeds and their protective action.a (Based on Kermode (1990). With permission from
CRC Press, Inc.)
Site or process
affected Protective component Protective action Possible mode of protection
Dessication - Chap 05

Membranes Carbohydrates: sucrose Prevent changes in selective permeability Hydroxyl groups of sucrose replace water on
plus raffinose and/or due to lateral phase separation of hydrophilic (polar) end groups of membrane
stachyose phospholipids in the bilayer and the phase phospholipids; oligosaccharide (raffinose and/or
transition from liquid crystalline to gel stachyose) inhibits sucrose crystallization during

drying, preventing loss of its protective potential

Lipid-soluble antioxidants As above; prevent de-esterification of Scavenging activity increases resistance to
(e.g. tocopherols) membrane phospholipid and free fatty free-radical-mediated desiccation injury
acid accumulation
2:07 pm

Structure/metabolism Reserves: carbohydrates, Prevent ‘whole scale’ mechanical Critical level of reserves in vacuoles/storage bodies
lipids, proteins disruption of cellular components confers mechanical strength to whole cell
Prevent loss of tightly bound (‘vital’) water Water-binding capacity of cells enhanced with
necessary for structural and functional increased number of sorption sites
integrity of biomolecules
Page 167

Hydrophilic, denaturation- As above Water-binding capacity of cells enhanced with

resistant proteins (e.g. LEAs, increased number and strength of sorption sites;
other desiccation-inducible native conformation of protective molecules maintained
polypeptides) throughout drying; bind ions and thereby counteract
damaging effects of increasing ionic strengths of
cytosol during drying
‘Repair’ proteins, proteases, Rapid re-establishment of structural Efficient repair of membranes and other cellular
ubiquitin and extension and metabolic integrity following components restores normal functioning; aid
protein, HSP/molecular imbibition proteins in recovering their native conformation;
chaperones, some LEAs degradation of damaged or denatured proteins
Desiccation Sensitivity in Relation to Seed Development

aRefer to review by Kermode (1990) and Ingram and Bartels (1996) and references therein.
Dessication - Chap 05 18/3/02 2:07 pm Page 168

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

ROLE OF SUGARS (see also Chapters 1, 10 and humidity (Blackman et al., 1992). However,
11). It is likely that the underlying basis an increase in the amount of raffinose is
of desiccation tolerance is diverse and is not correlated with the acquisition of des-
not simply restricted to the synthesis of iccation tolerance of wheat embryos (Black
specific proteins. The ability to withstand et al., 1999). Accumulation of fagopyritol
desiccation may also depend upon B1 (a galactopinitol) in buckwheat seeds is
increased amounts (or a heightened capac- temporally associated with the acquisition
ity to synthesize) molecules which stabi- of desiccation tolerance during develop-
lize membranes. Carbohydrates such as ment (Horbowicz et al., 1998). This major
trehalose (a non-reducing disaccharide of soluble carbohydrate, which comprises
glucose) are effective in preserving the 40% of the carbohydrate of the mature
structural and functional integrity of mem- buckwheat embryo, declines with the loss
branes in vitro at low water contents of desiccation tolerance following germina-
(reviewed by Crowe et al., 1992). Drying tion. Temperature conditions during seed
and rehydration of the model membrane development that have a favourable effect on
sarcoplasmic reticulum usually results in vigour and storability of buckwheat seeds
the fusion of vesicles and loss of the ability result in seeds having a lower sucrose-to-
to transport calcium. However, when disac- fagopyritol ratio as compared with those
charides such as trehalose are present in that develop under non-optimal tempera-
concentrations equivalent to those in desic- ture conditions (Horbowicz et al., 1998).
cation-tolerant organisms, functional vesi- Although trehalose is not abundant in
cles are preserved. Membrane fusion vascular plants, it has been identified as a
during desiccation is thought to be pre- major carbohydrate in more than 70
vented as a result of the sugars’ hydroxyl species of desiccation-tolerant lower plants
groups interacting (i.e. forming hydrogen (reviewed by Muller et al., 1995). There are
bonds) with the polar head groups of phos- recent reports of trehalose in relatively
pholipids and functional groups of pro- high amount in two desiccation-tolerant
teins (Crowe et al., 1992). Thus, the sugars angiosperms (reviewed by Muller et al.,
are thought to alter physical properties of 1995; see references therein). One is
dry membranes so that they resemble those Myrothammus flabellifolia, a dicotyledo-
of fully hydrated biomolecules (Crowe et nous plant living in arid, rocky regions in
al., 1992) (Table 5.1). southern Africa, the leaves of which con-
The occurrence of trehalose in high con- tain about 3% trehalose on a dry weight
centrations in anhydrobiotic organisms basis. The other is the grass Sporobolus
such as yeast and nematodes (up to 20% of stapfianus, in which trehalose comprises
their dry weight) suggests that this sub- 2–5% of the total soluble carbohydrates.
stance may be involved in their desiccation Interestingly, even though most higher
tolerance (Crowe et al., 1992). A role for plants contain low amounts of trehalose,
sucrose and raffinose (carbohydrates found high activities of trehalase, an enzyme
in much greater abundance in seed tissues which degrades trehalose, have been found
than trehalose) in the preservation of mem- (reviewed by Muller et al., 1995). A.
branes during drying has been suggested by thaliana possesses genes for at least one of
Leopold and Vertucci (1986) and several the enzymes required for trehalose synthe-
others. Orthodox seeds accumulate consid- sis, trehalose-6-phosphate phosphatase
erable amounts of soluble proteins and sug- (Vogel et al., 1998).
ars throughout maturation, and these As noted above, the protective effect of
collectively may be important in the acqui- sugars probably extends to preventing irre-
sition of a desiccation-tolerant state (Amuti versible changes to proteins. For example,
and Pollard, 1977). Stachyose accumulates phosphofructokinase is a tetrameric
in immature soybean seeds subjected to enzyme, which is irreversibly denatured
slow drying, but does not increase signifi- during desiccation, dissociating into inac-
cantly when seeds are maintained at high tive dimers. However, the disaccharides
Dessication - Chap 05 18/3/02 2:07 pm Page 169

Desiccation Sensitivity in Relation to Seed Development 169

sucrose, maltose and trehalose stabilize the dried embryos cannot account for their
activity of the enzyme (in vitro) during dry- enhanced viability as compared with
ing (Carpenter et al., 1987). Although the rapidly dried embryos; however, enhanced
evidence from these experiments carried synthesis of LEA proteins embedded in the
out in vitro is convincing, the role of sugars glassy matrix may be a contributing factor
(in vivo) in protecting cells during water (Wolkers et al., 1999).
deficit and during desiccation of orthodox The proteins of maturation-defective
seeds remains to be elucidated. mutants of Arabidopsis (e.g. abi-3 and lec
One way sugars may protect the cell mutants) appear to be more susceptible to
during severe desiccation is by glass forma- denaturation during heating (Wolkers et
tion (see Chapter 10); in the presence of al., 1998a). Proteins in dry wild-type seeds
sugars a supersaturated liquid is produced do not denature at temperatures up to
with the mechanical properties of a solid 150°C; those of dry desiccation-sensitive
(Koster, 1991). Only sugar mixtures equiva- seeds (lec1-1, lec1-3 and abi3-5) denature
lent in concentration and composition to at 68, 89 and 87°C, respectively. In con-
those of desiccation-tolerant embryos are trast, in desiccation-tolerant seeds (abi3-7
able to form glass at ambient temperature and abi3-1), denaturation commenced
(Koster, 1991) and this ability has been above 120 and 135°C, respectively. The dif-
associated with retention of viability of ferential sensitivity of the seed proteins of
maize embryos (Williams and Leopold, the mutants to denaturation has been
1989). Glass formation has been suggested attributed in part to differences in molecu-
to prevent cellular collapse during desicca- lar packing density, which is higher in dry
tion and to promote a state of metabolic desiccation-tolerant seeds than in dry des-
quiescence by restricting diffusion of sub- iccation-sensitive seeds (Wolkers et al.,
strates and products within cells (Koster, 1998a).
1991). Carrot somatic embryos, when pre-
treated with ABA, are able to tolerate slow OTHER MECHANISMS UNDERLYING DESICCATION TOLER-
drying, but are still intolerant of rapid dry- ANCE. The loss of desiccation tolerance dur-
ing. This appears to be due in part to the ing germination of soybean seeds is not
extent of protein denaturation, which is associated with any compositional changes
greater after rapid drying (Wolkers et al., in fatty acids, but is correlated with a
1999). In contrast to slowly dried embryos, decline in the quantity of lipid-soluble
which form a glassy state at room tempera- antioxidants in the membrane (Senaratna et
ture, no clearly defined glassy matrix is al., 1985a,b). These antioxidants may con-
formed in rapidly dried embryos. The aver- tribute to the desiccation tolerance of axes
age strength of hydrogen bonding is less in during the early stages of germination by
rapidly dried versus slowly dried embryos, preventing changes in membrane fluidity
which may be indicative of less extensive caused by free-radical attack on phospho-
‘molecular packing’ in the former. Sucrose lipids in response to drying (Senaratna et
accumulates following rapid drying of al., 1985a,b). The transition to desiccation
embryos; following slow drying, the trisac- sensitivity following germination of pea and
charide umbelliferose is accumulated at cucumber seeds is accompanied by a desic-
the expense of sucrose. In phospholipid cation-induced imbalance of metabolism
model systems, both carbohydrates are able (i.e. increased emission of CO2 and fermen-
to form a stable glass with drying; they tation products such as acetaldehyde) in the
depress the transition temperature of dry radicle, which precedes loss of membrane
liposomal membranes to an equal extent as integrity (Leprince et al., 2000). Imbalanced
well as preventing leakage from dry lipo- metabolism is significantly reduced when
somes upon subsequent rehydration. sensitive axes are dried in 50% O2 instead
Likewise, both exhibit an equal capacity to of air and it is suggested that a balance
protect a desiccation-sensitive protein. between down-regulated metabolism and O2
Thus, increased umbelliferose in slowly availability is associated with desiccation
Dessication - Chap 05 18/3/02 2:07 pm Page 170

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

tolerance. Products resulting from imbal- by facilitating the conversion of abnormal

anced metabolism (e.g. acetaldehyde) dis- L-isoaspartyl residues to normal L-aspartyl
turb the phase behaviour of phospholipid residues (Mudgett and Clarke, 1994). A
vesicles and thus may aggravate membrane summary of some of the possible compo-
damage induced by dehydration (Leprince nents of desiccation tolerance in seeds is
et al., 2000). presented in Table 5.1.
Within the developing seed, the thiol-
requiring (1-cysteine) peroxiredoxin family DIFFICULTIES IN ASSESSING THE ROLE OF PROTECTANTS
of antioxidants may protect tissues (e.g. the IN DESICCATION TOLERANCE. As indicated
embryo and aleurone layer of cereals) from above, a wealth of information has been
reactive oxygen species during desiccation derived from the study of desiccation-toler-
and early imbibition (Haslekas et al., 1998; ant systems, such as seeds and resurrection
Stacy et al., 1999). PER1, a protein belong- plants, and from the various molecular and
ing to this family, is maintained in imbibed biochemical analyses that have contributed
dormant barley seeds, but declines in the to our understanding of gene and protein
non-dormant seeds. In immature embryos function. In orthodox seeds, the metabolic
and aleurone layers, the protein resides in changes that occur either prior to or during
the nucleus and is most abundant within maturation drying (including the accumu-
the nucleolus (Stacy et al., 1999). In con- lation of oligosaccharides, sugars and LEA
trast, in mature imbibed dormant seeds, an proteins) may have functional significance
equivalent amount of protein is present in in protecting them against the rigours of
the cytosol. In Arabidopsis, the expression desiccation and/or subsequent rehydration.
of AtPer1, a gene encoding a protein with However, some of the changes in metabo-
similarity to barley PER1, is reduced in lism of orthodox seeds during the time of
seeds of the ABA-insensitive mutant, acquisition of desiccation tolerance may
abi3-1, but is unaltered in an ABA-defi- not directly contribute to the ability to
cient mutant of Arabidopis (aba-1) withstand water loss, but rather may be
(Haslekas et al., 1998). pre-programmed changes that are part of
Serotonin accumulation in walnut other seed developmental programmes ulti-
cotyledons is thought to protect seeds from mately important for seedling survival.
toxic ammonia concentrations following What are other research strategies that may
seed desiccation (Schroder et al., 1999). contribute to our understanding of the
In conclusion, the basis of desiccation underlying basis of desiccation tolerance?
tolerance of developing seeds is still a One strategy is the transfer of genes encod-
poorly understood phenomenon. What ing putative desiccation protectants into
emerges from the evidence available at pre- transgenic host plants with the ultimate
sent is a complex process involving various goal of testing protein function and
metabolic and/or structural adjustments, enhancing stress tolerance (see Chapter
which allow cells to undergo extensive 11). Another approach is the comparative
water loss with a minimum of damage analysis of LEA- and dehydrin-related pro-
(Table 5.1). However, while maturation teins and other putative desiccation protec-
drying may inflict limited damage on cells tants in recalcitrant versus orthodox seeds
of orthodox seeds, the capacity to reverse (discussed below). Both approaches have
such changes (i.e. to effect repair) upon limitations.
subsequent rehydration is probably an inte-
gral feature of desiccation tolerance Desiccation-tolerance mechanisms
(Bewley and Oliver, 1992; Ingram and
in sensitive seeds
Bartels, 1996; O’Mahony and Oliver,
1999a,b). An L-isoaspartyl protein methyl- In the previous sections we have shown
transferase that accumulates in wheat that development of orthodox seeds fol-
seeds during the late stages of caryopsis lows a largely predetermined sequence of
development may repair damaged proteins events that leads to desiccation and then
Dessication - Chap 05 18/3/02 2:07 pm Page 171

Desiccation Sensitivity in Relation to Seed Development 171

shedding of the seed in a quiescent (and sidered to be particularly sensitive to des-

sometimes dormant) state. In evolutionary iccation. The pattern of ABA concentration
terms, it is not known whether the ability in seeds of the tropical wetland species, A.
to develop full desiccation tolerance has marina, during seed expansion differs from
been lost in species with recalcitrant seeds, that of other recalcitrant species; low and
was never gained, or is just not fully decreasing concentrations of ABA are pre-
expressed. The progress of evolution is also sent in the axis during reserve accumula-
likely to have differed among taxa (von tion (Farrant et al., 1993a). Moreover, ABA
Teichman and van Wyk, 1994; see Chapter concentration does not increase with dry-
8). Despite increasing interest in recalci- ing and dehydrin proteins are not detected
trant seeds, it is clear from recent reviews (Farrant et al., 1996). In general, the pres-
(Berjak and Pammenter, 1997; Pammenter ence of dehydrins in recalcitrant seeds is
and Berjak, 1999) that the cause of their associated with those species that have
desiccation sensitivity is still far from high ABA concentrations and are most
understood. However, comparison of likely to be exposed to moisture stress
desiccation-sensitive seeds with tolerant (Farrant et al., 1996). In addition, an earlier
orthodox seeds can clarify our understand- peak in ABA concentration of recalcitrant
ing of desiccation-tolerance mechanisms. Q. robur seeds is associated with greater
In the following section, the occurrence of desiccation tolerance at shedding (Finch-
these putative mechanisms in desiccation- Savage and Farrant, 1997). However, in T.
sensitive seeds is reported. cacao embryos in vitro, ABA is associated
with maturation events as it is in orthodox
ABSCISIC ACID AND PROTEINS. As noted in the seeds, but does not influence desiccation
previous sections, ABA may play a role in tolerance (Pence, 1992).
the regulation of dehydrin and lea gene In orthodox cotton, lea mRNAs that
expression in orthodox seeds. In the recal- have protein homology with dehydrins
citrant seeds of Theobroma cacao (Pence, accumulate relatively slowly during the
1991) and Q. robur (Finch-Savage et al., period of cotyledon expansion, but then
1992; Finch-Savage and Blake, 1994), there increase rapidly at the point of vascular
is a clear pattern of ABA accumulation separation (Galau et al., 1991). Recalcitrant
during seed expansion, similar to that seeds are often shed at a time when dry
reported in orthodox species, such as P. weight is still increasing and may therefore
vulgaris (Prevost and Le Page-Degivry, lack the phase of rapid increase in lea
1985a,b). In both the axis and cotyledons, mRNAs that occurs at the end of orthodox
ABA increases to a maximum and then seed development. Desiccation sensitivity
decreases before shedding. However, the may therefore be due in part to an inability
decline in ABA concentration prior to to accumulate a sufficient quantity of dehy-
shedding in recalcitrant seeds is limited drins or other LEA proteins.
and consistent with a continuing role for Small HSPs have also been associated
ABA in preventing precocious germination with desiccation tolerance in orthodox
(Finch-Savage and Farrant, 1997). seeds (see earlier discussion; DeRocher and
Dehydrin proteins accumulate during seed Vierling, 1994; Wehmeyer et al., 1996), and
development, and in response to seed dry- have been shown, like dehydrins, to accu-
ing, in a number of recalcitrant species mulate to significant levels in recalcitrant
(Finch-Savage et al., 1994; Gee et al., 1994; C. sativa seeds during development and to
Farrant et al., 1996; Han et al., 1997; be present in seeds of other recalcitrant
Greggains et al., 2000a). However, dehy- species (Collada et al., 1997). As discussed
drin proteins are not detected in mature earlier, the presence of dehydrin and small
undried axes of a range of recalcitrant trop- HSPs in recalcitrant seeds supports the
ical wetland species (Farrant et al., 1996). view that they alone are not sufficient to
These species would not normally be confer desiccation tolerance. However,
exposed to significant drying and are con- Farrant et al. (1996) suggested that the con-
Dessication - Chap 05 18/3/02 2:07 pm Page 172

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

verse might be true, i.e. their absence may sue in recalcitrant seeds tends to have a
imply an inability to tolerate desiccation, much lower oligosaccharide:sucrose ratio
and the absence of a specific protective than that generally present in orthodox
protein cannot be ruled out as the cause of seeds (Steadman et al., 1996). This ratio is
desiccation sensitivity. For example, the therefore a potential indicator of seed
membranes surrounding oil bodies of seeds behaviour; however, seeds of cocoa and A.
contain integral proteins, called oleosins, marina are exceptions (Steadman et al.,
which may maintain the integrity of these 1996). Pammenter and Berjak (1999)
organelles during desiccation and subse- pointed out that the proposed mechanisms
quent imbibition (Murphy et al., 1995; for the involvement of sugars in desicca-
Leprince et al., 1998). Oleosins are pre- tion tolerance operate at moisture contents
sent in the membranes of oil bodies in below those at which most recalcitrant
desiccation-tolerant seeds, but are absent, or seeds can survive. It is therefore perhaps
their amount is diminished, in desiccation- not surprising that there is no clear rela-
sensitive seeds (Leprince et al., 1998). tionship between the degree of desiccation
Thus, a lack of oleosins may be an impor- tolerance in recalcitrant seeds and sugar
tant factor in the desiccation sensitivity of accumulation.
oil-storing recalcitrant seeds. High monosaccharide levels have been
linked with desiccation sensitivity and the
SUGARS. Studies with recalcitrant seeds potential for damage resulting from the
show that there is no clear link between Maillard reaction (Koster and Leopold,
the presence of sugars and the level of des- 1988). In the later stages of development in
iccation tolerance in seeds. For example, orthodox seeds, monosaccharide levels are
large amounts of sugars including sucrose reduced and this also occurs in some
and stachyose accumulate during develop- (Farrant and Walters, 1998), but not all,
ment in the highly desiccation-sensitive species with recalcitrant seeds (Farrant et
seeds of A. marina (Farrant et al., 1993b). al., 1992, 1993b; Finch-Savage et al., 1993).
In the more tolerant Q. robur, sucrose and Monosaccharide levels were generally low
raffinose accumulate in the cotyledons and in most of the 18 species studied by
axes during the later stages of reserve accu- Steadman et al. (1996), including the recal-
mulation (Finch-Savage et al., 1993; Finch- citrant ones.
Savage and Blake, 1994) and, in mature
axes of Quercus rubra, desiccation sensitiv- IS DESICCATION SENSITIVITY DUE TO RETENTION OF
ity is not caused simply by the absence of METABOLIC ACTIVITY AT SHEDDING? Recalcitrant
non-reducing sugars (Sun et al., 1994). In a seeds, perhaps because they remain moist,
more comprehensive study, Steadman et maintain active metabolism throughout
al. (1996) determined the sugar composi- development to the time of shedding. For
tion of a range of recalcitrant, intermediate example, respiration of A. marina seeds,
and orthodox species and combined this after a small decline at the start of reserve
with published data for additional species. accumulation, remains relatively constant
They found no simple relationship between until abscission (Farrant et al., 1992). High
seed type and total sugar content or sucrose respiration rates have also been recorded in
level; however, the content of raffinose and the seeds of other recalcitrant species at
stachyose was generally lower in recalci- shedding (Farrant et al., 1992, 1997;
trant than in orthodox seeds. These Poulsen and Eriksen, 1992; Finch-Savage
oligosaccharides were also found to be and Blake, 1994; Salmen Espindola et al.,
lower in seeds of recalcitrant A. pseudopla- 1994; Leprince et al., 1999). The absence of
tanus than in seeds of the orthodox A. pla- substantial developmental arrest as seeds
tanoides (Greggains et al., 2000a). In approach shedding is confirmed by ultra-
general, there are large variations in the structural and biochemical studies (Dodd
content of sugars between tissues of desic- et al., 1989; Berjak et al. 1992; Farrant et
cation-sensitive seeds, but at least one tis- al., 1992; Farrant and Walters, 1998).
Dessication - Chap 05 18/3/02 2:07 pm Page 173

Desiccation Sensitivity in Relation to Seed Development 173

Indeed, in A. marina the limited de-differ- like that thought to occur in orthodox
entiation of subcellular components species.
towards the end of development allows In most cases, the viability of recalci-
changes indicative of germination to begin trant seeds is lost during drying in region
immediately upon shedding (Farrant et al., three of the five hydration levels summa-
1992). Interestingly, in contrast to this and rized in Vertucci and Farrant (1995). In this
other recalcitrant species studied, respira- region (c. 3 to 11 MPa), seeds are meta-
tion in the dormant seeds of A. pseudopla- bolically active, respiration is measurable
tanus declines to a rate similar to that of and presumably membranes are still
the orthodox A. platanoides at shedding hydrated. However, at this level of hydra-
(Greggains et al., 2000a). tion, it is thought that metabolism becomes
There are differences in the activities of ‘unregulated’, repair processes become
respiratory enzymes between the recalci- inoperative and catabolic activities con-
trant Guilfoylia monostylis and the ortho- tinue unabated, but the processes utilizing
dox Erythrina caffra (Nkang and Chandler, the high-energy intermediates are impaired
1986). These differences may be indicative (Vertucci and Farrant, 1995). As Q. robur
of the seeds’ different germination strate- seeds are dried, increasing quantities of
gies; the recalcitrant seeds maintained a several harmful volatiles are produced,
balance of enzymes suitable for immediate including ethanol and acetaldehyde
germination, whereas, in the orthodox (Finch-Savage et al., 1993). These volatiles
seed, biosynthetic processes were drasti- are indicative of ‘unregulated’ respiration
cally reduced (Nkang and Chandler, 1986). and are a potential source of the free radi-
In contrast to recalcitrant seeds, the meta- cals that accumulate around the time of
bolic activity of orthodox seeds is thought viability loss in Q. robur (Hendry et al.,
to decline in a programmed way before or 1992). These free radicals could alter the
during the early stages of maturation dry- physical/chemical properties of mem-
ing, so that seeds are shed in a quiescent branes, causing them to lose liquid/crys-
state (Rogerson and Matthews, 1977; Miller talline structure (McKersie and Leshem,
et al., 1983; Farrant et al., 1997). This orga- 1994). Thus, it is reasonable to speculate
nized decline in metabolic activity, which that membrane damage and viability loss
presumably has a role in protection against during drying of recalcitrant seeds may
desiccation damage (reviewed by Vertucci result from unregulated metabolism
and Farrant 1995), does not occur in enhanced by inadequate protection by free-
species with the most sensitive seeds and radical scavengers.
is not completed in those species with
more tolerant recalcitrant seeds (Berjak and ANTIOXIDANT SYSTEMS. Lipid peroxidation and
Pammenter, 1997; Farrant et al., 1997). free-radical activity have been associated
Recent studies on seeds of a number of with seed viability loss in several recalci-
more tolerant temperate recalcitrant trant species during desiccation (Hendry et
species suggest that, although high at shed- al., 1992; Chaitanya and Naithani, 1994;
ding, respiration rates decline like those of Finch-Savage et al., 1996; Li and Sun,
orthodox seeds during desiccation (V. 1999). So far, it is difficult to tell whether
Greggains and W.E. Finch-Savage, unpub- the reported accumulation of free radicals
lished data). However, in the more sensi- is a cause or a consequence of viability
tive Araucaria angustifolia, respiration is loss. In either case, adequate protective
only reduced by levels of desiccation that systems to limit free-radical damage are
cause viability loss (Côme and Corbineau, likely to be essential for the maintenance of
1996). In temperate Castanea sativa seeds, viability (Côme and Corbineau, 1996) and a
disruption of the electron transport chain range of antioxidant systems is present in
occurs during drying (Leprince et al., recalcitrant seeds (Hendry et al., 1992;
1999), suggesting that the decline in respi- Chaitanya and Naithani, 1994; Li and Sun,
ration due to drying is not programmed, 1999; Tommasi et al., 1999). In Q. robur,
Dessication - Chap 05 18/3/02 2:07 pm Page 174

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

there appear to be different protective sys- differentiation that occurs in orthodox seeds
tems in the embryonic axis and cotyledons during maturation drying (reviewed by
(Hendry et al., 1992). In the axis, protec- Vertucci and Farrant, 1995).
tion occurs predominantly through the It is essential that there is effective
antioxidants ascorbic acid and -toco- maintenance of the integrity of DNA during
pherol, whereas in the cotyledons protec- desiccation and that any damage is
tion is largely enzymatic, with relatively repaired on rehydration for seed viability
high and increasing activities of superox- to be maintained (see Chapter 12). In A.
ide dismutase and glutathione reductase. marina, DNA repair processes are
Decreased levels of protection from lipid markedly compromised after limited dry-
peroxidation during desiccation may con- ing and DNA replication does not fully
tribute to the loss of seed viability (Hendry recover after only 8% water loss (Boubriak
et al., 1992). Increased lipid peroxidation et al., 2000). The arrest of cell cycle activ-
during desiccation, which precedes viabil- ity at the stage where DNA per nucleus is
ity loss in Shorea robusta (Chaitanya and lowest may render embryos more resistant
Naithani, 1994) and T. cacao (Li and to stress conditions (Deltour, 1985).
Sun, 1999) seeds, was associated with Desiccation tolerance may therefore be
decreased activities of free-radical-scaveng- related to the stage of cell cycle activity at
ing enzymes. In contrast to these findings, which desiccation occurs (Bino et al.,
in a comparison of orthodox and recalci- 1992). However, Sacandé et al. (1997) have
trant Acer species, it was concluded that presented data suggesting this is not true,
the limitation to desiccation tolerance does and further convincing evidence against
not result from inadequate free-radical the possibility comes from a comparison of
scavenging (Greggains et al., 2000a). the orthodox species A. platanoides and
However, A. pseudoplatanus used in the the recalcitrant species A. pseudoplatanus
study can be placed at the most tolerant (Finch-Savage et al., 1998). Both species
end of the continuum of desiccation sensi- produce seeds with stable high levels of 4C
tivity among recalcitrant species. Its respi- DNA during the later stages of develop-
ration rate at shedding is similar to that of ment, and both contain nuclei arrested at
the orthodox Acer species, and there is no the 2C and 4C levels at maturity.
evidence of increased lipid peroxidation as So far there has been little emphasis on
viability is lost. the study of post-desiccation repair mecha-
nisms in seeds and few studies have been
OTHER FACTORS. Differences in cellular struc- published on this topic in recalcitrant
ture could influence desiccation tolerance seeds; this may well be a limiting factor in
(reviewed by Ruhl, 1996) such that the ini- desiccation-sensitive seed tissues.
tial loss of water and consequent reduction
in cell volume in very sensitive seeds
causes mechanical damage (reviewed by 5.3. Conclusions
Berjak and Pammenter, 1997; Pammenter
and Berjak, 1999). For example, the seeds of Essential components of desiccation toler-
A. marina are highly vacuolated and this ance of seeds include the accumulation of
has been connected to their level of desicca- protective substances, which limit the
tion sensitivity compared with other species amount of damage that otherwise would be
(Farrant et al., 1997). However, Vertucci and induced by water loss, and the ability to
Farrant (1995) showed that the evidence for repair cellular components upon subse-
this is conflicting. Considerable disruption quent rehydration. Sugars (disaccharides,
of the cytoskeleton has also been observed such as sucrose, and oligosaccharides, such
during drying of Q. robur axes, and it is not as raffinose and stachyose) have been sug-
reassembled during rehydration (Mycock et gested to play a key protective role by
al., 1999), which may contrast with the accumulating under water deficit condi-
apparently organized intracellular de- tions and functioning to replace water, thus
Dessication - Chap 05 18/3/02 2:07 pm Page 175

Desiccation Sensitivity in Relation to Seed Development 175

stabilizing membranes and other sensitive 1998) for examining protein–protein inter-
systems. Another protective mechanism actions and the use of differential display
may involve dehydrins (Table 5.1). reverse-transcription PCR (Rodriguez-Uribe
Desiccation tolerance is acquired during et al., 2000), gene and enhancer trap tag-
development of orthodox seeds; tolerance to ging (Rojas-Pierce et al., 2000) and micro-
full desiccation is generally lost after germi- arrays (Nevarez et al., 2000) to identify
nation. Recalcitrant seeds, unlike orthodox water-deficit-regulated genes. Proteomics
seeds, are sensitive to desiccation when approaches could yield invaluable infor-
shed from the parent plant, and thus pro- mation concerning post-translational con-
vide a system to study temporal and stress- trols over desiccation-induced gene
induced changes in dehydrins and other expression. These and similar research
putative desiccation protectants. Although avenues may yield more decisive results
studies on recalcitrant seeds provide indi- than the traditional approaches.
rect evidence in relation to elucidating the Finally, it is noteworthy that desicca-
roles of putative desiccation protectants, tion tolerance of seeds is a complex and
more work needs to be done in this area. multifaceted property involving a multi-
It is currently an exciting time to under- tude of genes whose expression ultimately
take the challenges of understanding the leads to mechanisms of both cellular pro-
biochemical and genetic components of tection, to sustain limited damage during
desiccation tolerance of seeds. Several drying itself, and cellular repair, to
novel approaches are now available, includ- reverse any desiccation-induced changes
ing the yeast one- and two-hybrid systems when the appropriate hydrated conditions
(Ingram and Bartels, 1996; Frank et al., are re-established.

5.4. References

Adams, C.A. and Rinne, R.W. (1981) Seed maturation in soybeans (Glycine max L. Merr) is indepen-
dent of seed mass and of the parent plant, yet is necessary for production of viable seeds.
Journal of Experimental Botany 32, 615–620.
Adams, C.A., Fjerstad, M.C. and Rinne, R.W. (1983) Characteristics of soybean seed maturation:
necessity for slow dehydration. Crop Science 23, 265–267.
Akoroda, M.O. (1986) Seed desiccation and recalcitrance in Telfairia occidentalis. Seed Science and
Technology 14, 327–332.
Alamillo, J., Almoguera, C., Bartels, D. and Jordano, J. (1995) Constitutive expression of small heat
shock proteins in vegetative tissues of the resurrection plant Craterostigma plantagineum. Plant
Molecular Biology 29, 1093–1099.
Amuti, K.S. and Pollard, C.J. (1977) Soluble carbohydrates of dry and developing seeds.
Phytochemistry 16, 529–532.
Bartels, D., Singh, M. and Salamini, F. (1988). The onset of desiccation tolerance during development
of the barley embryo. Planta 175, 485–492.
Bartels, D., Schneider, K., Terstappen, G., Piatowski, D. and Salamini, F. (1990) Molecular cloning of
abscisic acid-modulated genes which are induced during desiccation of the resurrection plant
Craterostigma plantagineum. Planta 181, 27–34.
Baumlein, H., Braun, H., Kakhovskaya, I.A. and Shutov, A.D. (1995) Seed storage proteins of sper-
matophytes share a common ancestor with desiccation proteins of fungi. Journal of Molecular
Evolution 41, 1070–1075.
Berjak, P. and Pammenter, N.W. (1997) Progress in the understanding and manipulation of desicca-
tion-sensitive (recalcitrant) seeds. In: Ellis, R.H., Black, M., Murdoch, A.J. and Hong, T.D. (eds)
Basic and Applied Aspects of Seed Biology. Kluwer, Dordrecht, The Netherlands, pp. 689–703.
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 Development and Germination of Seeds. Plenum
Press, New York, pp. 89–108.
Dessication - Chap 05 18/3/02 2:07 pm Page 176

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

Berjak, P., Farrant, J.M., Macaque, 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. (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., Bradford, K.J., Kovach, D.A. and Pammenter, N.W. (1994) Differential effects of tempera-
ture on ultrastructural responses to dehydration in seeds of Zizania palustris. Seed Science
Research 4, 111–122.
Bewley, J.D. and Black, M. (1994) Seeds. Physiology of Development and Germination, 2nd edn.
Plenum Press, New York, 445 pp.
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: Somero, G.N., Osmond, C.B. and Bolis, C.L. (eds) Water and Life: Comparative
Analysis of Water Relationships at the Organismic, Cellular and Molecular Levels. Springer-
Verlag, Berlin, pp. 141–160.
Bino, R.J., de Vries, J.N., Kraak, H.L. and van Piljen, J.G. (1992) Flow cytometric determination of
nuclear replication stages in tomato seeds during priming and germination. Annals of Botany
69, 231–236.
Black, M., Corbineau, F., Gee, H. and Côme, D. (1999) Water content, raffinose, and dehydrins in the
induction of desiccation tolerance in immature wheat embryos. Plant Physiology 120, 463–472.
Blackman, S.A., Obendorf, R.L. and Leopold, A.C. (1992) Maturation proteins and sugars in desicca-
tion tolerance of developing soybean seeds. Plant Physiology 100, 225–230.
Bochicchio, A., Vazzana, C., Raschi, A., Bartels, D. and Salamini, F. (1988) Effect of desiccation on iso-
lated embryos of maize. Onset of desiccation tolerance during development. Agronomy 8, 29–36.
Boubriak, I., Dini, M., Berjak, P. and Osborne, D.J. (2000) Desiccation and survival in the recalcitrant
seeds of Avicennia marina: DNA replication, DNA repair and protein synthesis. Seed Science
Research 10, 307–315.
Bray, E.A. (1991) Regulation of gene expression by endogenous ABA during drought stress. In:
Davies, W.J. and Jones, H.G. (eds) Abscisic Acid Physiology and Biochemistry. BIOS, Oxford,
UK, pp. 81–98.
Bray, E.A. (1993) Molecular responses to water deficit. Plant Physiology 103, 1035–1040.
Butler, W.M. and Cuming, A.C. (1993) Differential molecular responses to abscisic acid and osmotic
stress in viviparous maize embryos. Planta 189, 47–54.
Carpenter, J.F., Crowe, L.M. and Crowe, J.H. (1987) Stabilization of phosphofructokinase with sugars
during freeze drying: characterization of enhanced protection in the presence of divalent
cations. Biochimica et Biophysica Acta 923, 109–115.
Carranco, R., Almoguera, C. and Jordano, J. (1997) A plant small heat shock protein gene expressed
during embryogenesis but noninducible by heat stress. Journal of Biological Chemistry 272,
Carranco, R., Almoguera, C. and Jordano, J. (1999) An imperfect heat shock element and different
upstream sequences are required for the seed-specific expression of a small heat shock protein
gene. Plant Physiology 121, 723–730.
Castillo, J., Rodrigo, M.I., Marquez, J.A., Zuniga, A. and Franco, L. (2000) A pea nuclear protein that
is induced by dehydration belongs to the vicilin superfamily. European Journal of Biochemistry
267, 2156–2165.
Chaitanya, K.S.K. and Naithani, S.C. (1994) Role of superoxide, lipid peroxidation and superoxide
dismutase in membrane pertubation during loss of viability in seeds of Shorea robusta Gaertn.f.
New Phytologist 126, 623–627.
Chandler, P.M. and Robertson, M. (1994) Gene expression regulated by abscisic acid and its relation
to stress tolerance. Annual Review of Plant Physiology and Plant Molecular Biology 45, 113–141.
Chandler, P.M., Walker-Simmons, M., King, R.W., Crouch, M. and Close, T.J. (1988) Expression of
ABA-inducible genes in water stressed cereal seedlings. Journal of Cellular Biochemistry (Suppl.
12C), 143.
Chin, H.F. and Roberts, E.H. (1980) Recalcitrant Crop Seeds. Tropical Press, Kuala Lumpur, Malaysia.
Close, T.J. (1996) Dehydrins: emergence of a biochemical role of a family of plant dehydration pro-
teins. Physiologia Plantarum 97, 795–803.
Dessication - Chap 05 18/3/02 2:07 pm Page 177

Desiccation Sensitivity in Relation to Seed Development 177

Close, T.J., Kortt, A.A. and Chandler, P.M. (1989) A cDNA-based comparison of dehydration induced
proteins (dehydrins) in barley and corn. Plant Molecular Biology 13, 95–108.
Coca, M.A., Almoguera, C. and Jordano, J. (1994) Expression of sunflower low-molecular-weight
heat-shock proteins during embryogenesis and persistence after germination: localization and
possible functional implications. Plant Molecular Biology 25, 479–492.
Cochrane, M.P. (1985) Assimilate uptake and water loss in maturing barley grains. Journal of
Experimental Botany 36, 770–782.
Cohen, A. and Bray, E.A. (1990) Characterization of three mRNAs that accumulate in wilted tomato
leaves in response to elevated levels of endogenous abscisic acid. Planta 182, 27–33.
Collada, C., Gomez, L., Casado, R. and Aragoncillo, C. (1997) Purification and in vitro chaperone
activity of a class I small heat-shock protein abundant in recalcitrant chestnut seeds. Plant
Physiology 115, 71–77.
Côme, D. and Corbineau, F. (1996) Metabolic damage related to desiccation sensitivity. In:
Ouedraogo, A.S., Poulsen, K. and Stubsgaard (eds) Intermediate/Recalcitrant Tropical Forest
Tree Seeds. Proceedings of a Workshop on Improved Methods of Handling and Storage of
Intermediate/Recalcitrant Tropical Forest Tree Seeds, 8–10 June, 1995, Humlebaek, Denmark.
IPGRI, Rome, pp. 107–120.
Crevecoeur, M., Deltour, R. and Bronchart, R. (1976) Cytological study of water stress during germi-
nation of Zea mays. Planta 132, 31–41.
Crowe, J.H., Hoekstra, F.A. and Crowe, L.M. (1992) Anhydrobiosis. Annual Review of Physiology 54,
Daniels, M.J., Chrispeels, M.J. and Yeager, M. (1999) Projection structure of a plant vacuole mem-
brane aquaporin by electron cryo-crystallography. Journal of Molecular Biology 294, 1337–1349.
Dasgupta, J., Bewley, J.D. and Yeung, E.C. (1982). Desiccation-tolerant and desiccation-intolerant
stages during development and germination of Phaseolus vulgaris seeds. Journal of
Experimental Botany 33, 1045–1057.
deKlerk, G.J. (1984) Changes in protein synthesis during the development of Agrostemma githago
seeds. Physiologia Plantarum 62, 607–614.
Deltour, R. (1985) Nuclear activation during early germination of the higher plant embryo. Journal of
Cell Science 75, 43–83.
DeRocher, A.E. and Vierling, E. (1994) Developmental control of small heat-shock protein expression
during pea seed maturation. Plant Journal 5, 93–102.
Dickie, J.B., May, K., Morris, S.V.A. and Titley, S.E. (1991) The effects of desiccation on seed survival
in Acer platanoides L. and Acer pseudoplatanus L. Seed Science Research 1, 149–162.
Dodd, M.C., van Staden, J. and Smith, M.T. (1989) Seed development in Podocarpus henkelii: an
ultrastructural and biochemical study. Annals of Botany 64, 297–310.
Dure, L.S. III (1993) The Lea proteins of higher plants. In: Verma, D.P.S. (ed.) Control of Plant Gene
Expression. CRC Press, Boca Raton, Florida, pp. 325–335.
Dussert, S., Chabrillange, N., Englemann, F. and Hamon, S. (1999) Quantitative estimation of seed
desiccation sensitivity using a quantal response model: application to nine species of the genus
Coffea L. Seed Science Research 9, 135–144.
Ellis, R.H., Hong, T.D. and Roberts, E.H. (1987) The development of desiccation-tolerance and maxi-
mum seed quality during seed maturation in six grain legumes. Annals of Botany 59, 23–29.
Ellis, R.H., Hong, T.D. and Roberts, E.H. (1990) An intermediate category of seed storage behaviour?
I. Coffee. Journal of Experimental Botany 41, 1167–1174.
Ellis, R.H., Hong, T.D. and Roberts, E.H. (1991a) An intermediate category of seed storage behaviour?
II. Effects of provenance, immaturity, and imbibition on desiccation-tolerance in coffee. Journal
of Experimental Botany 42, 653–657.
Ellis, R.H., Hong, T.D., Roberts, E.H. and Soetisna, U. (1991b) Seed storage behaviour in Elaeis
guineensis. Seed Science Research 1, 99–104.
Farrant, J.M. and Walters, C.W. (1998) Ultrastructural and biophysical changes in developing
embryos of Aesculus hippocastanum in relation to the acquisition of tolerance to drying.
Physiologia Plantarum 104, 513–524.
Farrant, J.M., Berjak, P. and Pammenter, N.W. (1985) The effect of drying rate on viability retention of
recalcitrant propagules of Avicennia marina. South African Journal of Botany 51, 432–438.
Farrant, J.M., Pammenter, N.W. and Berjak, P. (1986) The increasing sensitivity of recalcitrant
Avicennia marina seeds with storage time. Physiologia Plantarum 67, 291–298.
Dessication - Chap 05 18/3/02 2:07 pm Page 178

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

Farrant, J.M., Pammenter, N.W. and Berjak, P. (1988) Recalcitrance – a current assessment. Seed
Science and Technology 16, 155–166.
Farrant, J.M., Pammenter, N.W. and Berjak, P. (1992) Development of the recalcitrant (homoiohy-
drous) seeds of Avicennia marina: anatomical, ultrastructural and biochemical events associated
with development from histodifferentiation to maturation. Annals of Botany 70, 75–86.
Farrant, J.M., Berjak, P., Cutting, J.G.M. and Pammenter, N.W. (1993a) The role of plant growth regu-
lators in the development and germination of the desiccation-sensitive (recalcitrant) seeds of
Avicennia marina. Seed Science Research 3, 55–63.
Farrant, J.M., Pammenter, N.W. and Berjak, P. (1993b) Seed development in relation to desiccation
tolerance: a comparison between desiccation-sensitive (recalcitrant) seeds of Avicennia marina
and desiccation-tolerant types. Seed Science and Technology 3, 1–13.
Farrant, J.M., Pammenter, N.M., Berjak, P., Farnsworth, J. and Vertucci, C.W. (1996) Presence of dehy-
drin-like proteins and levels of abscisic acid in recalcitrant (desiccation sensitive) seeds may be
related to habitat. Seed Science Research 6, 175–182.
Farrant, J.M., Pammenter, N.M., Berjak, P. and Walters, C.W. (1997) Subcellular organisation and
metabolic activity during the development of seeds that attain different levels of desiccation tol-
erance. Seed Science Research 7, 135–144.
Finch-Savage, W.E. (1992) Seed water status and survival in the recalcitrant species Quercus robur
L.: evidence for a critical moisture content. Journal of Experimental Botany 43, 671–679.
Finch-Savage, W.E. (1996) The role of developmental studies in research on recalcitrant and interme-
diate seeds. In: Ouedraogo, A.S., Poulsen, K. and Stubsgaard (eds) Intermediate/Recalcitrant
Tropical Forest Tree Seeds. Proceedings of a Workshop on Improved Methods of Handling and
Storage of Intermediate/Recalcitrant Tropical Forest Tree Seeds, 8–10 June, 1995, Humlebaek,
Denmark. IPGRI, Rome, pp. 83–97.
Finch-Savage, W.E. and Blake, P.S. (1994) Indeterminate development in desiccation-sensitive seeds
of Quercus robur L. Seed Science Research 4, 127–133.
Finch-Savage, W.E. and Farrant, J.M. (1997) The development of desiccation-sensitive seeds in
Quercus robur L.: reserve accumulation and plant growth regulators. Seed Science Research 7,
Finch-Savage, W.E., Clay, H.A., Blake, P.S. and Browning, G. (1992) Seed development in the recalci-
trant species Quercus robur L.: water status and endogenous abscisic acid levels. Journal of
Experimental Botany 43, 671–679.
Finch-Savage, W.E., Grange, R.I., Hendry, G.A.F. and Atherton, N.M. (1993) Embryo water status and
loss of viability during desiccation in the recalcitrant species Quercus robur L. In: Côme, D. and
Corbineau, F. (eds) Fourth International Workshop on Seeds: Basic and Applied Aspects of Seed
Biology. AFSIS, Paris, pp. 723–730.
Finch-Savage, W.E., Pramanik, S.K. and Bewley, J.D. (1994) The expression of dehydrin proteins in
desiccation-sensitive (recalcitrant) seeds of temperate trees. Planta 193, 478–485.
Finch-Savage, W.E., Blake, P.S. and Clay, H.A. (1996) Desiccation stress in recalcitrant Quercus robur
L. seeds results in lipid peroxidation and increased synthesis of jasmonates and abscisic acid.
Journal of Experimental Botany 47, 661–667.
Finch-Savage, W.E., Bergervoet, J.H.W., Bino, R.J., Clay, H.A. and Groot, S.P.C. (1998) Nuclear replica-
tion activity during seed development, dormancy breakage and germination in three tree
species: Norway maple (Acer platanoides L.), sycamore (Acer pseudoplatanus L.) and cherry
(Prunus avium L.). Annals of Botany 81, 519–526.
Finkelstein, R.R. (1993) Abscisic acid-insensitive mutations provide evidence for stage-specific sig-
nal transduction pathways regulating expression of an Arabidopsis late embryogenesis-abundant
(Lea) gene. Molecular and General Genetics 238, 401–408.
Finkelstein, R.R. and Lynch, T.J. (2000) The Arabidopsis abscisic acid response gene ABI5 encodes a
basic leucine zipper transcription factor. Plant Cell 12, 599–610.
Frank, W., Phillips, J., Salamini, F. and Bartels, D. (1998) Two dehydration-inducible transcripts from
the resurrection plant Craterostigma plantagineum encode interacting homeodomain-leucine
zipper proteins. The Plant Journal 15, 413–421.
Fu, J.R., Jin, J.P., Peng, Y.F. and Xia, Q.H. (1994) Desiccation tolerance in two species with recalcitrant
seeds: Clausena lansium (Lour.) and Litchi chinensis (Sonn.). Seed Science Research 4, 257–261.
Galau, G.A., Jakobsen, K.S. and Hughes, D.W. (1991) The control of late dicot embryogenesis and
early germination. Physiologia Plantarum 81, 280–288.
Dessication - Chap 05 18/3/02 2:07 pm Page 179

Desiccation Sensitivity in Relation to Seed Development 179

Garay-Arroyo, A., Colmenero-Flores, J.M., Garciarrubio, A. and Covarrubias, A.A. (2000) Highly
hydrophilic proteins in prokaryotes and eukaryotes are common during conditions of water
deficit. Journal of Biological Chemistry 275, 5668–5674.
Gee, O.H., Probert, R.J. and Coomber, S.A. (1994) ‘Dehydrin-like’ proteins and desiccation tolerance
in seeds. Seed Science Research 4, 135–141.
Giordani, T., Natali, L., D-Ercole, A., Pugliesi, C., Frambrini, M., Vernieri, P., Vitagliano, C. and Cavallini,
A. (1999) Expression of a dehydrin gene during embryo development and drought stress in ABA-
deficient mutants of sunflower (Helianthus annuus L.). Plant Molecular Biology 39, 739–748.
Giraudat, J., Hauge, B.M., Valon, C., Smalle, J., Parcy, F. and Goodman, H.M. (1992) Isolation of the
Arabidopsis ABI3 gene by positional cloning. Plant Cell 4, 1251–1261.
Giraudat, J., Parcy, F., Bertauche, N., Gosti, F., Leung, J., Morris, P.-C., Bouvier-Durand, M. and
Vartanian, N. (1994) Current advances in abscisic acid action and signalling. Plant Molecular
Biology 26, 1557–1577.
Gomez, J., Sanchez-Martinez, D., Stiefel, V., Rigau, P., Puigdomenech, P. and Pages, M. (1988) A gene
induced by the plant hormone abscisic acid in response to water stress encodes a glycine-rich
protein. Nature 334, 262–264.
Goncharova, E.A., Udovenko, G.V., Nechiporenko, G.A. and Zholkevich, V.N. (1985) Water exchange
of vegetable marrow fruits as studied with the aid of a tritium label. Soviet Plant Physiology 31,
Greenwood, J.S. and Bewley, J.D. (1982) Seed development in Ricinus communis (castor bean). I.
Descriptive morphology. Canadian Journal of Botany 60, 1751–1760.
Greggains, V., Finch-Savage, W.E., Quick, W.P. and Atherton, N.M. (2000a) Putative desiccation toler-
ance mechanisms in orthodox and recalcitrant seeds of the genus Acer. Seed Science Research
10, 317–327.
Greggains, V., Finch-Savage, W.E., Quick, W.P. and Atherton, N.M. (2000b) Metabolism-induced free
radical activity does not contribute significantly to loss of viability in moist-stored recalcitrant
seeds of contrasting species. New Phytologist 148, 267–276.
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.
Han, B., Berjak, P., Pammenter, N., Farrant, J. and Kermode, A.R. (1997) The recalcitrant plant
species, Castanospermum australe and Trichillia dregeana, differ in their ability to produce
dehydrin-related polypeptides during seed maturation and in response to ABA or water-deficit-
related stresses. Journal of Experimental Botany 48, 1717–1726.
Haslekas, C., Stacy, R.A., Nygaard, V., Culianez-Macia, F.A. and Aalen, R.B. (1998) The expression of
a peroxiredoxin antioxidant gene, AtPer1, in Arabidopsis thaliana is seed-specific and related to
dormancy. Plant Molecular Biology 36, 833–845.
Hattori, T., Terada, T. and Hamasuna, S. (1995) Regulation of the Osem gene by abscisic acid and the
transcriptional activator VP1: analysis of cis-acting promoter elements required for regulation by
abscisic acid and VP1. The Plant Journal 7, 913–925.
Hendry, G.A.F., Finch-Savage, W.E., Thorpe, P.C., Atherton, N.M., Buckland, S.M., Nilsson, K.A. and
Seel, W.E. (1992) Free radical processes and loss of seed viability during desiccation in the
recalcitrant species Quercus robur L. New Phytologist 122, 273–279.
Hofmann, P. and Steiner, A.M. (1989) An updated list of recalcitrant seeds. Landwirtschaftliche
Forschung 42, 310–323.
Hong, T.H. and Ellis, R.H. (1990) A comparison of maturation drying, germination, and desiccation
tolerance between developing seeds of Acer pseudoplatanus L. and Acer platanoides L. New
Phytologist 116, 589–596.
Hong, T.D. and Ellis, R.H. (1992) The survival of germinating orthodox seeds after desiccation and
hermetic storage. Journal of Experimental Botany 43, 239–247.
Hong, T.D., Linington, S. and Ellis, R.H. (1996) Seed Storage Behaviour: a Compendium. IPGRI,
Rome, 656 pp.
Horbowicz, M., Brenac, P. and Obendorf, R.L. (1998) Fagopyritol B, O-alpha-D-galactopyranosyl-
(1→2)-D-chiro-inositol, a galactosyl cyclitol in maturing buckwheat seeds associated with desic-
cation tolerance. Planta 205, 1–11.
Hughes, D.W. and Galau, G.A. (1987) Translational efficiency of Lea mRNAs in cotton embryos:
minor changes during embryogenesis and germination. Plant Molecular Biology 9, 301–313.
Dessication - Chap 05 18/3/02 2:07 pm Page 180

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

Hughes, D.W. and Galau, G.A. (1989) Temporally modular gene expression during cotyledon devel-
opment. Genes and Development 3, 358–369.
Ingram, J. and Bartels, D. (1996) The molecular basis of dehydration tolerance in plants. Annual
Reviews of Plant Physiology and Plant Molecular Biology 47, 377–403.
Johnson, K.D., Herman, E.M. and Chrispeels, M.J. (1989) An abundant, highly conserved tonoplast
protein in seeds. Plant Physiology 91, 1006–1013.
Kermode, A.R. (1990) Regulatory mechanisms involved in the transition from seed development to
germination. Critical Reviews in Plant Sciences 9, 155–195.
Kermode, A.R. (1995) Regulatory mechanisms in the transition from seed development to germina-
tion: interactions between the embryo and the seed environment. In: Galili, G. and Kigel, J. (eds)
Seed Development and Germination. Marcel Dekker, New York, pp. 273–332.
Kermode, A.R. (1997) Approaches to elucidate the basis of desiccation-tolerance in seeds. Seed
Science Research 7, 75–95.
Kermode, A.R. and Bewley, J.D. (1985) The role of maturation drying in the transition from seed
development to germination. I. Acquisition of desiccation-tolerance and germinability during
development of Ricinus communis L. seeds. Journal of Experimental Botany 36, 1906–1915.
Kermode, A.R. and Bewley, J.D. (1986) Alteration of genetically regulated syntheses in developing
seeds by desiccation. In: Leopold, A.C. (ed.) Membranes, Metabolism and Dry Organisms.
Cornell University Press, Ithaca, New York, pp. 59–84.
Kermode, A.R., Bewley, J.D., Dasgupta, J. and Misra, S. (1986) The transition from seed develop-
ment to germination: a key role for desiccation? Horticultural Science 21 (special suppl.),
Kirik, V., Kolle, K., Misera, S. and Baumlein, H. (1998) Two novel MYB homologues with changed
expression in late embryogenesis-defective Arabidopsis mutants. Plant Molecular Biology 37,
Koornneef, M., Hanhart, C.J., Hilhorst, H.W.M. and Karssen, C.M. (1989) In vivo inhibition of seed
development and reserve protein accumulation in recombinants of abscisic acid biosynthesis
and responsiveness mutants in Arabidopsis thaliana. Plant Physiology 90, 463–469.
Koster, K.L. (1991) Glass formation and desiccation tolerance in seeds. Plant Physiology 96, 302–304.
Koster, K.L. and Leopold, A.C. (1988) Sugars and desiccation tolerance in seeds. Plant Physiology 88,
Lee, D.R. and Atkey, P.T. (1984) Water loss from the developing caryopsis of wheat (Triticum aes-
tivum). Canadian Journal of Botany 62, 1319–1326.
Leopold, A.C. and Vertucci, C.W. (1986) Physical attributes of desiccated seeds. In: Leopold, A.C.
(ed.) Membranes, Metabolism and Dry Organisms. Cornell University Press, Ithaca, New York,
pp. 22–34.
Leprince, O., van Aelst, A., Pritchard, H.W. and Murphy, D.J. (1998) Oleosins prevent oil-body coa-
lescence during seed imbibition as suggested by a low-temperature scanning electron micro-
scope study of desiccation-tolerant and -sensitive oilseeds. Planta 204, 109–119.
Leprince, O., Buitink, J. and Hoekstra, F.A. (1999) Axes and cotyledons of recalcitrant seeds of
Castanea sativa Mill. exhibit contrasting responses of respiration to drying in relation to desic-
cation sensitivity. Journal of Experimental Botany 50, 1515–1524.
Leprince, O., Harren, F.J., Buitink, J., Alberda, M. and Hoekstra, F.A. (2000) Metabolic dysfunction
and unabated respiration precedes the loss of membrane integrity during dehydration of germi-
nating radicles. Plant Physiology 122, 597–608.
Li, C. and Sun, W.Q. (1999) Desiccation sensitivity and activities of free radical-scavenging enzymes
in recalcitrant Theobroma cacao seeds. Seed Science Research 9, 209–217.
Luerssen, H., Kirik, V., Herrmann, P. and Misera, S. (1998) FUSCA3 encodes a protein with a con-
served VP1/ABI3-like B3 domain which is of functional importance for the regulation of seed
maturation in Arabidopsis thaliana. The Plant Journal 15, 755–764.
Mariaux, J.B., Bockel, C., Salamini, F. and Bartels, D. (1998) Desiccation- and abscisic acid-respon-
sive genes encoding major intrinsic proteins (MIPs) from the resurrection plant Craterostigma
plantagineum. Plant Molecular Biology 38, 1089–1099.
Maurel, C., Chrispeels, M., Lurin, C., Tacnet, F., Geelen, D., Ripoche, P. and Guern, J. (1997) Function
and regulation of seed aquaporins. Journal of Experimental Botany 48, 421–430.
McCarty, D.R. (1995) Genetic control and integration of maturation and germination pathways in
seed development. Annual Reviews of Plant Physiology and Plant Molecular Biology 46, 71–93.
Dessication - Chap 05 18/3/02 2:07 pm Page 181

Desiccation Sensitivity in Relation to Seed Development 181

McKersie, B.D. and Leshem, Y.Y. (1994) Desiccation. In: McKersie, B.D. and Leshem, Y.Y. (eds) Stress
and Stress Coping in Cultivated Plants. Kluwer Academic Publishers, Dordrecht, The
Netherlands, pp. 132–147.
Meredith, P. and Jenkins, L.D. (1975) Loss of moisture from developing and ripening cereal grains.
New Zealand Journal of Science 18, 501–509.
Meurs, C., Basra, A.S., Karssen, C.M. and van Loon, L.C. (1992) Role of abscisic acid in the induction