DESICCATION TOLERANCE AND NUCLEIC ACID CHANGES DURING SEED MATURATION

Introduction

Anhydrobiosis (‘life without water’) is the remarkable ability of certain organisms to

survive almost total dehydration. ‘Drought tolerance’ can be considered as the tolerance of

moderate dehydration, down to a moisture content below which there is no bulk cytoplasmic

water present [~23% water on a fresh weight basis, or ~0.3 (g H2O) (g dry weight)−1].

‘Desiccation tolerance’ generally refers to the tolerance of further dehydration, when the

hydration shell of molecules is gradually lost. Desiccation tolerance includes also the ability of

cells to rehydrate successfully. In nature, anhydrobiosis often bridges periods of adverse

conditions. Desiccation tolerance, as opposed to drought tolerance, which involves surviving

moderate water loss (e.g. 90% relative water content [RWC]), is the ability to survive absolute

water contents of 0.1 g H2O g_1

Occurrence.

The phenomenon of desiccation tolerance is found throughout the microbial, fungal,

animal and plant kingdoms . In the plant kingdom, it is mainly seeds and non-tracheophytes,

such as mosses, that commonly display tolerance to desiccation. Desiccation tolerance is

widespread in the plant kingdom, including: ferns, mosses and their spores; pollen and seeds of

higher plants; and, rarely, even whole angiosperm, but not gymnosperm, plants. The

phenomenon also occurs in prokaryotes, protists and fungi and animals such as tardigrades,

nematodes and crustaceans. Many mosses, lichens and ferns can survive dehydration of their

vegetative organs (e.g. leaves), whereas this is uncommon in tracheophytes . Although there are

no gymnosperms that show vegetative desiccation tolerance, there are several angiosperm

families that contain desiccation. Desiccation tolerant members . These individual species are

collectively referred to as ‘resurrection plants’ .

Evolution of desiccation tolerance

Inordertocolonizetheland,theearliestterres-trialplantsmusthavebeendesiccation-tolerantat

every stage of the life cycle(Oliveretal.2005).With the evolution of more complex vascular

plants, desiccation tolerance was lost invegetativet issues but was retained in reproductive tissues

(Oliveretal.2000).The acquisition of desiccation tolerance is part of amaturation program during

normal seed development.The majority of terrestrial plants today are capable of producing

desiccation-tolerant structures such as spores, seeds and pollen which can remain viable in the

desiccated state for decades, or centuries, in the case of the ancient Nelumbonucifera (sacred

lotus)seed from China (Shen-Milleretal.1995). The structural genes required for desiccation

tolerance are not uniquet or esurrection plants, but are also present in the genomes of desicca-

tion-sensitiveplants(BartelsandSalamini 2001). However, resurrection plants may haven listed

Genes normally expressed in seed tissue for expression in vegetative tissue to allow these plants

to withstand desiccation(Illingetal. 2005). For example, genes encoding late embryogenesis

abundant(LEA) proteins that are normally expressed in the seeds of desiccation- sensitive plants

during embryo maturation(Close 1996) have been isolated from drought-stressed vegetative

tissues of resurrection plant ssuch as Sporobolus stapfianus (Nealeetal. 2000) and

Craterostigma plantagineum (Bartels 2005). Recent phylogenetic evidence suggests that these

vascularplants gained the ability to withstand desiccation of their vegetative tissues froma

mechanism present first in spores, and that this evolution(orre-evolution)has occurred on at

Leastten in dependent occasions withinthe angiosperms(Oliveretal. 2005).

When is desiccation tolerance acquired?

The desiccation tolerance program can be switched on by dehydration and the plant

hormone abscisic acid3,4. In the green vegetative tissues of mosses, ferns and angiosperm

resurrection plants, it is often a slight dehydration that triggers gene expression associated with

desiccation tolerance. In anhydrobiotic (orthodox) seeds, this gene expression occurs during

development as a part of the maturation program. As a result, seed embryos become desiccation

tolerant considerably before maturation drying.

Signaling to switch on the desiccation tolerance program in developing seeds occurs via abscisic

acid, which also inhibits premature germination. Desiccation tolerance in seeds can also be

induced by premature, slow drying, which leads to the development of dwarf, anhydrobiotic

embryos. However, even if embryos do not develop to the germination-competent stage, the cells

in the proembryos nevertheless acquire desiccation tolerance. In both desiccation-tolerant and

desiccation-sensitive plants, the expression of drought-responsive genes is mediated by ABA-

independent and -dependent signal transduction pathways.

MECHANISMS INVOLVED

It is most expedient to consider the processes or mechanisms listed below, which might

confer protection against desiccation, and their deficiency or absence, which could contribute to

the relative degrees of desiccation sensitivity.

Intracellular physical characteristics such as

— Reduction of the degree of vacuolation

— Amount and nature of insoluble reserves accumulated

— Integrity of the cytoskeleton,

— Conformation of the DNA, chromatin, and nuclear architecture.

Intracellular de-differentiation .

“‘Switching off” of metabolism

Presence, and efficient operation of, antioxidant systems.

Deployment of certain amphipathic molecules.

An effective peripheral oleosin layer around lipid bodies.

Accumulation and roles of putatively protective molecules

- LEA’s

-Sucrose and

-Certain oligosaccharides

- Galactosyl cyclitols.

The presence and operation of repair mechanisms during rehydration.

Intra cellular physical characteristics.

Vacuole volume reduction, whether by the shrinkage of the space occupied by these

usually fluid-filled organelles or by their becoming filled with insoluble reserve material, is one

of the mechanisms that would contribute to increased mechanical resilience of cells to

dehydration. The vacuoles ultimately occupy almost 60 percent on average of the volume across

the cells of all axis tissues and 90 percent of the cotyledonary cells when mature. At no stage do

either the axial or cotyledonary vacuoles contain insoluble reserves, the little insoluble reserve

material occurring as plastid starch.

Vacuoles ultimately constitute only a small fraction of the intracellular volume, particularly in

the axis cells at maturity. The cotyledonary cells contain many large, starch-filled plastids and

protein bodies and are considerably less vacuolated. orthodox and able to tolerate low water

contents, vacuolar volume is reduced to an insignificant proportion in axis cells, and vacuoles in

cotyledonary cells accumulate an amorphous, presumably insoluble, material. The differential

degree of vacuolation and insoluble reserve deposition. in both developing and mature seeds,

correlates with their degree of desiccation sensitivity. This is in accord with the concept that a

high degree of vacuolation can lead to lethal mechanical damage upon dehydration

(Farrant ,1997).

Cell wall changes during desiccation

When water availability to roots decreases, plants tend to limit water loss from

transpiration by closing the stomata, and thereby reducing the water flux through the plant, and

by reducing leaf growth, which results in a smaller transpiring leaf area (Tardieu 2005). The

reduction of growth under water deficit is accomplished by a reduction in cell division rate and

an increase in cell wall stiffening, which inhibits cell wall expansion(Cosgrove 2000). This

stiffening of the cell wall poses a problem for drying cells. In order to tolerate desiccation, any

cell with a large, water-filled vacuole must overcome or limit the mechanical stress caused by its

shrinking during drying. The cell walls of some resurrection plants have special adaptations that

promote folding to reduce the mechanical stress caused by desiccation. In C. wilmsii, a

significant increase in xyloglucans and unesterified pectins is observed in the cell wall during

drying (Vicre et al. 1999). Dehydration also induces a considerable reduction ofglucose in the

hemicellulosic fraction of C. wilmsii cell walls (Vicre et al. 2004). These changes are thought to

enhance the tensile strength of the C.wilmsii cell wall, allowing it to contract and to fold without

collapsing in the dried tissue. Thus, cell wall flexibility is an important factor in tolerance to

injury caused by desiccation. In C.plantagineum, an increase in expansin activity during

desiccation is associated with a rise in cell wall flexibility and folding (Jones and McQueen-

Mason 2004). Similarly, desiccation-sensitive maize plants adapted to low water potentials are

able to maintain root growth by increasing the extensibility of their cell walls (Wu et al. 1996).

This increase in cell wall flexibility is correlated with an increase in expansin activity and

transcript accumulation (Wu et al. 2001). Expansins were first identified as cell wall-loosening

factors in acid-induced cell expansion (McQueen- Mason et al. 1992). They are thought to act by

disrupting the hydrogen bonds between cellulose and hemicellulose polymers in the cell wall

(McQueen-Mason and Cosgrove 1995).

Reaction of cytoskeleton.

The cytoskeleton, the major components of which are microtubules and microfilaments,

is not only an integrated intracellular support system, it also plays a major role in imposing

organization on the cytoplasm and also thenucleus.

In the hydrated

state, there is an extensive microfilamentous network in the cells of the root tip, which becomes

dismantled as the seeds are increasingly dehydrated–a feature that is expected for orthodox seeds

as well. The resultant lack of the intracellular support and structural organization afforded by the

cytoskeleton would obviously be a major damaging factor upon rehydration of recalcitrant seeds.

Additionally, certain cytomatrical (cytoplasmic) enzyme systems exist as multienzyme particles

in plant cells (Hrazdina , 1992), the formation of which could occur because of the binding of

key or anchor enzymes to the microfilaments of the cytoskeleton, as illustrated for glycolysis

(Masters ,1984). Thus, failure of the cytoskeleton to reassemble following deleteriously low

levels of dehydration would have physiological as well as structural consequences in the cells of

desiccation-sensitive seed tissues.

DNA, Chromatin, and Nuclear Architecture Conformation.

Maintenance of the integrity of the genetic DNA material in the desiccated condition in

orthodox seeds, and/or its rapid repair when seeds are rehydrated, is considered to be a

fundamental requirement for desiccation tolerance. DNA assumes different conformational states

depending on water activity and, although this has not yet been demonstrated for seeds, it is

considered that as water is lost (i.e. water activity is lowered) such conformational changes will

occur (Osborne and Boubriak 1994). there is an increase in the number of base pairs per turn of

the DNA helix as water is lost from the individually hydrated phosphate groups, and water

bridges are formed instead as the conformation changes from the B to the Z form.

Osborne and Boubriak (1994) have suggested that protein glycation (i.e. the nonenzymic

addition of reducing sugars to histone proteins) is likely to occur, which could increase the

incidence of DNA conformations appropriate to the dehydrated state. Those authors also discuss

the possibility of nonenzymic methylation of cytosine occurring, which would favor the Z form

of the DNA.

The highly condensed state of the chromatin in dry, orthodox seeds (e.g. Crévecoeur and others

1976, Sargent and others 1981), which is reversed at the stage in germination when desiccation

sensitivity ensues (Deltour,1985), is thought to be a visible manifestation of its stabilized

condition. A major factor in chromatin stabilization in the dry state in orthodox seeds might be

the change in the H1-histone: nucleosome ratio to 2:1 from the 1:1 ratio that typifies the hydrated

condition (Ivanov and Zlatanova 1989).

Nuclear architecture is a further factor that is probably involved in chromatin stability.

The structural framework of the nucleus has been convincingly demonstrated for plant cells and

is based on intermediate-type filaments called lamins (Moreno Díaz de la Espina 1995) It is

implicit that during dehydration and in the desiccated state of orthodox seeds, orderly

reorganization of the nucleoskeleton should occur with its restitution as a functional framework

upon rehydration. Maintenance of the integrity of the nucleus as a whole, and the genome in

particular, may be imperfectly expressed, or the ability for this may even be totally lacking, in

recalcitrant seeds.

Intracellular dedifferentiation.

De-differentiation, a characteristic of maturing desiccation tolerant seeds, is essentially a

means by which intracellular structures are simplified and minimized (Vertucci and Farrant

1995), which strongly suggests that membranes and cytoskeletal elements are vulnerable to

dehydration. This

phenomenon is reversed in orthodox seeds when water istaken up early during germination

(Bewley 1979) It seems that retention of organelles in the highly differentiated state is a major

factor in the desiccation sensitivity of recalcitrant species, whereas the ability for ordered de-

differentiation is, in fact, a prerequisite for seed survival in the dehydrated state. There has long

been uncertainty as to whether dehydration causes de-differentiation, or this intracellular

minimization actually precedes the initiation of maturation drying (e.g. Vertucci and Farrant

1995). However, the observations on P. vulgaris reported by Farrant and others (1997),

indicating that mitochondrial de-differentiation occurs, and that respiratory rate declines

markedly before maturation drying, support the idea that substantial qualitative and quantitative

change actually occurs in advance of water loss.

“SWITCHING OFF” OF METABOLISM

Electron transport, albeit at a low level, has been recorded for dehydrated plant tissues,

and respiration is measurable even at seed water contents as low as 0.25 g g-1 [20 percent, wmb]

(Vertucci 1989, Vertucci and Farrant 1995). However, in the water content range 0.45 to 0.25 g

g-1 (30 to 20 percent [wmb]), unbalanced metabolism may lead to the generation, and essentially

uncontrolled activity, of free radicals (Savage and others 1994) It is therefore imperative that,

during maturation drying, desiccation- tolerant seeds be able to pass through this water content

range with the minimum of damage. The efficient operation of antioxidant systems (Leprince

and others 1993, Puntarulo and others 1991), as well as the “switching off” of metabolism,

would reduce such damage.

In desiccation-sensitive seeds, lethal damage occurs in the water content range 0.45 to 0.25 g g1

(Vertucci and Farrant 1995) and, in some species, at considerably higher levels (Pammenter and

others 1993). Death of relatively hydrate recalcitrant seeds (at c. 0.7 g g-1, or higher [ 40 percent,

wmb]) occurs when water is lost slowly. However, rapid dehydration rates allow survival to

lower water contents (Farrant and others 1985).

Another critical aspect of ongoing metabolism is cell cycling. The cell cycle describes the

nuclear DNA content as 2C in cells that are not preparing for nuclear division, and as 4C in cells

in which DNA replication has occurred, where the constant, C, denotes the DNA content of the

haploid condition. During the cell cycle four distinct phases can be identified, viz. the G1 phase

(2C), which is followed by the S phase, during which DNA replication occurs; after this the cells

enter the G2 phase, during which the amount of DNA remains doubled (i.e. 4C) as a result of

events in the S phase, and this is followed by the phase known as G2M, when mitosis reduces the

DNA content to the 2C level typical of somatic cells in the next G1 phase. Brunori (1967) found

that in orthodox Vicia faba seeds, most of the cells were arrested in G1, and that DNA

replication was one of the first events to be curtailed as the embryo cells lost water. S-phase

replication is resumed only after several hours of imbibition, when water again becomes

available to postharvest, orthodox seeds, as shown by Sen and Osborne (1974) for Secale cereale

(rye): as soon as replication to 4C values occurs and the cells enter G2M, desiccation tolerance is

lost.

Presence and efficient operation of antioxidant system

A range of antioxidant processes operate in orthodox seeds (e.g. Hendry 1993, Leprince

and others 1993), and the role of such processes under conditions of water deficit and desiccation

stress in plants has been reviewed by McKersie (1991) and Smirnoff (1993). it is particularly in

the water content range from 0.45 to 0.25 g g-1 (30 to 20 percent, wmb), that unregulated

metabolic events resulting in the first wave of free-radical generation are likely to occur

(Vertucci and Farrant 1995). This implies that antioxidant systems (i.e. free-radical scavenging

systems) should be maximally effective during maturation drying of orthodox seeds, and again

when seeds take up water upon imbibition. protective mechanisms appear to become impaired

under conditions of water stress

(Senaratna and McKersie 1986, Smith and Berjak 1995). Rapid formation of free radicals and

decreasing activity of antioxidant systems have been reported as occurring during dehydration of

the seeds of the temperate recalcitrant species.

Lipid peroxidation, which is a major consequence of uncontrolled free-radical generation, with

the ultimate accumulation of a stable free radical in the embryonic axes. damage ascribable to

uncontrolled free-radical generation occurs during dehydration in the recalcitrant seeds of a

range of species that show differing degrees and manifestations of nonorthodox behavior. This

implies not only that free radicals are produced as a consequence of water stress in these

desiccation- sensitive seeds, but also that antioxidant systems are ineffective at curbing them.

Together, then, these factors must be seriously considered as constituting one of the major causes

of desiccation sensitivity.

Accummulation and roles of putatively protective molecules

Late Embryogenic Accumulating/Abundant Proteins (LEA’s)

LEA’s (Galau and others 1986) comprise a set of hydrophilic, heat-resistant proteins

associated with the acquisition of desiccation tolerance in developing orthodox seeds Their

synthesis appears to be associated with the high ABA levels that peak during the later stages of

seed development (Kermode 1990). The characteristics of LEA’s and the conditions under which

they appear have led to suggestions that they function as protectants, perhaps stabilizing

subcellular structures in the desiccated condition (Close and others 1989).

The position of LEA’s (or dehydrin-like proteins, as they may be termed) in nonorthodox seeds

appears at first sight to be anomalous, as some species do not express these proteins while others

express them to variable extents. Dehydrin-like proteins were shown to be expressed in a range

of temperate, recalcitrant species (Finch-Savage and others 1994b, Gee and others 1994), but the

absence of such proteins correlated with low ABA levels was found to characterize the mature,

recalcitrant seeds of 10 tropical, wetland species (Farrant and others 1996). Those authors

showed the presence of dehydrin-like proteins in other temperate and tropical recalcitrant

(nonwetland) species, and suggested that their occurrence may be habitat-related, perhaps also

providing protection against low-temperature stress.

it seems that the ability to express LEA’s or dehydrin- type proteins cannot be taken as an

indication that the seeds of a particular species will or will not withstand dehydration. This

indicates clearly that desiccation tolerance must be the outcome of the interplay of more than one

(and probably many) mechanisms or processes. Details of this, particularly npertaining to

LEA’s/dehydrins, sugars, and various stresses, have been reviewed by Kermode (1997).

However, the variable expression of LEA’s/dehydrins in recalcitrant seeds on a species basis

may, in association with the presence or absence of other factors, account for the degree of

nonorthodox behavior exhibited under a particular set of circumstances.

Sucrose, oligosaccharides or galactosyl cyclitolsSugar contents

Water deficit results in a reduction of photosynthesis and starch content in leaf tissues. There is a

rapid conversion of starch into sugars, the most common of which are sucrose and trehalose

(Crowe et al. 1998). Conversion of starch, which is usually stored in plastids, into sucrose is

attributed primarily to activation of sucrose phosphate synthase by reversible protein

phosphorylation upon perception of osmotic stress.

The activation of sucrose synthesis may also be associated with a concurrent inhibition of starch

synthesis. Sucrose biosynthetic genes have been shown to be induced by both desiccation and

ABA in the resurrection plant C. plantagineum (Kleines et al. 1999).

Sucrose and trehalose have been proposed to contribute towards the maintenance of turgor

during stress, and the prevention of protein denaturation and membrane fusions in the cell

(Crowe et al. 1998). Both sugars are capable of forming biological glasses (a process called

vitrification) within the dried cell. Vitrification of the cytoplasm may not be due to the effects of

sugars only, but probably results from the interaction of sugars with other molecules, most likely

proteins (Hoekstra 2005). The formation of an intracellularglass phase is believed to be

indispensable to survival during desiccation, and cells of many desiccation-tolerant plants and

animals undergo vitrification upon drying to protect organelles from damage (Buitink and

Leprince 2004). Vitrification is thought to limit the production of free radicals through the

slowing down of chemical reaction rates and molecular diffusion in the cytoplasm (Hoekstra

2005). It may also help to preserve protein structure. Thus, vitrification be an important

protective mechanism for resurrection plants against oxidative damage during desiccation

Accumulation of nonreducing sugars, particularly of the raffinose series (Blackman and

others 1992, Koster and Leopold 1988, Leprince and others 1990a) and/or galactosyl cyclitols

(Horbowicz and Obendorf 1994, Obendorf 1997) has been implicated in the acquisition and

maintenance of the desiccated state in orthodox seeds, generally in two major ways. These are in

terms of the “Water Replacement Hypothesis” (Clegg 1986, Crowe and others 1992) and

vitrification, otherwise referred to as glassy state formation (Koster and Leopold 1988, Leopold

and others 1994, Williams and Leopold 1989).

Orthodox seed maturation invariably seems to be accompanied by the accumulation of

nonreducing oligosaccharides which coincides with the reduction of monosaccharides, and

maintenance of the desiccated state is associated with high levels of sucrose and other

oligosaccharides. Evidence for the replacement of membrane-associated water (the Water

Replacement Hypothesis, i.e. the replacement of water by sucrose to maintain lipid head-group

spacing, thereby preventing gel-state transformation) is equivocal, and a recent critique questions

its relevance in the desiccated state of orthodox seeds (Hoekstra and others 1997). However, the

role of sucrose in the formation of intracellular glasses (i.e. vitrification) is more convincing.

Mechanisms of protein structure stabilization at different stages of water loss

Significant proportion of the sugars may be tightly associated with LEA’s–these

complexes acting to control and optimize the rate of water loss during dehydration of orthodox

seeds. It should be noted, however, that this should not obviate the participation of either the

LEA’s or the sugars in the maintenance of orthodox seed viability in the desiccated state. The

formation of intracellular oligosaccharides occurs at the expense of monosaccharides, and

confers the advantage that immediately available respiratory substrates are removed (Koster and

Leopold 1988, Leprince and others 1992, Rogerson and Matthews 1977). This would serve to

reduce the spectrum of damaging reactions that can occur as orthodox seeds pass through critical

water content ranges favoring unbalanced metabolism, during maturation drying. Whatever the

role(s) of sucrose and oligosaccharides or galactosyl cyclitols may be in orthodox seeds, seeking

parallels for desiccation-sensitive seeds is entirely inappropriate. While sucrose and other

oligosaccharides are produced in some of the few recalcitrant seed species that have been

assayed (Farrant and others 1993, Finch-Savage and Blake 1994), glass formation will occur

only at water contents well below the lethal limit. When recalcitrant seeds are dehydrated under

ambient conditions (which is what would occur in the natural habitat), they lose viability at

relatively high water contents–in the region of 0.7 g (or more) water per g dry mass [ 40 percent,

wmb] (Pammenter and others 1991), which are far higher that those required for glass formation

to occur (Bruni and Leopold 1992, Leopold and others 1994, Sun and others 1994, Williams and

Leopold 1989). The same argument holds if water replacement by sugars is an operative

phenomenon in orthodox seeds; this too would occur only at water contents of 0.3 g per g dry

material (Hoekstra and Van Roekel 1988), which is well below the lethal limit for slowly drying

recalcitrant seeds.

Partitioning of amphipathic molecules

In fully hydrated cells (a), membrane lipids are in an undisturbed liquidcrystalline state.

Upon water loss (intermediate water contents), cytoplasmic amphiphilic compounds increase in

concentration and partition into membranes, which can be considered as a preferential

interaction. This causes membrane disturbance in both tolerant (b) and sensitive (c) cells.

Amphiphile partitioning into membranes is complete with the dissipation of bulk water. In the

intermediate water range, the presence of preferentially excluded solutes (sugars) in tolerant cells

(b) keeps the membrane surface preferentially hydrated (indicated by the blue band) and prevents

membrane fusion. The absence of these solutes in the sensitive cells (c) might result in

membrane fusion, as in model membrane systems. On further drying below 0.3 (g H2O) (g dry

weight)−1, the sugar molecules in tolerant cells replace water in the hydration shell of the

membranes, thereby maintaining the spacing between phospholipid molecules. The bilayer

remains in the liquid crystalline phase. In sensitive cells, the removal of water from the hydration

shell in the absence of sugars results in packing of the phospholipid molecules, which leads to a

phase transition into the gel phase. This might lead to lateral phase separations and irreversible

membrane damage. Below 0.1 (g H2O) (g dry weight)−1, cytoplasmic components are

immobilized in a glassy matrix, which might differ in properties between tolerant (d) and

sensitive (e) cells. Whether preferential exclusion has an influence on amphiphile partitioning

into membranes is not known. The arrows indicate the reversibility of the processes during

rehydration. The slow repartitioning of amphiphiles from membranes into the cytoplasm on

rehydration might causetransient leakage of cytoplasmic solutes from tolerant cells.

In highly desiccation-sensitive recalcitrant seeds, it is possible that phase changes of the

membrane bilayers might not be reversible, for example, if nonbilayer structures or hexagonal

phases result (reviewed by Vertucci and Farrant 1995). Partitioning of endogenous amphipaths

into the bilayer upon dehydration is unlikely to act in isolation; thus, even if such molecules are

present in cells of recalcitrant seeds, they may well depend on another mechanism or process to

achieve their reversible migration

THE POSSIBLE ROLE OF OLEOSINS

The term oleosin refers to a unique protein type that surrounds the lipid (oil) droplets in

plant cells (Huang 1992). Oleosins have a central, hydrophobic domain that interacts with the

periphery of the lipid, and an amphipathic N-terminal domain that, with the C-terminal domain,

facilitates interaction with the aqueous cytomatrix. The oleosin boundary of lipid bodies allows

these hydrophobic masses to be accommodated as discrete entities in the aqueous cytomatrix

under hydrated conditions, and it has been suggested that their role during dehydration

prevents the bodies from coalescing in desiccation-tolerant seeds (Leprince and others 1997).

Lack (or inadequate amount) of oleosins in desiccation-sensitive seeds of some species, and

although little obvious change in the integrity of the bodies as a consequence of dehydration was

observed, rehydration appeared to have deleterious effects on their stability. Coalescence of lipid

bodies is a common abnormality accompanying deterioration, even in cells of stored, orthodox

seeds (Smith and Berjak 1995). Although the effects of fungi associated with both stored

orthodox and recalcitrant seeds in bringing about lipid body coalescence cannot be ruled out, the

occurrence of this phenomenon could well be, at least partly, a consequence of some deficiency

in desiccation-sensitive seeds. In view of the findings of Leprince and others (1997), the

deficiency of an adequate oleosin sheath around the lipid bodies may underlie the inherent

instability of these organelles during rehydration following damaging levels of desiccation of

some recalcitrant seeds. However, it must be stressed that the presence of fully functional

oleosins cannot, in itself, account for desiccation tolerance. Rather, it must be viewed as one of

the mechanisms contributing to the spectrum of properties necessary if orthodox seeds are to

survive extreme dehydration.

THE PRESENCE AND OPERATION OF REPAIR MECHANISMS DURING REHYDRATION

There is both indirect and direct evidence that repair mechanisms do come into play when

dry orthodox seeds are rehydrated. For example, seeds that have been stored under adverse

conditions, but are still 100-percent viable, typically show a lag before there are visible signs of

germination, during which it is commonly accepted that repair processes are taking place.

Ultrastructural studies on maize seeds have provided evidence supporting this contention, where

mitochondrial repair was observed during the lag period (Berjak and Villiers 1972). Studies on

rye seeds have shown that even in the dry state there is progressive deterioration of the DNA as a

result of endo- and exonuclease activity during storage (Elder and others 1987), which cannot be

repaired until the seeds are rehydrated (Boubriak and others 1997).

Much of the evidence for the operation of repair processes during rehydration comes from

osmopriming experiments on low-vigor seeds. This process involves controlled rehydration to

the end of phase II, which achieves a hydration level that facilitates repair but precludes

germination proper (Bray 1995, Bray and others 1993). Those authors have shown that

replacement of damaged rRNA occurs, and lesions in the DNA and protein-synthesizing systems

are repaired, during priming. It is generally agreed that free radical generation (see above)

continues in air-dried orthodox seeds during storage (reviewed by Smith and Berjak 1995) and

the ensuing damage obviously must be repaired on rehydration, arguing strongly for the presence

and efficient operation of antioxidant systems at this stage. During dehydration of desiccation-

sensitive seeds and seedlings, however, such systems have been shown to fail (Hendry and others

1992, Leprince and others 1992) and are assumed to remain ineffective when water is once again

provided (Côme and Corbineau 1996a, 1996b). When recalcitrant seeds or axes excised from

such seeds are subjected to nonlethal dehydration, it is generally observed that there is an

increase in the time taken for the onward growth of germination, which might be interpreted as

facilitating repair. However, this is likely to be strictly limited; present studies have shown that

after 22 percent of the water is lost from hypocotyl tips of Avicennia marina, dehydration-

associated DNA damage can no longer be repaired when water is once again provided. DNA

instability to dehydration is also shown by seedlings produced from orthodox seeds, once they

have reached the stage when desiccation tolerance has been lost (Boubriak and others 1997).

Very little work that targets the aspect of possible repair of mature, dehydration-damaged

recalcitrant seeds has yet been done. It is presently tacitly assumed that the necessary repair

systems are present, but are themselves damaged by dehydration beyond certain limits–limits

that might vary among seed species of markedly differing desiccation sensitivity.

Case study

Membrane phospholipid composition was investigated in seeds of two species from the genus

Acer Norway maple (Acer platanoides L.) - tolerant to desiccation, and sycamore (Acer

pseudoplatanus L.) - intolerant to desiccation, during their Maturation,. Phospholipid

composition revealed marked differences between studied seeds Norway maple after acquiring

tolerance to desiccation contained much more phosphatidylcholine (PC) and

phosphatidylethanolamine (PE), compared to sycamore.

PC/PE RATIO DURING MATURATION

T

he ratio of 18:2/16:0 in PC and PE was also higher for Norway maple seeds This ratio is a good

indicator of the degree of unsaturation of phospholipids. An increased level of unsaturated fatty

acids can have beneficial effects by decreasing the likelihood of liquid crystalline to gel phase

transmission of membrane (Hoekstra and van Roekel, 1988, Crowe et al. 1992) during

desiccation Membrane composition is not a sole factor of seed tolerance to desiccation. More

important is the contribution of carbohydrates, especially sucrose, raffinose and stachyose. As

was found by Pukacka and Pukacki (1997) the acquisition of desication tolerance by Norway

maple seeds is coincident with the accumulation of sucrose, raffinose and stachyose during seed

maturation. These sugars are acknowledged to be important in membranes and protein protection

in a dry state (Crowe et al. 1987, Cafrey et al. 1989, Bruni and Leopold 1991, Tettetoo et al.

1994). Nevertheless, some membrane constituents may also help to survive severe desiccation

before seeds maturation. The differences in lipid membrane composition between the two studie

seeds may confirm it.

Conclusion

The ability of vegetative tissues to survive near complete desiccation is shared by about

400species of resurrection plants, and appears to haveevolved from reproductive tissues at least

10 timesindependently during the evolution of vascular plants. Genes involved in desiccation

tolerance may be constitutively expressed, as in mosses, or induced during dehydration stress, as

in most resurrection angiosperms, a process that may or may not involve abscisic acid as a

signal. In order to survive in dry environments, plants must limit damage from desiccation and/or

rehydration to a minimum, maintain cellular integrity in the dried state, and activate repair

mechanisms upon rehydration. This is accomplished by the synthesis of compatible solutes

including sugars (sucrose in most plants), and proteins with putative protective functions such as

LEAs. Sugars such as sucrose and trehalose are particularly important in vitrification of the cell

cytoplasm during desiccation, which may decrease chemical reaction rates and molecular

diffusion, and limit oxidative damage. Oxidative stress damage is further reduced by the

induction of anti-oxidant enzymes such as glutathione reductase, and the inhibition of

photosynthesis, with some resurrection plants losing all oftheir chlorophyll during desiccation

altogether. Dehydrating plant cells must also prevent or minimize mechanical damage to the cell

wall and membranes due to the shrinking of the vacuole. The increase in cell wall flexibility that

promotes wall folding may be due to the increase in activity and transcript accumulation of cell

wall-loosening proteins such as expansins. Thus, the ability of resurrection plants to survive in

extremely dry environments is a multigenic trait that relies on systems of cellular protection

during dehydration, and recovery and repair upon rehydration (Rascio and La Rocca 2005).

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