Dehydration-induced redistribution of amphiphilic molecules between cytoplasm and lipids is associated with desiccation tolerance in seeds

This study establishes a relationship between desiccation tolerance and the transfer of amphiphilic molecules from the cytoplasm into lipids during drying, using electron paramagnetic resonance spectroscopy of amphiphilic spin probes introduced into imbibed radicles of pea (Pisum sativum) and cucumber (Cucumis sativa) seeds. Survival following drying and a membrane integrity assay indicated that desiccation tolerance was present during early imbibition and lost in germinated radicles. In germinated cucumber radicles, desiccation tolerance could be re-induced by an incubation in polyethylene glycol (PEG) before drying. In desiccation-intolerant radicles, partitioning of spin probes into lipids during dehydration occurred at higher water contents compared with tolerant and PEG-induced tolerant radicles. The difference in partitioning behavior between desiccation-tolerant and -intolerant tissues could not be explained by the loss of water. Consequently, using a two-phase model system composed of sunflower or cucumber oil and water, physical properties of the aqueous solvent that may affect the partitioning of amphiphilic spin probes were investigated. A significant relationship was found between the partitioning of spin probes and the viscosity of the aqueous solvent. Moreover, in desiccation-sensitive radicles, the rise in cellular microviscosity during drying commenced at higher water contents compared with tolerant or PEG-induced tolerant radicles, suggesting that the microviscosity of the cytoplasm may control the partitioning behavior in dehydrating seeds.     

The role of sugars and hexose phosphorylation in regulating the re-establishment of desiccation tolerance in germinated radicles of Cucumis sativa and Medicago truncatula

This study examined whether sugars and hexose phosphorylation participate in the regulatory mechanisms that induce desiccation tolerance (DT) in seeds. In germinated desiccation-sensitive radicles of Cucumis sativa and Medicago truncatula , DT was re-established by an osmotic treatment using polyethylene glycol (PEG) for several days. In cucumber, Glc kinase activity (EC 2.7.1.1) transiently peaked early during PEG incubation before the induction of DT in protruded radicles, whereas Fru kinase activity (EC 2.7.1.4) increased progressively during the re-establishment of DT. Glucosamine (GAM, a competitive inhibitor of HXK) was able to repress the PEG-induced DT in both species, whereas hexose and poorly metabolizable hexose analogues had no effect. GAM was effective in repressing DT only early during PEG incubation, indicating that this effect is transient. Both Glc and Man fully rescued GAM-inhibited DT. PEG-induced accumulation of Suc was not affected by GAM. Isocitrate lyase (ICL) gene expression, which is known to be regulated by hexoses, responded to the re-establishment of DT and GAM feeding. In cucumber, expression of ICL was repressed after 6 h of PEG incubation whereas GAM feeding led to ICL de-repression. When GAM could no longer inhibit the re-establishment of DT, neither were steady-state levels of ICL influenced. The implication of HXK as a catalytic regulator and sugar-sensor in DT is discussed.     

NMR metabolite profiling analysis reveals changes in phospholipid metabolism associated with the re-establishment of desiccation tolerance upon osmotic stress in germinated radicles of cucumber

The adaptation of metabolism is thought to play a role in the acquisition of desiccation tolerance (DT). However, the importance of such a role and whether specific regulatory pathways exist remain to be assessed. Using in vitro 31P and 13C nuclear magnetic resonance (NMR) spectroscopy and biochemical assays, we analysed metabolite profiles of perchloric extracts from germinating radicles of cucumber to identify changes in carbon and phosphate metabolism associated with DT. Emerged radicles measuring 2 mm long can be rendered tolerant to desiccation by incubation in a polyethylene glycol (PEG) solution with a water potential of 1.5 MPa. However, in 4-mm-long emerged radicles, this treatment was ineffective. This manipulable system enabled the discrimination of changes in metabolites associated with DT from those associated with the response to osmotic stress. Independent of radicle length, the PEG treatment resulted in an increase in sucrose (Suc) content, whereas glucose (Glc), fructose (Fru) and the hexose phosphate pool, as well as phosphoenolpyruvate decreased three- to fourfold. In addition, three derivatives arising early during phospholipid catabolism (glycerylphosphorylcholine, glycerylphosphorylethanolamine and glycerylphosphorylinositol) appeared in the PEG-treated radicles. Interestingly, phospholipid degradation was much more pronounced in osmotically challenged radicles that remain sensitive to drying. This was proved by the appearance of catabolites, such as phosphocholine and phosphoethanolamine, solely in 4 mm PEG-treated radicles. Furthermore, glycerol-3-phosphate and its derivative 3-phosphoglycerate increased significantly. Our data suggest that the metabolic response leading to the re-establishment of DT is not entirely identical to that of an osmotic response. It is inferred that membrane remodelling and/or increased phospholipid catabolism is an adaptive response common to osmotic adjustment and DT but is controlled differently in tolerant and sensitive radicles.     

Stress-associated factors increase after desiccation of germinated seeds of <Emphasis Type="Italic">Tabebuia impetiginosa</Emphasis> Mart.

Improved re-establishment of desiccation tolerance was studied in germinated seeds of Tabebuia impetiginosa Mart. by exposing to a polyethylene glycol solution prior to desiccation. The effects of different osmotic potentials and drying rates were studied. In addition, temporary temperature stress and exogenous abscisic acid were applied to evaluate their effect on desiccation tolerance of the protruded radicle. An osmotic potential of −1.7 MPa at 5°C followed by slow drying was most effective in the re-establishment of desiccation tolerance in protruded radicles with a length up to 3 mm. An osmotic potential of −1.4 or −2.0 MPa was less effective. Fast drying completely prevented the re-induction of desiccation tolerance. Cold shock or heat shock prior to osmotic treatment as well as abscisic acid added to the osmotic solution improved desiccation tolerance of protruded radicles. Surprisingly, survival of the germinated seed did not depend on re-establishment of desiccation tolerance in the protruded radicle. Even after the protruded radicle became necrotic and died, the production of adventitious roots from the hypocotyls allowed for survival and the development of high quality seedlings. Thus, T. impetiginosa appeared to be well adapted to the seasonally dry biome in which the species thrives via mechanisms that offer protection against desiccation in the young seedling stage.     

Acquisition of desiccation tolerance in developing wheat embryos correlates with appearance of a fluid phase in membranes

Membrane behaviour in developing wheat (Triticum aestivum cv Priokskaya) embryos was studied in relation to the acquisition of desiccation tolerance, using spin probe techniques. Fresh embryos were able to develop into seedlings at day 15 after anthesis, but it took 18 d before fast‐dried, isolated embryos could germinate. On the basis of membrane integrity measurements it was estimated that between 14 and 18 d after anthesis the proportion of embryonic cells surviving fast drying increased and the critical moisture content, to which embryonic cells could be dehydrated, decreased. Apparently, embryonic cells do not acquire the same level of desiccation tolerance simultaneously. Only when all cells had become desiccation tolerant was germination of air‐dried embryos possible. Using 5‐doxylstearic acid as the probe molecule, an approximately similar lipid–water interface ordering of membranes was observed in all hydrated embryos, irrespective of age. Dehydration had a dual effect on the lipid interface: further ordering of the major part of the interface and the appearance of additional, disturbed regions. The proportion of these regions correlated with the proportion of desiccation‐tolerant cells. We propose that the membrane surface disturbance be caused by endogenous amphiphiles that partition from the cytoplasm into membranes during drying. The absence of such disturbed regions in dried, desiccation‐sensitive embryos might reflect a lack of sufficient amphiphiles. The relevance of membrane surface disturbance for desiccation tolerance is discussed.     

Increased drying rate lowers the critical water content for survival in embryonic axes of English oak (Quercus robur L.) seeds

The potential to cryopreserve embryonic axes of desiccation-sensitive (recalcitrant) seeds is limited by damage during the desiccation necessary for low temperature survival, but the basis of this injury and how to reduce it is not well understood. The effects of drying rate on the viability, respiratory metabolism and free radical-mediated processes were therefore investigated during dehydration of Quercus robur L. embryonic axes. Viability, assessed by evidence of germination and tetrazolium staining, showed a sharp decline at 0.27 and 0.8 g/g during rapid (<12 h) or slow (3 d) dehydration, respectively. Rapid dehydration therefore lowered the critical water content for survival. At any given water content rapid dehydration was associated with higher activities of the free radical processing enzymes, superoxide dismutase, catalase and glutathione reductase and lower levels of hydroperoxide and membrane damage. Rapid dehydration was also associated with lower malate dehydrogenase activity, and a reduced decline in phosphofructokinase activity and in levels of the oxidized form of nicotinamide dinucleotide. Ageing may have contributed to increased damage during slow dehydration, since viability declined even in hydrated storage after 3 d. The results presented are consistent with rapid dehydration reducing the accumulation of damage resulting from desiccation induced aqueous-based deleterious reactions.     

Ecological correlates of seed desiccation tolerance in tropical African dryland trees

In the tropics, species with recalcitrant or desiccation-sensitive, Type III seeds are largely restricted to regions with comparatively high rainfall, because desiccation-induced seed death will be minimal in these environments. However, species with recalcitrant seeds do occur in drylands, although little is known about ecological adaptations to minimize seed death in these environments. Here we present data for the seed desiccation tolerance of 10 African dryland species and examine the relationships between seed size, rainfall at the time of seed shed, and desiccation tolerance for these and a further 70 species from the scientific literature. The combined data set encompasses species from 33 families. Three species (Syzygium cumini, Trichilia emetica, and Vitellaria paradoxa) had desiccation-sensitive seeds, and the remaining seven species investigated were desiccation-tolerant. The desiccation-sensitive species had large (>0.5 g) seeds, germinated rapidly, and had comparatively small investments in seed physical defenses. Furthermore, seed was shed in months of high rainfall (>60 mm). In comparison, for species with desiccation-tolerant seeds, seed mass varied across five orders of magnitude, and seed was shed in wet and dry months. Although infrequent in dryland environments (approximately 11% of the species examined here), species with desiccation-sensitive seeds do occur; large size, rapid germination, and the timing of dispersal all reduce the likelihood of seed drying. Furthermore, desiccation-sensitivity may be advantageous for large-seeded species by increasing the efficiency of resource use in seed provisioning.     

The competence to acquire cellular desiccation tolerance is independent of seed morphological development.

Acquisition of desiccation tolerance and the related changes at the cellular level in wheat (Triticum aestivum cv. Priokskaya) kernels during normal development and premature drying on the ear were studied using a spin probe technique and low temperature scanning electron microscopy. During normal development, the ability of embryos to germinate after rapid drying and rehydration was acquired after completion of morphological development, which is a few days before mass maturity. The acquisition of desiccation tolerance, as assessed by germination, was associated with an upsurge in cytoplasmic viscosity, the onset of accumulation of protein and oil bodies, and the retention of membrane integrity upon dehydration/rehydration. These features were also used to assess cellular desiccation tolerance in the cases when germination could not occur. Slow premature drying was used to decouple the acquisition of cellular desiccation tolerance from morphogenesis. Upon premature drying of kernels on the ears of plants cut at 5 d after anthesis, desiccation-tolerant dwarf embryos were formed that were able to germinate. When plants were cut at earlier stages poorly developed embryos were formed that were unable to germinate, but cellular desiccation tolerance was nevertheless acquired. In such prematurely dried kernels, peripheral meristematic endosperm cells had already passed through similar physiological and ultrastructural changes associated with the acquisition of cellular desiccation tolerance. It is concluded that despite the apparent strong integration in seed development, desiccation tolerance can be acquired by the meristematic cells in the developing embryo and cambial layer of endosperm, independently of morphological development.     

Postgenomic analysis of desiccation tolerance

Desiccation tolerance is the capacity to survive complete drying. It is an ancient trait that can be found in prokaryotes, fungi, primitive animals (often at the larval stages), whole plants, pollens and seeds. In the dry state, metabolism is suspended and the duration that anhydrobiotes can survive ranges from years to centuries. Whereas genes induced by drought stress have been successfully enumerated in tissues that are sensitive to cellular desiccation, we have little knowledge as to the adaptive role of these genes in establishing desiccation tolerance at the cellular level. This paper reviews postgenomic approaches in a variety of desiccation tolerant organisms in which the genetic responses have been investigated when they acquire the capacity of tolerating extremes of dehydration or when they are dry. Accumulation of non-reducing sugars, LEA proteins and a coordinated repression of metabolism appear to be the essential and universal attributes that can confer desiccation tolerance. The protective mechanisms of these attributes are described. Furthermore, it is most likely that other mechanisms have evolved since the function of about 30% of the genes involved in desiccation tolerance remains to be elucidated. The question of the overlap between desiccation tolerance and drought tolerance is briefly addressed.     

Proteomics of desiccation tolerance during development and germination of maize embryos

Maize seeds were used to identify the key embryo proteins involved in desiccation tolerance during development and germination. Immature maize embryos (28N) during development and mature embryos imbibed for 72 h (72HN) are desiccation sensitive. Mature maize embryos (52N) during development are desiccation tolerant. Thiobarbituric acid reactive substance and hydrogen peroxide contents decreased and increased with acquisition and loss of desiccation tolerance, respectively. A total of 111 protein spots changed significantly (1.5 fold increase/decrease) in desiccation-tolerant and -sensitive embryos before (28N, 52N and 72HN) and after (28D, 52D and 72HD) dehydration. Nine pre-dominantly proteins, 17.4 kDa Class I heat shock protein 3, late embryogenesis abundant protein EMB564, outer membrane protein, globulin 2, TPA:putative cystatin, NBS-LRR resistance-like protein RGC456, stress responsive protein, major allergen Bet v 1.01C and proteasome subunit alpha type 1, accumulated during embryo maturation, decreased during germination and increased in desiccation-tolerant embryos during desiccation. Two proteins, Rhd6-like 2 and low-molecular-weight heat shock protein precursor, showed the inverse pattern. We infer that these eleven proteins are involved in seed desiccation tolerance. We conclude that desiccation-tolerant embryos make more economical use of their resources to accumulate protective molecules and antioxidant systems to deal with maturation drying and desiccation treatment.     

The signature of seeds in resurrection plants: a molecular and physiological comparison of desiccation tolerance in seeds and vegetative tissues

Desiccation-tolerance in vegetative tissues of angiosperms hasa polyphyletic origin and could be due to 1) appropriation ofthe seed-specific program of gene expression that protects orthodoxseeds against desiccation, and/or 2) a sustainable version ofthe abiotic stress response. We tested these hypotheses by comparingmolecular and physiological data from the development of orthodoxseeds, the response of desiccation-sensitive plants to abioticstress, and the response of desiccation-tolerant plants to extremewater loss. Analysis of publicly-available gene expression dataof 35 LEA proteins and 68 anti-oxidant enzymes in the desiccation-sensitiveArabidopsis thaliana identified 13 LEAs and 4 anti-oxidantsexclusively expressed in seeds. Two (a LEA6 and 1-cys-peroxiredoxin)are not expressed in vegetative tissues in A. thaliana, buthave orthologues that are specifically activated in desiccatingleaves of Xerophyta humilis. A comparison of antioxidant enzymeactivity in two desiccation-sensitive species of Eragrostiswith the desiccation-tolerant E. nindensis showed equivalentresponses upon initial dehydration, but activity was retainedat low water content in E. nindensis only. We propose that theseantioxidants are housekeeping enzymes and that they are protectedfrom damage in the desiccation-tolerant species. Sucrose isconsidered an important protectant against desiccation in orthodoxseeds, and we show that sucrose accumulates in drying leavesof E. nindensis, but not in the desiccation-sensitive Eragrostisspecies. The activation of "seed-specific" desiccation protectionmechanisms (sucrose accumulation and expression of LEA6 and1-cys-peroxiredoxin genes) in the vegetative tissues of desiccation-tolerantplants points towards acquisition of desiccation tolerance fromseeds.     

In vivo characterization of the effects of abscisic acid and drying protocols associated with the acquisition of desiccation tolerance in alfalfa (Medicago sativa L.) somatic embryos

Although somatic embryos of alfalfa (Medicago sativa L.) had acquired some tolerance to desiccation at the cotyledonary stage of development (22 d after plating), additional culturing in 20 microm abscisic acid (ABA) for 8 d induced greater desiccation tolerance, as determined by increased germination. Compared with fast drying, slow drying of the ABA-treated embryos improved desiccation tolerance. However, slow drying of non-ABA-treated embryos led to the complete loss of germination capacity, while some fast-dried embryos survived. An electron paramagnetic resonance spin probe technique and in vivo Fourier transform infrared microspectroscopy revealed that cellular membrane integrity and a-helical protein secondary structure were maintained during drying in embryos cultured in media enriched with 20 microM ABA, but not in embryos cultured in the absence of ABA. Slow-dried, non-ABA-treated embryos had low oligosaccharide to sucrose ratios, an increased proportion of beta-sheet protein secondary structures and broad membrane phase transitions extending over a temperature range of more than 60 degrees C, suggestive of irreversible phase separations. The spin probe study showed evidence of imbibitional damage, which could be alleviated by prehydration in humid air. These observations emphasize the importance of appropriate drying and prehydration protocols for the survival and storage of somatic embryos. It is suggested that ABA also plays a role in suppressing metabolism, thus increasing the level of desiccation tolerance; this is particularly evident under stressful conditions such as slow drying.     

Changing desiccation tolerance of pea embryo protoplasts during germination

Protoplasts were isolated from pea (Pisum sativum L. cv. Alaska) embryonic axes during and after germination to determine whether the loss of desiccation tolerance in the embryos also occurs in the protoplasts. At all times studied, protoplast survival decreased as water content decreased; however, the sensitivity to dehydration was less when the protoplasts were isolated from embryos that were still desiccation-tolerant (12 h and 18 h of imbibition) than when protoplasts were derived from axes that were sensitive (24 h and 36 h of imbibition). The water content at which 50% of the population was killed (WC50) increased throughout germination and early seedling growth for both the intact tissue and the protoplasts derived from them. Prior to radicle emergence, protoplasts were less desiccation-tolerant than the intact axes; however, protoplasts isolated from radicles shortly after emergence had lower WC50s than the intact radicles. A comparison of protoplast survival after isolation and dehydration in either 500 mM sucrose/raffinose or 700 mM sucrose revealed no difference in tolerance except at 24 h of imbibition, when protoplasts treated in the more concentrated solution had improved tolerance of dehydration. Although intact epicotyls are generally more desiccation-tolerant than radicles, protoplasts isolated separately from epicotyls and radicles did not differ in tolerance. Collectively, these data suggest that protoplasts gradually lose desiccation tolerance during germination, as do the orthodox embryos from which they were derived. However, even prior to radicle emergence, protoplasts display a sensitivity to progressive dehydration that is similar to that shown by recalcitrant and ageing embryos.