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.     

Survival of dried eukaryotes (anhydrobiotes) after exposure to very high temperatures

Organisms that tolerate essentially complete dehydration are said to be in anhydrobiosis, and can be referred to as anhydrobiotes. Those organisms are of great ecological and medical importance, but also provide models for the study of a variety of biological phenomena. We examined the tolerance of selected eukaryotic anhydrobiotes to high temperatures using slow (∼4 °C min⁻¹) and rapid (∼100 °C min⁻¹) heating to 110, 120, 130, and 140 °C. Test organisms were then either returned to storage temperatures close to 22 °C (preheating), or held at those high temperatures for an additional 10 min. Some anhydrobiotes survived slow heating to 130 °C, whereas rapid heating led to a dramatic reduction in survival. None of these organisms encounter anywhere near these high temperatures in nature, so tolerance is not an obvious result of adaptation to current or recent conditions. We speculate that tolerance could have been achieved during the much earlier evolution of these organisms, and has been retained up to the present.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 93 , 15–22.     

Differential mechanisms to induce dehydration tolerance by abscisic acid and sucrose in Spathoglottis plicata (Orchidaceae) protocorms

Abscisic acid (ABA) and sucrose are known to induce dehydration tolerance of in vitro plant cells and tissues. The present study reports the presence of different mechanisms by which sucrose and ABA improve dehydration tolerance of Spathoglottis plicata (orchid) protocorms. Orchid protocorms were generated aseptically from seeds on Murashig and Skoog medium, and then treated for 7 d in medium containing 10 mg L^?1 ABA and/or 10% (w/v) sucrose. Dehydration tolerance of protocorms was determined at ～25 °C under various drying conditions at relative humidity from 7 to 93%. The actual rate of water loss (i.e. drying rate) was determined using the rate constant of tissue water loss during drying according to the first‐order kinetics. Drying rate affected dehydration tolerance. ABA treatment reduced drying rate and increased dehydration tolerance of protocorms at all relative humidity values tested. However, when compared on the basis of actual drying rates, there was no difference in dehydration tolerance between control and ABA‐treated protocorms, suggesting that ABA‐induced tolerance was correlated with the drying rate reduction. Sucrose treatment was more effective than ABA treatment for the induction of dehydration tolerance. Interestingly, sucrose only slightly affected drying rate. ABA treatment significantly enhanced the synthesis of dehydrin, whereas sucrose treatment primarily resulted in sucrose accumulation. Sucrose treatment also affected protein turnover during drying, causing a significant decrease in protein content in protocorms. Slow drying promoted the degradation of high molecular weight proteins and enhanced the synthesis of low molecular weight dehydrin. The data suggest that different physiological mechanisms are probably involved in the induction of dehydration tolerance by ABA and sucrose treatment.     

Glass formation in plant anhydrobiotes: survival in the dry state

Anhydrobiotes can resist complete dehydration and survive the dry state for extended periods of time. During drying, cytoplasmic viscosity increases dramatically and in the dry state, the cytoplasm transforms into a glassy state. Plant anhydrobiotes possess large amounts of soluble non-reducing sugars and their state diagrams resemble those of simple sugar mixtures. However, more detailed in vivo measurements using techniques such as Fourier transform infrared spectroscopy and electron paramagnetic resonance spectroscopy reveal that these intracellular glasses are complex systems with properties quite different from those of simple sugar glasses. Intracellular glasses exhibit a high molecular packing and slow molecular mobility, resembling glasses made of mixtures of proteins and sugars, which potentially interact with additional cytoplasmic components such as salts, organic acids, and amino acids. Above the glass transition temperature, the cytoplasm of biological systems still exhibits a high stability and low molecular mobility, which could serve as an ecological advantage. All desiccation-tolerant organisms form glasses upon drying, but desiccation-sensitive organisms generally lose their viability during drying at water contents at which the glassy state has not yet been formed, suggesting that other factors are necessary for desiccation tolerance. Nevertheless, the formation of intracellular glasses is indispensable to survive the dry state. Storage stability of seeds and pollens is related to the molecular mobility and packing density of the intracellular glass, suggesting that the characteristic properties of intracellular glasses provide stability for long-term survival.     

Rate of dehydration and cumulative desiccation stress interacted to modulate desiccation tolerance of recalcitrant cocoa and ginkgo embryonic tissues

Rate of dehydration greatly affects desiccation tolerance of recalcitrant seeds. This effect is presumably related to two different stress vectors: direct mechanical or physical stress because of the loss of water and physicochemical damage of tissues as a result of metabolic alterations during drying. The present study proposed a new theoretic approach to represent these two types of stresses and investigated how seed tissues responded differently to two stress vectors, using the models of isolated cocoa (Theobroma cacao) and ginkgo (Ginkgo biloba) embryonic tissues dehydrated under various drying conditions. This approach used the differential change in axis water potential (DeltaPsi/Deltat) to quantify rate of dehydration and the intensity of direct physical stress experienced by embryonic tissues during desiccation. Physicochemical effect of drying was expressed by cumulative desiccation stress [integralf(psi,t)], a function of both the rate and time of dehydration. Rapid dehydration increased the sensitivity of embryonic tissues to desiccation as indicated by high critical water contents, below which desiccation damage occurred. Cumulative desiccation stress increased sharply under slow drying conditions, which was also detrimental to embryonic tissues. This quantitative analysis of the stress-time-response relationship helps to understand the physiological basis for the existence of an optimal dehydration rate, with which maximum desiccation tolerance could be achieved. The established numerical analysis model will prove valuable for the design of experiments that aim to elucidate biochemical and physiological mechanisms of desiccation tolerance.     

Drying rate and dehydrin synthesis associated with abscisic acid-induced dehydration tolerance in Spathoglottis plicata orchidaceae protocorms

Dehydration tolerance of in vitro orchid protocorms was investigated under controlled drying conditions and after abscisic acid (ABA) pretreatment. Protocorms were obtained by germinating seeds on Murashige and Skoog (MS) medium containing 10% (v/v) coconut water, 2% (w/v) sucrose and 0.8% (w/v) agar, and were dehydrated in relative humidities (RH) ranging from 7% to 93% at 25 degrees C. The critical water content of dehydration tolerance was determined, using the electrolyte leakage method. Drying rate affected the critical water content. Slow drying under high RH conditions achieved the greatest tolerance to dehydration. ABA pretreatment decreased the drying rate of protocorms, and increased dehydration tolerance. Improved tolerance to dehydration after ABA treatment was correlated with the effect of ABA on drying rate of protocorms. When critical water content of protocorms dried under different RH was plotted as a function of actual drying rate, no significant difference in tolerance to dehydration was observed between ABA-treated and control protocorms. ABA pretreatment and dehydration of orchid protocorms induced the synthesis of dehydrin, especially under the slow drying conditions. ABA pretreatment also promoted dry matter accumulation such as carbohydrates and soluble proteins and increased the concentration of K(+) and Na(+) ions in protocorms. The ABA-induced decrease in drying rate was correlated with lower osmotic potential, the enhanced maturity of protocorms and the accumulation of dehydrin in protocorms during pretreatment.     

Desiccation tolerance in Physcomitrella patens: Rate of dehydration and the involvement of endogenous abscisic acid (ABA)

The moss Physcomitrella patens , a model system for basal land plants, tolerates several abiotic stresses, including dehydration. We previously reported that Physcomitrella patens survives equilibrium dehydration to ?13 MPa in a closed system at 91% RH. Tolerance of desiccation to water potentials below ?100 MPa was only achieved by pretreatment with exogenous abscisic acid (ABA). We report here that gametophores, but not protonemata, can survive desiccation below ?100 MPa after a gradual drying regime in an open system, without exogenous ABA. In contrast, faster equilibrium drying at 90% RH for 3–5 days did not induce desiccation tolerance in either tissue. Endogenous ABA accumulated in protonemata and gametophores under both drying regimes, so did not correlate directly with desiccation tolerance. Gametophores of a Ppabi3a/b/c triple knock out transgenic line also survived the gradual dehydration regime, despite impaired ABA signaling. Our results suggest that the initial drying rate, and not the amount of endogenous ABA, may be critical in the acquisition of desiccation tolerance. Results from this work will provide insight into ongoing studies to uncover the role of ABA in the dehydration response and the underlying mechanisms of desiccation tolerance in this bryophyte.     

Effects of abscisic acid on growth and dehydration tolerance of <Emphasis Type="Italic">Cynanchum komarovii</Emphasis> seedlings

Cynanchum komarovii is well adapted to hot and dry adverse environments. To determine if exogenous abscisic acid (ABA) affects the growth and dehydration tolerance of this wild plant, ABA was added into the hydroponic solution at a final concentration of 10 μM for 14 days. Root growth is less inhibited than shoot growth under well-watered condition by ABA treatment. ABA reduced the drying rate of seedlings, indicating the acquisition of increased dehydration tolerance. Increased dehydration tolerance is associated with osmotic adjustment and with accumulation of soluble sugars and dehydrins. Results from immunological detection showed that a band of dehydrins with molecular mass of 28.3 kDa appeared in ABA-treated fresh seedlings, which coincided with that in nontreated seedling dessicated for 4 h in 75% relative humidity. The increased ratio of higher to lower amide I bands in Fourier transform infrared spectroscopy analysis indicated that the proportion of unordered structures in proteins was higher after ABA treatment. We concluded that exogenous ABA improved dehydration tolerance of C. komarovii seedlings.     

The role of amphiphiles

This paper reviews our work on the partitioning of amphiphilic compounds from the cytoplasm into membranes during drying of plant systems, and discusses how relevant this phenomenon might be for anhydrobiosis. Amphiphilic guest molecules do partition into membranes and oil bodies, as demonstrated by the results of in vivo electron paramagnetic resonance spectroscopy on incorporated spin probes. Arguments for the likelihood of endogenous cytoplasmic amphiphiles behaving similarly during dehydration and rehydration of plant systems are presented. Negative and positive aspects of the partitioning are summarized. Positive aspects are the automatic insertion of amphiphilic antioxidants into membranes of the dehydrating organism, and the control of membrane fluidity and the phase transition temperature. A negative aspect is the perturbation of membrane structure, leading to increased permeability and loss of function. The finding that after an initial fluidization during dehydration, the membrane surface becomes immobilized in desiccation-tolerant systems and not in desiccation-sensitive systems, is discussed in the light of a strict control of the effect of partitioning. The adaptive significance of amphiphile partitioning into the membranes of anhydrobiotes is discussed.     

Mechanisms to reduce dehydration stress in larvae of the Antarctic midge, Belgica antarctica

The Antarctic midge, Belgica antarctica, is exposed to frequent periods of dehydration during its prolonged larval development in the cold and dry Antarctic environment. In this study, we determined the water requirements of the larvae and the mechanisms it exploits to reduce the stress of drying. Larvae lost water at an exceptionally high rate (>10%/h) and tolerated losing a high portion (>70%) of their water content. Larvae were unable to absorb water from subsaturated water vapor (< or = 0.98 a(v)) to replenish their water stores, thus this midge relies exclusively on the intake of liquid water to increase its pool of body water and maintain water balance. To reduce dehydration stress, the midge employed a variety of mechanisms. Behaviorally, the larvae suppressed water loss by clustering. In response to slow dehydration, glycerol concentration increased 2-fold and trehalose concentration increased 3-fold, responses that are known to decrease the rate of water loss and increase dehydration tolerance. No changes in the mass of cuticular lipids occurred in response to desiccation, but the observed shift to longer hydrocarbons likely contributes to reduced water loss as the larvae dehydrate. As the larvae dehydrated, their oxygen consumption rate dropped, resulting in a reduction of water loss by respiration. Lastly, one bout of slow dehydration also enhanced the larva's ability to survive subsequent dehydration, suggesting that the larvae have the capacity for drought acclimation. Thus, these hydrophilic midge larvae prevent dehydration by multiple mechanisms that collectively reduce the water loss rate and increase dehydration tolerance.     

Desiccation tolerance of recalcitrant Theobroma cacao embryonic axes: the optimal drying rate and its physiological basis

Recalcitrant seed axes were reported to survive to lower water contents under fast-drying conditions. The present study was to examine the hypothesis that drying rate and dehydration duration could interact to determine desiccation tolerance through different physico-chemical mechanisms. The effect of drying rate on desiccation tolerance of Theobroma cacao seed axes at 16 degrees C was examined. Rapid-drying at low relative humidity (RH) and slow-drying at high RH were more harmful to cocoa axes, because electrolyte leakage began to increase and axis viability began to decrease at high water contents. Maximum desiccation tolerance was observed with intermediate drying rates at RH between 88% and 91%, indicating the existence of an optimal drying rate or optimal desiccation duration. This maximum level of desiccation tolerance for cocoa axes (corresponding to a critical water potential of -9 MPa) was also detected using the equilibration method, in which axes were dehydrated over a series of salt solutions or glycerol solutions until the equilibrium. These data confirmed that the physiological basis of the optimal drying rate is related to both mechanical stress during desiccation and the length of desiccation duration during which deleterious reactions may occur. The optimal drying rate represents a situation where combined damages from mechanical and metabolic stresses become minimal.     

Hardening by partial dehydration and ABA increase desiccation tolerance in the cyanobacterial lichen Peltigera polydactylon

* BACKGROUND AND AIMS: The ability of partial dehydration and abscisic acid pretreatments to increase desiccation tolerance in the cyanobacterial lichen Peltigera polydactylon was tested. * METHODS: Net photosynthesis and respiration were measured using infrared gas analysis during a drying and rehydration cycle. At the same time, the efficiency of photosystem two was measured using chlorophyll fluorescence, and the concentrations of chlorophyll a were spectrophotometrically assayed. Heat production was also measured during a shorter drying and rehydration cycle using differential dark microcalorimetry. * KEY RESULTS: Pretreating lichens by dehydrating them to a relative water content of approx. 0.65 for 3 d, followed by storing thalli hydrated for 1 d in the light, significantly improved their ability to recover net photosynthesis during rehydration after desiccation for 15 but not 30 d. Abscisic acid pretreatment could substitute for partial dehydration. The improved rates of photosynthesis during the rehydration of pretreated material were not accompanied by preservation of photosystem two activity or chlorophyll a concentrations compared with untreated lichens. Partial dehydration and ABA pretreatments appeared to have little direct effect on the desiccation tolerance of the mycobiont, because the bursts of respiration and heat production that occurred during rehydration were similar in control and pretreated lichens. * CONCLUSIONS: Results indicate that the photobiont of P. polydactylon possesses inducible tolerance mechanisms that reduce desiccation-induced damage to carbon fixation, and will therefore improve the supply of carbohydrates to the whole thallus following stress. In this lichen, ABA is involved in signal transduction pathways that increase tolerance of the photobiont.     

Metabolic dysfunction and unabated respiration precede the loss of membrane integrity during dehydration of germinating radicles

This study shows that dehydration induces imbalanced metabolism before loss of membrane integrity in desiccation-sensitive germinated radicles. Using a photoacoustic detection system, responses of CO(2) emission and fermentation to drying were analyzed non-invasively in desiccation-tolerant and -intolerant radicles of cucumber (Cucumis sativa) and pea (Pisum sativum). Survival after drying and a membrane integrity assay showed that desiccation tolerance was present during early imbibition and lost in germinated radicles. However, tolerance could be re-induced in germinated cucumber radicles by incubation in polyethylene glycol before drying. Tolerant and polyethylene glycol (PEG)-induced tolerant radicles exhibited a much-reduced CO(2) production before dehydration compared with desiccation-sensitive radicles. This difference was maintained during dehydration. In desiccation-sensitive tissues, dehydration induced an increase in the emission of acetaldehyde and ethanol that peaked well before the loss of membrane integrity. Acetaldehyde emission from sensitive radicles was significantly reduced when dehydration occurred in 50% O(2) instead of air. Acetaldehyde/ethanol were not detected in dehydrating tolerant radicles of either species or in polyethylene glycol-induced tolerant cucumber radicles. Thus, a balance between down-regulation of metabolism during drying and O(2) availability appears to be associated with desiccation tolerance. Using Fourier transform infrared spectroscopy, acetaldehyde was found to disturb the phase behavior of phospholipid vesicles, suggesting that the products resulting from imbalanced metabolism in seeds may aggravate membrane damage induced by dehydration.     

Hydroxyectoine is superior to trehalose for anhydrobiotic engineering of Pseudomonas putida KT2440

Anhydrobiotic engineering aims to increase the level of desiccation tolerance in sensitive organisms to that observed in true anhydrobiotes. In addition to a suitable extracellular drying excipient, a key factor for anhydrobiotic engineering of gram-negative enterobacteria seems to be the generation of high intracellular concentrations of the nonreducing disaccharide trehalose, which can be achieved by osmotic induction. In the soil bacterium Pseudomonas putida KT2440, however, only limited amounts of trehalose are naturally accumulated in defined high-osmolarity medium, correlating with relatively poor survival of desiccated cultures. Based on the enterobacterial model, it was proposed that increasing intracellular trehalose concentration in P. putida KT2440 should improve survival. Using genetic engineering techniques, intracellular trehalose concentrations were obtained which were similar to or greater than those in enterobacteria, but this did not translate into improved desiccation tolerance. Therefore, at least for some populations of microorganisms, trehalose does not appear to provide full protection against desiccation damage, even when present at high concentrations both inside and outside the cell. For P. putida KT2440, it was shown that this was not due to a natural limit in desiccation tolerance since successful anhydrobiotic engineering was achieved by use of a different drying excipient, hydroxyectoine, with osmotically preconditioned bacteria for which 40 to 60% viability was maintained over extended periods (up to 42 days) in the dry state. Hydroxyectoine therefore has considerable potential for the improvement of desiccation tolerance in sensitive microorganisms, particularly for those recalcitrant to trehalose.     

玉米胚发育过程中脱水耐性的变化

对离体玉米胚脱水耐性的变化以及不同脱水速率对其脱水耐性的影响进行了研究。授粉后16d的玉米胚能耐轻微脱水,含水量从1.45降低到0.28gH2Og-1DW时胚的萌发率为100%,但含水量低于0.1gH2Og-1DW时胚死亡。胚的脱水耐性随着发育逐渐加强,表现为电解质渗漏速率逐渐降低,萌发率和幼苗干重逐渐增加。授粉后20d胚内超氧化物歧化酶(SOD)和抗坏血酸过氧化物酶(APX)活性较高,过氧化氢酶(CAT)活性较低;授粉后24d,这些酶的活性与授粉后20d的正好相反。脂质过氧化产物丙二醛(MDA)在种子发育过程中呈下降趋势。不同脱水速率明显地影响胚的脱水耐性:在慢速脱水到含水量0.1~0.18gH2Og-1DW时,胚的萌发率和幼苗干重比快速脱水高,电解质渗漏速率比快速脱水低;在快速脱水条件下胚中的SOD、APX活性和MDA含量也比慢速脱水高;CAT活性的变化不明显。     

Recovery of photosynthesis after exposure of intertidal algae to osmotic and temperature stresses: comparative studies of species with differing distributional limits

Summary This study examines possible relationships between stress tolerance by marine algae and distributions of these species. The ability to recover photosynthetic activity following dehydration or temperature treatments was the assay used to evaluate stress tolerance, and Porphyra perforata, Rhodoglossum affine, Gelidium coulteri, and Smithora naiadum differed in thresholds of tolerance, even though plants were collected from low tidal sites.Limits of dehydration tolerance were well correlated with limits of tidal distribution for these species. Additionally, other high tidal species tolerated severe dehydration while subtidal and low tidal species were sensitive to dehydration. High tidal individuals of P. perforata were also more tolerant of dehydration than were low tidal thalli of P. perforata.Limits of high or low temperature tolerance were not well correlated with tidal elevation for any groups of algae studied. However, cold-tolerant species had more northerly extensions, and warm-tolerant species had more southerly distributions. Thus, differential tolerance to temperature extremes may be an important influence for latitudinal ranges of species.By comparing the experimentally determined thresholds of stress with distributions of species, we test the role of stress in influencing photosynthesis and ultimately distributions of marine algae.     

Differential proteomics of dehydration and rehydration in bryophytes: evidence towards a common desiccation tolerance mechanism

All bryophytes evolved desiccation tolerance (DT) mechanisms during the invasion of terrestrial habitats by early land plants. Are these DT mechanisms still present in bryophytes that colonize aquatic habitats? The aquatic bryophyte Fontinalis antipyretica Hedw. was subjected to two drying regimes and alterations in protein profiles and sucrose accumulation during dehydration and rehydration were investigated. Results show that during fast dehydration, there is very little variation in protein profiles, and upon rehydration proteins are leaked. On the other hand, slow dehydration induces changes in both dehydration and rehydration protein profiles, being similar to the protein profiles displayed by the terrestrial bryophytes Physcomitrella patens (Hedw.) Bruch and Schimp. and, to what is comparable with Syntrichia ruralis (Hedw.) F. Weber and D. Mohr. During dehydration there was a reduction in proteins associated with photosynthesis and the cytoskeleton, and an associated accumulation of proteins involved in sugar metabolism and plant defence mechanisms. Upon rehydration, protein accumulation patterns return to control values for both photosynthesis and cytoskeleton whereas proteins associated with sugar metabolism and defence proteins remain high. The current results suggest that bryophytes from different ecological adaptations may share common DT mechanisms.     

Hydroxyectoine Is Superior to Trehalose for Anhydrobiotic Engineering of Pseudomonas putida KT2440

Anhydrobiotic engineering aims to increase the level of desiccation tolerance in sensitive organisms to that observed in true anhydrobiotes. In addition to a suitable extracellular drying excipient, a key factor for anhydrobiotic engineering of gram-negative enterobacteria seems to be the generation of high intracellular concentrations of the nonreducing disaccharide trehalose, which can be achieved by osmotic induction. In the soil bacterium Pseudomonas putida KT2440, however, only limited amounts of trehalose are naturally accumulated in defined high-osmolarity medium, correlating with relatively poor survival of desiccated cultures. Based on the enterobacterial model, it was proposed that increasing intracellular trehalose concentration in P. putida KT2440 should improve survival. Using genetic engineering techniques, intracellular trehalose concentrations were obtained which were similar to or greater than those in enterobacteria, but this did not translate into improved desiccation tolerance. Therefore, at least for some populations of microorganisms, trehalose does not appear to provide full protection against desiccation damage, even when present at high concentrations both inside and outside the cell. For P. putida KT2440, it was shown that this was not due to a natural limit in desiccation tolerance since successful anhydrobiotic engineering was achieved by use of a different drying excipient, hydroxyectoine, with osmotically preconditioned bacteria for which 40 to 60% viability was maintained over extended periods (up to 42 days) in the dry state. Hydroxyectoine therefore has considerable potential for the improvement of desiccation tolerance in sensitive microorganisms, particularly for those recalcitrant to trehalose.