干燥-再吸水条件下爪哇伪枝藻生理特性和超微结构特征

以典型荒漠丝状蓝藻爪哇伪枝藻为材料,在温室中设置水合(对照)、轻微干燥、中度干燥和极度干燥4种处理,研究干燥胁迫对藻体光合活性、膜脂过氧化、细胞可溶性物质含量、抗氧化酶活性以及细胞超微结构的影响,并采用不同促进剂和抑制剂对干燥藻体进行再吸水处理,测定藻体光合活性的恢复情况。结果显示:(1)爪哇伪枝藻在干燥胁迫下PSⅡ最大光化学效率(Fv/Fm)显著降低,并与其藻体水分含量之间呈极显著性相关(r=0.97、P<0.000 1);(2)随着干燥胁迫程度增加,藻体MDA含量、SOD和CAT活性随之升高,细胞可溶性蛋白和可溶性糖含量增加;(3)在藻体再吸水条件下,培养液(BG-110)、胞外多糖和蔗糖对藻体Fv/Fm的恢复具有重要作用,N-乙酰半胱氨酸和脯氨酸对Fv/Fm有一定的恢复效果,氯霉素和敌草隆则抑制Fv/Fm;(4)与水合状态下的细胞结构相比,干燥藻体细胞结构发生明显的变化,如细胞壁增厚,原生质粘稠浓缩、呈紧密分层排列,细胞内出现大量细小黑色颗粒物等。(5)采用不同外源物质对干燥藻体进行再吸水时,藻体的光合活性呈现不同的恢复效果。研究表明,干燥胁迫下爪哇伪枝藻的光合活性受到明显抑制,细胞质膜过氧化程度加剧,细胞出现可溶性小分子物质积累,抗氧化酶活性增强,并造成细胞结构出现适应性变化。     

Tolerance to oxidative stress induced by desiccation in Porphyra columbina (Bangiales, Rhodophyta)

Unravelling the mechanisms underlying desiccation tolerance is crucial in order to understand the position of algal species in the intertidal zone. The alga Porphyra columbina lives in the uppermost part of the rocky intertidal zones around the world and was selected as a model for this study. Naturally desiccated plants were collected during low tide and studied for morphological changes, oxidative burst induction, biomolecule oxidation, antioxidant responses, and photosynthetic status. Naturally hydrated plants collected during high tides were used for comparative purposes. In addition, changes induced by desiccation were assessed in vitro and the capacity to recover from desiccation was determined by rehydrating the fronds in seawater. The global results show that desiccation induces morphological and cellular alterations accompanied by a loss of ～96% of the water content. Overproduction of reactive oxygen species (ROS) was induced by desiccation and two peaks of H(2)O(2) were detected at 1 and 3 h of desiccation. However, during in vitro rehydration post-desiccation, the ROS quickly returned to the basal levels. At the biomolecular level, only a low production of oxidized proteins was recorded during desiccation, whereas the activity of diverse antioxidant enzymes increased. However, this activity diminished to near basal levels during rehydration. The photosynthetic efficiency (F(v)/F(m)) during desiccation declined by 94-96% of the values recorded in hydrated plants. This reduction was generated by the low levels of trapped energy flux per cross-section (TRo/CS), electron transport flux per CS (ETo/CS), and density of reaction centres (RC/SCo) as well as the chlorophyll content. The inverse pattern was observed for the levels of phycocyanin and phycoerythrin content. F(v)/F(m) and the photosynthetic indicators were restored to normal levels after only 5 min of rehydration. The results indicate that desiccation in P. columbina causes overproduction of ROS that is efficiently attenuated. The morphological and photosynthetic changes could be operating as tolerance mechanisms due to the fact that these responses principally prevent biomolecular alteration and cellular collapse. Thus, the activation of different physiological mechanisms helps to explain the high tolerance to desiccation of P. columbina and, at least in part, the position of this species at the highest level in the intertidal zone.     

Membrane phase behavior of Escherichia coli during desiccation, rehydration, and growth recovery

The membrane lipid bilayer is one of the primary cellular components affected by variations in hydration level, which cause changes in lipid packing that may have detrimental effects on cell viability. In this study, Fourier transform infrared (FTIR) spectroscopy was used to quantify changes in the membrane phase behavior, as identified by membrane phase transition temperature (T_m), of Escherichia coli during desiccation and rehydration. Extensive cell desiccation (1 week at 20%-40% RH) resulted in an increase in T_m from 8.4 ± 1.7 °C (in undried control samples) to 16.5 ± 1.3 °C. Fatty acid methyl ester analysis (FAME) on desiccated samples showed an increase in the percent composition of saturated fatty acids (FAs) and a decrease in unsaturated FAs in comparison to undried control samples. However, rehydration of E. coli resulted in a gradual regression in T_m, which began approximately 1 day after initial rehydration and plateaued at 12.5 ± 1.8 °C after approximately 2 days of rehydration. FAME analysis during progressive rehydration revealed an increase in the membrane percent composition of unsaturated FAs and a decrease in saturated FAs. Cell recovery analysis during rehydration supported the previous findings that showed that E. coli enter a viable but non-culturable (VBNC) state during desiccation and recover following prolonged rehydration. In addition, we found that the delay period of approximately 1 day of rehydration prior to membrane reconfiguration (i.e. decrease in T_m and increase in membrane percent composition of unsaturated FAs) also preceded cell recovery. These results suggest that changes in membrane structure and state related to greater membrane fluidity may be associated with cell proliferation capabilities.     

Dehydration rate determines the degree of membrane damage and desiccation tolerance in bryophytes

Desiccation tolerant (DT) organisms are able to withstand an extended loss of body water and rapidly resume metabolism upon rehydration. This ability, however, is strongly dependent on a slow dehydration rate. Fast dehydration affects membrane integrity leading to intracellular solute leakage upon rehydration and thereby impairs metabolism recovery. We test the hypothesis that the increased cell membrane damage and membrane permeability observed under fast dehydration, compared with slow dehydration, is related to an increase in lipid peroxidation. Our results reject this hypothesis because following rehydration lipid peroxidation remains unaltered, a fact that could be due to the high increase of NO upon rehydration. However, in fast‐dried samples we found a strong signal of red autofluorescence upon rehydration, which correlates with an increase in ROS production and with membrane leakage, particularly the case of phenolics. This could be used as a bioindicator of oxidative stress and membrane damage.     

Distinct contractile and cytoskeletal protein patterns in the Antarctic midge are elicited by desiccation and rehydration

Desiccation presents a major challenge for the Antarctic midge, Belgica antarctica. In this study, we use proteomic profiling to evaluate protein changes in the larvae elicited by dehydration and rehydration. Larvae were desiccated at 75% relative humidity (RH) for 12 h to achieve a body water loss of 35%, approximately half of the water that can be lost before the larvae succumb to dehydration. To evaluate the rehydration response, larvae were first desiccated, then rehydrated for 6 h at 100% RH and then in water for 6 h. Controls were held continuously at 100% RH. Protein analysis was performed using 2‐DE and nanoscale capillary LC/MS/MS. Twenty‐four identified proteins changed in abundance in response to desiccation: 16 were more abundant and 8 were less abundant; 84% of these proteins were contractile or cytoskeletal proteins. Thirteen rehydration‐regulated proteins were identified: 8 were more abundant and 5 were less abundant, and 69% of these proteins were also contractile or cytoskeletal proteins. Additional proteins responsive to desiccation and rehydration were involved in functions including stress responses, energy metabolism, protein synthesis, glucogenesis and membrane transport. We conclude that the major protein responses elicited by both desiccation and rehydration are linked to body contraction and cytoskeleton rearrangements.     

Comparative Quantitative Proteomics Reveals the Desiccation Stress Responses of the Intertidal Seaweed NEOPORPHYRA haitanensis

Neoporphyra haitanensis is an economically important red seaweed that inhabits upper intertidal zones. The thallus tolerates extreme fluctuating environmental stresses (e.g., surviving more than 80% water loss during low tides). To elucidate the global molecular responses relevant to this outstanding desiccation tolerance, a quantitative proteomics analysis of N. haitanensis under different desiccation treatments as well as rehydration was performed. According to the clustering of expression patterns and the functional interpretation of the 483 significantly differentially expressed proteins, a three-stage cellular response to desiccation stress and subsequent rehydration was proposed. Stage I: at the beginning of water loss, multiple signal transduction pathways were triggered including lipid signaling, protein phosphorylation cascades, and histone acetylation controlling acetate biosynthesis to further modulate downstream hormone signaling. Protein protection by peptidyl-prolyl isomerase and ROS scavenging systems were also immediately switched on. Stage II: with the aggravation of stress, increases in antioxidant systems, the accumulation of LEA proteins, and the temporary biosynthesis of branched starch were observed. Multiple enzymes involved in redox homeostasis, including peroxiredoxin, thioredoxin, ascorbate peroxidase, superoxide dismutase, glutathione peroxidase, and glutathione reductase, were hypothesized to function in specific cellular compartments. Stage III: when the desiccated thalli had rehydrated for 30 mins, photosynthesis and carbon fixation were recovered, and antioxidant activities and protein structure protection were maintained at a high level. This work increases the understanding of the molecular responses to environmental stresses via a proteomic approach in red seaweeds and paves the way for further functional studies and genetic engineering.     

Desiccation sensitivity and tolerance in the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>: assessing limits and damage

The moss Physcomitrella patens is becoming the model of choice for functional genomic studies at the cellular level. Studies report that Physcomitrella survives moderate osmotic and salt stress, and that desiccation tolerance can be induced by exogenous ABA. Our goal was to quantify the extent of dehydration tolerance in wild type moss and to examine the nature of cellular damage caused by desiccation. We exposed Physcomitrella to humidities that generate water potentials from −4 (97% RH) to −273 MPa (13% RH) and monitored water loss until equilibrium. Water contents were measured on a dry matter basis to determine the extent of dehydration because fresh weights (FW) were found to be variable and, therefore, unreliable. We measured electrolyte leakage from rehydrating moss, assessed overall regrowth, and imaged cells to evaluate their response to drying and rehydration. Physcomitrella did not routinely survive water potentials <−13 MPa. Upon rehydration, moss dried to water contents >0.4 g g dm⁻¹ maintained levels of leakage similar to those of hydrated controls. Moss dried to lower water contents leaked extensively, suggesting that plasma membranes were damaged. Moss protonemal cells were shrunken and their walls twisted, even at −13 MPa. Moss cells rehydrated after drying to −273 MPa failed to re-expand completely, again indicating membrane damage. ABA treatment elicited tolerance of desiccation to at least −273 MPa and limited membrane damage. Results of this work will form the basis for ongoing studies on the functional genomics of desiccation tolerance at the cellular level.     

Membrane Organization of the Desiccation-Tolerant Moss Tortula ruralis in Dehydrated States

Membrane organization of the desiccation tolerant moss Tortula ruralis was studied in several intensely dehydrated states (75% relative humidity [RH], 90% RH, plasmolysis in molar salt, freezing to −20°C) by ³¹P nuclear magnetic resonance and ultrastructural analyses. Both methods revealed that even at 75% RH (−400 bars), the moss cellular membranes retained extended phospholipid bilayers. Ultrastructural analyses of the fully hydrated moss showed an extensive proliferation of membrane vesicles in the endoplasmic reticulum. During dehydration, these vesicles form layers of membrane under the plasmalemma and in some cases appear to fuse with the surface membrane. This suggests that these vesicles may serve as a reservoir of membranes to accommodate for membrane surface area changes during desiccation and subsequent rehydration.     

The impact of dehydration rate on the production and cellular location of reactive oxygen species in an aquatic moss

Background and Aims

The aquatic moss Fontinalis antipyretica requires a slow rate of dehydration to survive a desiccation event. The present work examined whether differences in the dehydration rate resulted in corresponding differences in the production of reactive oxygen species (ROS) and therefore in the amount of cell damage.

Methods

Intracellular ROS production by the aquatic moss was assessed with confocal laser microscopy and the ROS-specific chemical probe 2,7-dichlorodihydrofluorescein diacetate. The production of hydrogen peroxide was also quantified and its cellular location was assessed.

Key Results

The rehydration of slowly dried cells was associated with lower ROS production, thereby reducing the amount of cellular damage and increasing cell survival. A high oxygen consumption burst accompanied the initial stages of rehydration, perhaps due to the burst of ROS production.

Conclusions

A slow dehydration rate may induce cell protection mechanisms that serve to limit ROS production and reduce the oxidative burst, decreasing the number of damaged and dead cells due upon rehydration.     

Maintenance or Collapse: Responses of Extraplastidic Membrane Lipid Composition to Desiccation in the Resurrection Plant Paraisometrum mileense

Resurrection plants usually grow in specific or extreme habitats and have the capacity to survive almost complete water loss. We characterized the physiological and biochemical responses of Paraisometrum mileense to extreme desiccation and found that it is a resurrection plant. We profiled the changes in lipid molecular species during dehydration and rehydration in P. mileense, and compared these with corresponding changes in the desiccation-sensitive plant Arabidopsis thaliana. One day of desiccation was lethal for A. thaliana but not for P. mileense. After desiccation and subsequent rewatering, A. thaliana showed dramatic lipid degradation accompanied by large increases in levels of phosphatidic acid (PA) and diacylglycerol (DAG). In contrast, desiccation and rewatering of P. mileense significantly decreased the level of monogalactosyldiacylglycerol and increased the unsaturation of membrane lipids, without changing the level of extraplastidic lipids. Lethal desiccation in P. mileense caused massive lipid degradation, whereas the PA content remained at a low level similar to that of fresh leaves. Neither damage nor repair processes, nor increases in PA, occurred during non-lethal desiccation in P. mileense. The activity of phospholipase D, the main source of PA, was much lower in P. mileense than in A. thaliana under control conditions, or after either dehydration or rehydration. It was demonstrated that low rates of phospholipase D-mediated PA formation in P. mileense might limit its ability to degrade lipids to PA, thereby maintaining membrane integrity following desiccation.     

快速脱水与复水过程中2种藓类植物的活性氧代谢与脂质过氧化特征

以来自不同水分生境的金发藓和湿地匐灯藓为材料,对二者在脱水与复水胁迫条件下的活性氧代谢、脂质过氧化损伤程度及其抗氧化系统应答的差异进行比较研究。结果显示:在脱水与复水过程中,(1)硅胶快速脱水更接近阳光直射条件下藓类植物的水分丧失。(2)随着含水量的变化,湿地匐灯藓虽然能够在复水后迅速修复细胞的完整性,但变化剧烈;金发藓则能够始终维持较低的膜透性。(3)2种藓类植物的丙二醛(MDA)含量变化均呈先升后降趋势,但金发藓的MDA含量明显低于湿地匐灯藓。(4)2种藓类植物的超氧阴离子自由基(O2.-)产生速率和过氧化氢含量(H2O2)的变化均与MDA含量变化相似,且金发藓活性氧水平明显高于湿地匐灯藓。(5)2种藓类植物的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性受活性氧诱导亦呈先升后降的趋势,但金发藓抗氧化酶对活性氧迸发的应答更快,活性更强。(6)2种藓类植物的抗坏血酸(AsA)含量呈先降后升态势,但金发藓的含量低于湿地匐灯藓。研究表明,来自不同生境的2种藓类植物对脱水胁迫所致的氧化胁迫均具有很强的适应能力,尤其是复水过程中的修复能力,但不同藓类可能通过不同途径和机制来适应脱水所致的氧化胁迫;来自易发生水分亏缺生境的金发藓可能因具有更强抗氧化能力,从而获得比来自水分充沛生境的湿地匐灯藓更高的脱水耐性。     

Desiccation-tolerant plants in dry environments

The majority of terrestrial plants are unable to survive in very dry environments. However, a small group of plants, called ‘resurrection’ plants, are extremely desiccation-tolerant and are capable of losing more than 90% of the cellular water in vegetative tissues. Resurrection plants can remain dried in an anabiotic state for several years and, upon rehydration, are able to resume normal growth and metabolism within 24 h. Vegetative desiccation tolerance is thought to have evolved independently several times within the plant kingdom from mechanisms that allow reproductive organs to survive air-dryness. Resurrection plants synthesise a range of compounds, either constitutively or in response to dehydration, that protect various components of the cell wall from damage during desiccation and/or rehydration. These include sugars and late embryogenesis abundant (LEA) proteins that are thought to act as osmoprotectants, and free radical-scavenging enzymes that limit the oxidative damage during dehydration. Changes in the cell wall composition during drying reduce the mechanical damage caused by the loss of water and the subsequent shrinking of the vacuole. These include an increase in expansin or cell wall-loosening activity during desiccation that enhances wall flexibility and promotes folding.     

A modulating role for antioxidants in desiccation tolerance

Most organisms depend on the availability of water. However, some life-forms, among them plants and fungi, but very few animals, can survive in the desiccated state. Here we discuss biochemical mechanisms that confer tolerance to desiccation in photosynthetic and non-photosynthetic organisms. We first consider damage caused by water removal and point out that free radicals are a major cause of death in intolerant tissue. Free radicals impair metabolism and necessitate protection and repair during desiccation and rehydration, respectively. As a consequence, desiccation tolerance and prolonged longevity in the desiccated state depend on the ability to scavenge free radicals, using antioxidants such as glutathione, ascorbate, tocopherols and free radical-processing enzymes. Some 'classic' antioxidants may be absent in lower plants and fungi. On the other hand, lichens and seeds often contain secondary phenolic products with antioxidant properties. The major intracellular antioxidant consistently found in all life forms is glutathione, making it essential to survive desiccation. We finally discuss the role of glutathione to act as a signal that initiates programmed cell death. The failure of the antioxidant system during long-term desiccation appears to trigger programmed cell death, causing ageing and eventual death of the organism. In turn, this suggests that a potent antioxidant machinery is one of the underlying mechanisms of desiccation tolerance.     

Human selenium-containing single-chain variable fragment with glutathione peroxidase activity protects NIH3T3 fibroblast against oxidative damage

Ultraviolet B (UVB medium wave, 280–315 nm) induces cellular oxidative damage and apoptosis by producing reactive oxygen species (ROS). Glutathione peroxidase functions as an antioxidant by catalyzing the reduction of hydrogen peroxide, the more important member of reactive oxygen species. A human selenium-containing single-chain variable fragment (se-scFv-B3) with glutathione peroxidase activity of 1288 U/μmol was generated and investigated for its antioxidant effects in UVB-induced oxidative damage model. In particular, cell viability, lipid peroxidation extent, cell apoptosis, the change of mitochondrial membrane potential, caspase-3 activity and the levels of intracellular reactive oxygen species were assayed. Human se-scFv-B3 protects NIH3T3 cells against ultraviolet B-induced oxidative damage and subsequent apoptosis by prevention of lipid peroxidation, inhibition of the collapse of mitochondrial membrane potential as well as the suppression of the caspase-3 activity and the level of intracellular ROS. It seems that antioxidant effects of human se-scFv-B3 are mainly associated with its capability to scavenge reactive oxygen species, which is similar to that of the natural glutathione peroxidase.     

REACTIVE OXYGEN PRODUCTION AND DAMAGE IN INTERTIDAL FUCUS SPP. (PHAEOPHYCEAE)

The research described in this paper was designed to test the hypothesis that the differential stress tolerance associated with the vertical zonation of intertidal seaweeds is attributable to reactive oxygen metabolism. To do so, we measured the production of, and damage caused by, reactive oxygen in three species of intertidal brown seaweeds— Fucus spiralis L., F. evanescens C. Ag., and F. distichus L.— that differ in their ability to withstand freezing, desiccation, and high light stress. Fucus spiralis is the most stress-tolerant species and F. distichus the least. Reactive oxygen production was determined by measuring the production of H₂O₂ and the oxidation of dichlorohydrofluorescein diacetate to dichlorofluorescein. Damage caused by freezing, desiccation, and high-light stress was assessed by measuring variable fluorescence (F_v/F_m) and lipid peroxidation. Production of reactive oxygen increased following freezing, desiccation, or high-light stress. In general, the data were consistent with the hypothesis that reactive oxygen metabolism is involved in stress tolerance. The production of reactive oxygen was relatively low in unstressed seaweeds, and there was little difference between species. Fucus distichus showed the greatest increase in reactive oxygen production after desiccation and freezing stress. Fucus evanescens produced more reactive oxygen production after desiccation than F. spiralis. Although F. evanescens and F. spiralis produced similar amounts of reactive oxygen after freezing, this treatment resulted in an increase in lipid peroxidation only in F. evanescens (and F. distichus ).     

An imbalance in antioxidant defense affects cellular function: the pathophysiological consequences of a reduction in antioxidant defense in the glutathione peroxidase-1 (Gpx1) knockout mouse

Aerobic cells are subjected to damaging reactive oxygen species (ROS) as a consequence of oxidative metabolism and/or exposure to environmental toxins. Antioxidants limit this damage, yet peroxidative events occur when oxidant stress increases. This arises due to increased radical formation or decreased antioxidative defenses. The two-step enzymatic antioxidant pathway limits damage to important biomolecules by neutralising superoxides to water. However, an imbalance in this pathway (increased first-step antioxidants relative to second-step antioxidants) has been proposed as etiological in numerous pathologies. This review presents evidence that a shift in favor of hydrogen peroxide and/or lipid peroxides has pathophysiological consequences. The involvement of antioxidant genes in the regulation of redox status, and ultimately cellular homeostasis, is explored in murine transgenic and knockout models. The investigations of Sod1 transgenic cell-lines and mice, as well as Gpx1 knockout mice (both models favor H(2)O(2) accumulation), are presented. Although in most instances accumulation of H(2)O(2) affects cellular function and leads to exacerbated pathology, this is not always the case. This review highlights those instances where, for example, increased Sod1 levels are beneficial, and indicates a role for superoxide radicals in pathogenesis. Studies of Gpx1 knockout mice (an important second-step antioxidant) lead us to conclude that Gpx1 functions as the primary protection against acute oxidative stress, particularly in neuropathological situations such as stroke and cold-induced head trauma, where high levels of ROS occur during reperfusion or in response to injury. In summary, these studies clearly highlight the importance of limiting ROS-induced cellular damage by maintaining a balanced enzymatic antioxidant pathway.     

Reactive oxygen species metabolism in desiccation-stressed thalli of the liverwort Dumortiera hirsuta

Rates of extracellular superoxide radical formation were estimated in the liverwort Dumortiera hirsuta (SW) Nees. Initial experiments showed that D. hirsuta produced extracellular superoxide at high rates, even when unstressed, and that production increased considerably during rehydration following mild desiccation stress. Experiments in which desiccation was artificially induced using polyethylene glycol showed that superoxide was produced during rehydration rather than desiccation. Incubation of plants in water induced the release about 23% of the superoxide-producing activity, although most appeared to be tightly bound to the cell surface. Experiments with metabolic inhibitors indicated that superoxide production was insensitive to the flavoprotein inhibitor DPI, but inhibited by cyanide, suggesting that peroxidases may produce the superoxide. Despite producing large amounts of superoxide, D. hirsuta only produced small quantities of hydrogen peroxide, the natural product of superoxide dismutation. However, experiments showed that D. hirsuta can reduce the concentration of exogenously supplied hydrogen peroxide from 50 µ M to zero within 1 h, suggesting that any hydrogen peroxide produced is rapidly metabolized. The physiological significance of superoxide production is discussed, with special reference to its possible role as a defence against pathogenic fungi and bacteria.     

Effects of starvation and desiccation on energy metabolism in desert and mesic Drosophila

Energy availability can limit the ability of organisms to survive under stressful conditions. In Drosophila, laboratory experiments have revealed that energy storage patterns differ between populations selected for desiccation and starvation. This suggests that flies may use different sources of energy when exposed to these stresses, but the actual substrates used have not been examined. We measured lipid, carbohydrate, and protein content in 16 Drosophila species from arid and mesic habitats. In five species, we measured the rate at which each substrate was metabolized under starvation or desiccation stress. Rates of lipid and protein metabolism were similar during starvation and desiccation, but carbohydrate metabolism was several-fold higher during desiccation. Thus, total energy consumption was lower in starved flies than desiccated ones. Cactophilic Drosophila did not have greater initial amounts of reserves than mesic species, but may have lower metabolic rates that contribute to stress resistance.     

Basis of coffee seed sensitivity to liquid nitrogen exposure: oxidative stress or imbibitional damage?

Two hypotheses, namely the occurrence of post‐thaw oxidative stress or imbibitional damage, were tested to explain the high sensitivity of coffee seeds to liquid nitrogen (LN) exposure. Oxidative stress was studied by measuring primary and secondary products of lipid peroxidation in seeds during the desiccation and rehydration periods. The 4‐hydroxynonenal (4‐HNE) content of seeds remained constant throughout the desiccation step. No significant difference was observed between desiccated seeds and seeds desiccated and exposed to LN for the evolution of their 4‐HNE and hydroperoxide contents during rehydration. In both cases, an increase in 4‐HNE and hydroperoxide contents of seeds was observed during the first hours of culture under germination conditions, followed by a progressive decrease down to values comparable to those observed in desiccated seeds. The hydroperoxide composition of frozen seeds was not significantly different from that of control seeds. The (S)/(R) enantiomeric ratios of 9‐ and 13‐hydroxy‐octadecadienoic acid extracted from rehydrating seeds were chiral, suggesting that they originated from lipoxygenase activity. These results suggest that the high sensitivity of coffee seeds to LN exposure is not directly associated with the occurrence of an oxidative stress during post‐thaw rehydration. The effect on seed viability of different rehydration procedures previously identified to reduce membrane imbibitional injury was studied after desiccation and LN exposure. Desiccation tolerance increased with, by increasing order, seed osmoconditioning, pre‐heating and pre‐humidifying prior to their culture under germination conditions. Among the four combinations of pre‐humidification durations (24 or 48 h) and temperatures (25 or 37°C) tested, pre‐humidification for 24 h at 37°C gave the highest level of desiccation tolerance. This rehydration procedure also dramatically increased seed viability after LN exposure. Seed desiccation sensitivity modelling in combination with the calculation of the decrease in seed water activity during cooling facilitated the explanation of the beneficial effect of controlled rehydration after desiccation and LN exposure. These results support the hypothesis that imbibitional membrane damage is involved in the sensitivity of coffee seeds to LN exposure.