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.     

Drying Increases Intracellular Partitioning of Amphiphilic Substances into the Lipid Phase : Impact on Membrane Permeability and Significance for Desiccation Tolerance

Previously we proposed that endogenous amphiphilic substances may partition from the aqueous cytoplasm into the lipid phase during dehydration of desiccation-tolerant organ(ism)s and vice versa during rehydration. Their perturbing presence in membranes could thus explain the transient leakage from imbibing organisms. To study the mechanism of this phenomenon, amphiphilic nitroxide spin probes were introduced into the pollen of a model organism, Typha latifolia, and their partitioning behavior during dehydration and rehydration was analyzed by electron paramagnetic resonance spectroscopy. In hydrated pollen the spin probes mainly occurred in the aqueous phase; during dehydration, however, the amphiphilic spin probes partitioned into the lipid phase and had disappeared from the aqueous phase below 0.4 g water g⁻¹ dry weight. During rehydration the probes reappeared in the aqueous phase above 0.4 g water g⁻¹ dry weight. The partitioning back into the cytoplasm coincided with the decrease of the initially high plasma membrane permeability. A charged polar spin probe was trapped in the cytoplasm during drying. Liposome experiments showed that partitioning of an amphiphilic spin probe into the bilayer during dehydration caused transient leakage during rehydration. This was also observed with endogenous amphipaths that were extracted from pollen, implying similar partitioning behavior. In view of the fluidizing effect on membranes and the antioxidant properties of many endogenous amphipaths, we suggest that partitioning with drying may be pivotal to desiccation tolerance, despite the risk of imbibitional leakage.     

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.     

Membrane behavior as influenced by partitioning of amphiphiles during drying: a comparative study in anhydrobiotic plant systems

During cellular desiccation, reduction in volume can in principle cause amphiphilic compounds to partition from the cytoplasm into membranes, with structural perturbance as the result. Here, we studied the effect of partitioning of endogenous amphiphiles on membrane surface dynamics in desiccation-tolerant and -intolerant, higher and lower plant systems, using electron paramagnetic resonance (EPR) spin probe techniques. Labeling cells with the amphiphilic spin probe perdeuterated TEMPONE (PDT) enabled partitioning into the various phases to be followed. During drying, PDT molecules preferentially partitioned from the aqueous cytoplasm into the membrane surface and, at advanced stages of water loss, also into oil bodies. There was no specific partition behavior that could be correlated with lower/higher plants or with desiccation-tolerance. In vivo labeling with 5-doxylstearate (5-DS) enabled membrane surface fluidity to be characterized. In hydrated plants, the 5-DS spectra contained an immobile and a fluid component. The characteristics of the immobile component could not be specifically correlated with either lower or higher plants, or with desiccation tolerance. The relative contribution of the fluid component to the 5-DS spectra was higher in lower plants than in higher plants, but considerably decreased with drying in all desiccation-tolerant organisms. In contrast, the proportion of the fluid component in desiccation-sensitive wheat seedling root was higher than that in desiccation-tolerant wheat axis and considerably increased at the onset of water loss. We suggest that partitioning of amphipaths fluidize the membrane surface, but that in desiccation-tolerant systems the membranes are protected from excessive fluidization.     

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.     

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.     

Effect of laser irradiation on microviscosity of aqueous medium in imbibing maize seeds as studied with a spin probe method

A spin probe method was used to study microviscosity of aqueous medium in embryos and endosperm of maize (Zea mays L.) seeds after their irradiation with a Lvov-1 Elektronika laser device. Based on parameters of electron spin resonance (ESR) spectra of nitroxyl radicals (probes) absorbed by imbibing seeds during water uptake, correlation times τ_C were determined for the rotational diffusion of probes in embryos and endosperm of seeds. The τ_C values were found smaller in embryos of irradiated seeds than in untreated seeds; the dependence of τ_C on the duration of seed imbibition was determined. It is concluded that laser irradiation of seeds decreases the microviscosity of aqueous medium in embryo cells and elevates the mobility of the probes. Effect of laser irradiation on τ_C in seed endosperm was less pronounced but also led to the increase in probe mobility.     

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.     

成熟脱水对种子发育和萌发的作用

成熟脱水是正常性种子发育的末端事件。种子在成熟时胚的脱水耐性增加;当种子萌发时胚变得不耐脱水。当种子获得脱水耐性时,糖、蛋白质和抗氧化防御系统等保护性物质积累;当脱水耐性丧失时,这些物质被降解。成熟脱水是种子从发育过程向萌发过程转变的“开关”,它降低发育的蛋白质和ｍＲＮＡ的合成,终止发育事件和促进萌发事件。顽拗性种子不经历成熟脱水的发育阶段,对脱水高度敏感。     

Disappearance of desiccation tolerance of imbibed crop seeds is not associated with the decline of oligosaccharides

The relationship between sugar content and loss of desiccation tolerance of hydrated crop seeds (tomato, okra, snow pea, mung bean, and cucumber) was evaluated by imbibing seeds with or without ABA, followed by dehydration and germination. During the process of hydration, but before the seeds lost desiccation tolerance, monosaccharide content increased only slightly, sucrose increased in snow peas, mung bean and cucumber, but maintained its original level in other species and the oligosaccharides declined dramatically. At the time of losing desiccation tolerance, the sucrose content of imbibed seeds was 2-3 times higher than the original level in most species. Positive significant correlation coefficients (r) were found in many, but not all crop seeds between desiccation tolerance and the oligosaccharide mass, or oligo/sucrose ratio. The ratio of oligo/sucrose in intact seeds at the time of losing desiccation tolerance, however, was not a fixed value and varied among species. Oligosaccharides declined significantly in different seed parts of imbibed cucumber seeds while sucrose increased to a higher level in the radicle than in the hypocotyl. Radicles were far more sensitive to desiccation than hypocotyls. The same observation was found for cucumber seeds imbibed in 100 M ABA, yet desiccation tolerance was largely maintained in hypocotyls and cotyledons. It is concluded that sucrose and oligosaccharides are not the determinants of the loss of desiccation tolerance in hydrated seeds.Imbibed seeds did not show any differences between seed parts in their ability to resynthesize sugars during the process of slow dehydration. Differences in sensitivity to desiccation among seed parts were not due to differences in the initial water content or to the rate of water content increase among seed parts. Physiological regulation of the loss of desiccation tolerance in crop seeds during hydration is discussed.     

Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.): II. DEVELOPMENT OF GERMINABILITY, VIGOUR, AND DESICCATION TOLERANCE

The germinability, vigour, and desiccation tolerance of muskmelon(Cucumis melo L. cv. Top Mark) seeds was studied in relationto changes in seed water content during development within thefleshy fruit. Seed water content (fresh weight basis) declinedfrom 91% to 42% between 10 d and 35 d after anthesis (DAA) (whenmaximum dry weight was attained), then declined more slowlyto a minimum of 35% at 50 DAA before increasing again to 43%at 65 DAA. Fresh intact seeds were first germinable at 25 to30 DAA and attained maximum germination percentages at 45 DAA.Between 15 and 35 DAA, cotyledons, hypocotyls, radicles andepicotyls of isolated embryos (testa and perisperm enveloperemoved) sequentially developed the ability to grow when incubatedon water. Dehydration to water contents less that those attainedwithin the fleshy fruit is not a requirement for developmentof germination capacity of muskmelon seeds. Seeds became tolerantof rapid desiccation after 25 DAA, and drying of immature seeds(25 to 40 DAA) increased their germination percentages uponsubsequent imbibition. Washing, drying, or washing followedby drying increased seedling vigour (root length) as comparedto fresh seeds, which had very poor vigour. Water absorptionisotherms were constructed to test whether changes in water-bindingcomponents were correlated with the development of desiccationtolerance. Isotherms for seeds older than 25 DAA fit well tothe D'Arcy/Watt model, which postulates the existence of high-affinity,low-affinity and multi-molecular water-binding sites. Desiccation-intolerantseeds younger than 25 DAA lacked the component of the absorptionisotherm characteristic of the high-affinity water-binding siteswhich have been hypothesized to confer desiccation tolerance.However, we were unable to determine whether the absence ofhigh-affinity binding characteristics was specifically relatedto desiccation intolerance or was artifactual due to the lossof volatiles when immature seed samples were dried at high temperatures. Key words: Muskmelon, embryo, germination, development, vigour, desiccation     

与种子耐脱水性有关的基础物质研究进展

耐脱水的获得和维持与种子的类型有关,正常型种子耐脱水,而顽拗形种子对脱水高度敏感,正常型种子的脱水耐性随发良[过程而变化,种子成熟时胚的脱水耐性增强,其萌发时胚变为不耐脱水,当种子获得脱水耐性时,糖,LEA蛋白质和抗氧化防御系统等保护性物质积累,但脱水耐性是一种复杂的数量的特性,任何一种单一的机制都不能 充分地解释脱水耐性,各种保护性物质协同调节脱水耐性,本文综述了近几年来关于种子耐脱水性与保护性物质相关性的研究进展。     

与种子耐脱水性有关的基础物质研究进展

耐脱水的获得和维持与种子的类型有关,正常型种子耐脱水,而顽拗形种子对脱水高度敏感。正常型种子的脱水耐性随发育过程而变化,种子成熟时胚的脱水耐性增强,而萌发时胚变为不耐脱水。当种子获得脱水耐性时,糖、LEA蛋白质和抗氧化防御系统等保护性物质积累。但脱水耐性是一种复杂的数量的特性,任何一种单一的机制都不能充分地解释脱水耐性,各种保护性物质协同调节脱水耐性。本文综述了近几年来关于种子耐脱水性与保护性物质相关性的研究进展。     

Developmental Changes in Relation to Desiccation Tolerance of Archontophoenix alexandrae (Palmae) Seeds

Germination of Archontophoenix alexandrae seeds and embryos were studied under gradient water content treatments throughout the seed development phases of maturation in 2005 to investigate seed desiccation tolerance and storage characteristics. During the maturation process, seed water content decreased gradually from55 DAF (days after flowering) to 70 DAF, and seeds reached the maximum dry-weight at 90 DAF. Seed germinability appeared after 60 DAF. Seeds germinated with a temperature range from15℃- 40℃ under alternating photoperiod (14 h light, 10 h dark, 12μmol m^{- 2}s ^{- 1} ), while the best germination percentage was obtained between 30℃- 35℃. A maximum germination capacity reached at 70 DAF. However, seed germination was greatly inhibited by light. Desiccation tolerance of seeds and embryos increasedgradually from 55 DAF to 90 DAF and reached the maximum at 90 DAF with a semilethal water content of 0.18 g/g ( seed) and 0.3 g/g ( embryo) respectively. Rapid dehydration maintained higher seed germination percentage than thatof slow dehydration when drying to the same water content. Seeds with without water content treatments failed to germinate after 1 month storage under - 18℃, whereas appropriate desiccation treatment prolonged seed longevity under 4℃, 10℃ and 15℃ storage temperatures. It revealed obviously the recalcitrant characteristics of Archontophoenix alexandrae seeds torage behaviour which are tolerant toward neither deep desiccation nor low temperatures.     

成熟脱水对种子发育和萌发的作用

成熟脱水是正常性种子发育的末端事件。种子在成熟时胚的脱水耐性增加;当种子萌发时胚变得不耐脱水。当种子获得脱水耐性时,糖、蛋白质和抗氧化防御系统等保护性物质积累;当脱水耐性丧失时,这些物质被降解。成熟脱水是种子从发育过程向萌发过程转变的“开关”,它降低发育的蛋白质和mRNA的合成,终止发育事件和促进萌发事件。顽拗性种子不经历成熟脱水的发育阶段,对脱水高度敏感。     

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.     

脱水导致的胞内溶质变化与植物耐干性的获得

植物耐干性是指许多植物个体和部分植物种子能在含水量极低的条件下存活,在回水的过程中迅速启动修复机制,细胞经重新水合修复所受损伤的能力。在脱水过程中,植物会合成和积累某些小分子物质、碳水化合物和特殊的蛋白质;在极度脱水状态下,多组分参与的玻璃化的形成和两性物质的重新分配、耐干性植物中特有的抗氧化机制都是植物获得耐干性的重要条件。复苏植物(resurrection plant)和部分被子植物种子是当前研究植物耐干性的模式材料。     

Phospholipase Dα1‐mediated phosphatidic acid change is a key determinant of desiccation‐induced viability loss in seeds

High sensitivity of seeds to water loss is a widespread phenomenon in the world's plant species. The molecular basis of this trait is poorly understood but thought to be associated with critical changes in membrane function. We profiled membrane lipids of seeds in eight species with varying levels of desiccation tolerance and found a close association between reducing seed viability and increasing phosphatidic acid (PA). We applied hydration–dehydration cycles to Arabidopsis seeds, which are normally desiccation tolerant, to mimic the onset of desiccation sensitivity with progression towards germination and examined the role of phospholipase D (PLD) in desiccation stress‐induced production of PA. We found that PLDα1 became more abundant and migrated from the cytosol to the membrane during desiccation, whereas PLDδ did not change, and that all desiccation‐induced PA was derived from PLDα1 hydrolysis. When PLDα1 was suppressed, the germination level after each hydration–dehydration cycle improved significantly. We further demonstrated that PLDα1‐mediated PA formation modulates desiccation sensitivity as applying its inhibitor improved seed desiccation tolerance and its suppression in protoplasts enhanced survival under dehydration. The insights provided by comparative lipidomics enable us to propose a new membrane‐based model for seed desiccation stress and survival.     

Homeohydrous (Recalcitrant) Seeds: Dehydration, the State of Water and Viability Characteristics in Landolphia kirkii

Differential scanning calorimetry was used to study the relationships among drying rate, desiccation sensitivity, and the properties of water in homeohydrous (recalcitrant) seeds of Landolphia kirkii. Slow drying of intact seeds to axis moisture contents of approximately 0.9 to 0.7 gram/gram caused lethal damage, whereas very rapid (flash) drying of excised embryonic axes permitted removal of water to approximately 0.3 gram/gram. The amount of nonfreezable water in embryonic axes (0.28 gram H₂O/gram dry mass) did not change with drying rate and was similar to that of desiccation-tolerant seeds. These results suggest that the amount of nonfreezable water per se is not an important factor in desiccation sensitivity. However, flash drying that removed all freezable water damaged embryonic axes. Differences between desiccation-sensitive and -tolerant seeds occur at two levels: (a) tolerant seeds naturally lose freezable water, and sensitive seeds can lose this water without obvious damage only if it is removed very rapidly; (b) tolerant seeds can withstand the loss of a substantial proportion of nonfreezable water, whereas sensitive seeds are damaged if nonfreezable water is removed.