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.     

Characterization of Membrane Properties in Desiccation-Tolerant and -Intolerant Carrot Somatic Embryos

In previous studies, we have shown that carrot (Daucus carota L.) somatic embryos acquire complete desiccation tolerance when they are treated with abscisic acid during culture and subsequently dried slowly. With this manipulable system at hand, we have assessed damage associated with desiccation intolerance. Fast drying caused loss of viability, and all K+ and carbohydrates leached from the somatic embryos within 5 min of imbibition. The phospholipid content decreased by about 20%, and the free fatty acid content increased, which was not observed after slow drying. However, the extent of acyl chain unsaturation was unaltered, irrespective of the drying rate. These results indicate that, during rapid drying, irreversible changes occur in the membranes that are associated with extensive leakage and loss of germinability. The status of membranes after 2 h of imbibition was analyzed in a freeze-fracture study and by Fourier transform infrared spectroscopy. Rapidly dried somatic embryos had clusters of intramembraneous particles in their plasma membranes, and the transition temperature of isolated membranes was above room temperature. Membrane proteins were irreversibly aggregated in an extended [beta]-sheet conformation and had a reduced proportion of [alpha]-helical structures. In contrast, the slowly dried somatic embryos had irregularly distributed, but non-clustered, intramembraneous particles, the transition temperature was below room temperature, and the membrane proteins were not aggregated in a [beta]-sheet conformation. We suggest that desiccation sensitivity of rapidly dried carrot somatic embryos is indirectly caused by an irreversible phase separation in the membranes due to de-esterification of phospholipids and accumulation of free fatty acids.     

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.     

Desiccation tolerance of protoplasts isolated from pea embryos

To facilitate studies of desiccation tolerance at the cellular level, a technique to isolate protoplasts from desiccation-tolerant pea (Pisum sativum L. cv. Alaska) embryos has been developed. Using FDA (fluorescein diacetate) as a probe, viability of the protoplasts was investigated before and after drying to determine whether the protoplasts could survive desiccation in a manner similar to the tissue from which they were isolated. Protoplasts were isolated from 12 h imbibed pea axes, suspended in several different sugar solutions, then dried to water contents less than 0.2 g H(2)O g(-1) DW. Protoplasts only survived drying if the rate was rapid (<2 h), while slow drying (24 h) was lethal. Maximal survival (75%) was obtained after drying protoplasts with a mixture of sucrose and raffinose, while pure sucrose and trehalose were somewhat less effective protectants. Low survival was obtained after drying protoplasts with monosaccharides and pure raffinose. Protoplasts isolated from germinated seedlings did not survive dehydration below 0.2 g H(2)O g(-1) DW. Transmission electron microscopy revealed that dried desiccation-tolerant protoplasts appeared shrunken, with folded membranes, while dried protoplasts from sensitive tissue had disrupted membranes. While isolated protoplasts maintained some of the desiccation tolerance of orthodox seeds, their inability to survive complete drying and their sensitivity to drying rate is similar to the behaviour of recalcitrant embryos.     

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.     

Fourier Transform Infrared Microspectroscopy Detects Changes in Protein Secondary Structure Associated with Desiccation Tolerance in Developing Maize Embryos

Isolated immature maize (Zea mays L.) embryos have been shown to acquire tolerance to rapid drying between 22 and 25 d after pollination (DAP) and to slow drying from 18 DAP onward. To investigate adaptations in protein profile in association with the acquisition of desiccation tolerance in isolated, immature maize embryos, we applied in situ Fourier transform infrared microspectroscopy. In fresh, viable, 20- and 25-DAP embryo axes, the shapes of the different amide-I bands were identical, and this was maintained after flash drying. On rapid drying, the 20-DAP axes had a reduced relative proportion of α-helical protein structure and lost viability. Rapidly dried 25-DAP embryos germinated (74%) and had a protein profile similar to the fresh control axes. On slow drying, the α-helical contribution in both the 20- and 25-DAP embryo axes increased compared with that in the fresh control axes, and survival of desiccation was high. The protein profile in dry, mature axes resembled that after slow drying of the immature axes. Rapid drying resulted in an almost complete loss of membrane integrity in the 20-DAP embryo axes and much less so in the 25-DAP axes. After slow drying, low plasma membrane permeability ensued in both the 20- and 25-DAP axes. We conclude that slow drying of excised, immature embryos leads to an increased proportion of α-helical protein structures in their axes, which coincides with additional tolerance of desiccation stress.     

Effects of Drying Rates on the Desiccation Tolerance of Citrus maxima ‘Feizhouyou’ Seeds

The effects of drying rates on the desiccation tolerance of Citrus maxima ‘Feizhouyou’ seeds at different developmental stages were studied in this paper. For seeds harvested at 130 days after anthesis (DAA), 190 DAA, 245 DAA and 275 DAA, slow dried seeds had higher desiccation tolerance than those rapid dried, with difference at significant level (P < 005). However, such improvement was little for seeds harvested at 155 DAA and 220 DAA, indicating that effect of drying rate on desiccation tolerance depends on seed developmental stages. These results accorded with previous reports on orthodox soybean seeds and maize embrys. It was suggested that the effects of drying rate on desiccation tolerance of intermediate Citrus maxima ‘Feizhouyou’ seeds mainly resulted from expression and accumulation of some desiccation related proteins induced by slow drying. On the required genetic basis, desiccation tolerance in seeds can be induced only at suitable seed developmental stages.     

Water Content, Raffinose, and Dehydrins in the Induction of Desiccation Tolerance in Immature Wheat Embryos

Desiccation tolerance is initiated in wheat (Triticum aestivum L.) embryos in planta at 22 to 24 d after anthesis, at the time that the embryo water content has decreased from about 73% fresh weight (2.7 g water/g dry weight) to about 65% fresh weight (1.8 g water/g dry weight). To determine if desiccation tolerance is fully induced by the loss of a relatively small amount of water, detached wheat grains were treated to reduce the embryo water content by just a small amount to approximately 69% (2.2 g water/g dry weight). After 24 h of such incipient water loss, subsequently excised embryos were able to withstand severe desiccation, whereas those embryos that had not previously lost water could not. Therefore, a relatively small decrease in water content for only 24 h acts as the signal for the development of desiccation tolerance. Embryos that were induced into tolerance by a 24-h water loss had no detectable raffinose. The oligosaccharide accumulated at later times even in embryos of detached grains that had not become desiccation tolerant, although tolerant embryos (i.e. those that previously had lost some water) contained larger amounts of the carbohydrate. It is concluded that desiccation tolerance and the occurrence of raffinose are not correlated. Immunodetected dehydrins accumulated in embryos in planta as desiccation tolerance developed. Detachment of grains induced the appearance of dehydrins at an earlier age, even in embryos that had not been made desiccation tolerant by incipient drying. It is concluded that a small reduction in water content induces desiccation tolerance by initiating changes in which dehydrins might participate but not by their interaction with raffinose.     

黄皮种子发育过程中脱水敏感性与细胞膜透性的关系

黄皮（Ｃｌａｕｓｅｎａ ｌａｎｓｉｕｍ （Ｌｏｕｒ．） Ｓｋｅｅｌｓ）胚轴与完整种子的发育模式以及发育中电解质渗漏率变化有些不同． 种子生理成熟前、后的胚轴对脱水的反应也不同,前者经轻微脱水可提高萌发率和活力指数,后者不耐任何程度的脱水．活力指数的急剧下降伴随着电解质渗漏率的迅速上升．实验表明,黄皮种子在发育过程中没有形成耐脱水性． 细胞膜透性变化可反映脱水对种子的伤害程度     

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。     

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.     

Survival of root‐lesion nematodes (Pratylenchus thornei) after wheat growth in a vertisol is influenced by rate of progressive soil desiccation

The root‐lesion nematode (Pratylenchus thornei) is a major pathogen of wheat in the subtropical grain region of eastern Australia. Experiments were conducted to learn whether soil desiccation can account for the rapid fall in peak P. thornei population densities noted in the field after wheat matures. The decline in population densities of P. thornei after growth of wheat was measured on progressive desiccation of soil with roots by fast and slow drying methods. The vertisolic soil of initial moisture content 45% w/w (or matric potential of pF 3.3) was dried in 5% decrements to an air‐dried gravimetric moisture content of 15% (pF 5.6) taking 10.7 h for fast drying and 91.5 h for slow drying. After drying, live nematodes were extracted with Whitehead trays for 2 and 7 days and counted in four life stages (adults and juvenile stages J2, J3 and J4). Fast drying resulted in a sigmoidal decline in total P. thornei with only 5% of the population alive in soil at 15% moisture content, but slow drying had no significant effect on the population density. The percentage of nematodes extracted at 2 days compared with the total extracted over 7 days in undried soil (～89% of total) declined quadratically on desiccation to be 48% (fast drying) and 78% (slow drying) at 15% moisture content. With fast drying, the proportion of adults and J2 decreased whereas the proportion of J4 increased as the soil dried. With slow drying, the proportion of J2 and J3 stages decreased while the proportion of J4 increased. Thus the J4 or pre‐adult was the life stage most tolerant of soil desiccation. Time is required for P. thornei to go into a state of anhydrobiosis as a soil dries and this information can be used to model P. thornei survival in the field based on environmental parameters.     

Changes in starch and soluble sugars in relation to the acquisition of desiccation tolerance during maturation of Brassica campestris seed

Abstract. We investigated the onset of desiccation tolerance in developing embryos of Brassica campestris seeds and possible correlated ultrastructural modifications in the radiele cells. Since the acquisition of desiccation tolerance is a long asynchronous process which took 9 d to be achieved, we determined criteria allowing us to separate freshly intact harvested seeds into desiccation intolerant and desication tolerant batches that differed in age by only 2 d. No particular structural modifications were found except a strong depletion of intraplastidial starch (-90%) coincident with the appearance of stachyose and an increase of sucrose (+30%) on the acquisition of desiccation tolerance. As we did not observe an increase of lipid reserves as a consequence, we suggest that these metabolic events can be a key factor towards the acquisition of desiccation tolerance.     

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。     

Changes in oligosaccharide content and antioxidant enzyme activities in developing bean seeds as related to acquisition of drying tolerance and seed quality.

Seeds of bean (Phaseolus vulgaris cv. Vernel) were collected throughout their development on the plant and dried at 15 degrees C and 75% relative humidity to a final moisture content of about 16% (fresh weight basis) to determine whether the onset of tolerance to this drying condition was related to changes in soluble sugars or the activities of the main antioxidant enzymes, namely superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR). Measurements of soluble sugars and enzyme activities were made after drying the seeds, and drying tolerance was evaluated by the ability of dried seeds to germinate and to produce normal seedlings. Seeds became tolerant to drying at 45 d after anthesis, a time marking physiological maturity. At physiological maturity, the moisture content of seeds was about 50-55% (fresh weight basis) and seed dry matter reached about 190 mg per seed. Seed vigour, evaluated by controlled deterioration and conductivity measurements, continued to increase after seed mass maturity, but decreased when seeds remained thereafter for more than 7 d on the plant. Acquisition of drying tolerance was coincident with an accumulation of raffinose and stachyose. Dried-tolerant seeds were also characterized by a high amount of sucrose, the most abundant sugar, and by a low content of monosaccharides. The (raffinose+stachyose)/sucrose ratio increased during seed filling, reaching a value close to 1 when all the seeds became tolerant to drying, and maintaining this proportion during the final stages of maturation. Acquisition of drying tolerance was also related to a reorientation of the enzymatic antioxidant defence system. Drying-tolerant dried seeds displayed high CAT and GR activities and low SOD and APX activities, while the opposite condition was observed in immature dried seeds. The shift in antioxidant enzymes corresponded to the beginning of the maturation-drying phase. These results suggest that oligosaccharide metabolism and enzymatic antioxidant defences may be involved in acquisition of drying tolerance during bean seed development, but are not related to seed vigour.     

Desiccation of microspore derived embryos of oilseed rape (Brassica napus L.)

Summary Microspore-derived embryos from Brassica napus L. were dried to less than 15% moisture and stored dry for a minimum of 7 days. Successful plant regeneration was observed when embryos at the cotyledonary stage of development were treated with 50 uM ABA for 7 days prior to desiccation. Solid agar or liquid medium gave similar results. The rate of drying of embryos after ABA pretreatment had only minor effects on embryo survival, but for untreated embryos, slow drying gave a small degree of survival. These results are very comparable to those with alfalfa somatic embryos, suggesting that the ABA treatment of cotyledonary stage embryos may be broadly used as a pretreatment for inducing the expression of desiccation tolerance in plant embryos.     

Desiccation tolerance in embryonic stages of the tardigrade

Desiccation tolerance commonly found among tardigrades allows them to cope with temporal variation of available water. Although the long-term survival of adults has been demonstrated in several species, desiccation tolerance of eggs and embryos is less well studied, however it is an important aspect from an ecological and evolutionary point of view. For the first time we evaluated the desiccation tolerance and subsequent hatching success of five different developmental stages of the tardigrade species Milnesium tardigradum , when rehydrated following drying at eight different humidity levels (10, 20, 31, 40, 54, 59, 72, 81%). Humidity level and developmental stage are significant factors in determining successful hatch rates. The results showed that the less developed stages were quite sensitive to desiccation. Low humidity levels during the first 3 days of development lead to a decrease in hatch rates following rehydration. Later developmental stages showed higher hatch rates than embryos dried at earlier stages. However, fast drying at low humidity levels resulted in delayed development and lower hatch rates following rehydration. In general, further developed embryos exhibit a better survival capacity compared with younger stages.     

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.