Acquisition of desiccation tolerance in soybeans

The entry into a desiccation-tolerant state is a major developmental component of seed maturation. Development of desiccation tolerance of embryonic axes of soybean [Glycine max (L.) Merrill cv. Chippewa 64] was studied by measuring changes in electrolyte leakage. germination and relative growth rate after axes were rapidly air-dried to various water contents. Axes acquired the full capacity for germination at 34 days after flowering (DAF). and reached physiological maturity (maximum dry weight) at 48 DAF. When dried to water content h = 0. 08 (g water g⁻¹ dry weight). few axes germinated before 42 DAF. but more than 90% germinated after 48 DAF. However, electrolyte leakage of rehydrated axes showed a linear decline from 30 to 55 DAF. For developing axes there was a critical water content or desiccation threshold. which could be estimated by using the electrolyte leakage method. The threshold of desiccation tolerance decreased gradually from h = 1. 10 to 0. 18 as axes matured from 28 to 55 DAF. The development of desiccation tolerance continued after physiological maturity at 48 DAF. We conclude that the acquisition of desiccation tolerance of soybean axes is a gradual event, rather than an abrupt transition.     

Desiccation tolerance acquisition in developing beech (Fagus sylvatica L.) seeds: the contribution of dehydrin-like protein

The acquisition of desiccation tolerance (DT) in developing beech (Fagus sylvatica L.) seeds and the role of a dehydrin protein in this process were investigated. DT was determined by measurement of electrolyte leakage and germination capacity after drying to 10–12% moisture content (MC). In addition to mass maturity, the presence of heat-stable proteins, dehydrin accumulation and the peak of ABA content were measured in relation to the acquisition of DT. Mass maturity was achieved at 16 weeks after flowering (WAF). The germination capacity increased from 8% at 12 WAF to 80–90% after 16 WAF. Cell membrane integrity, measured as a decrease in electrolyte leakage after desiccation, was acquired at 16 WAF. Additionally, the ratio of heat-stable to soluble proteins was the highest at 16 WAF. One dehydrin-like protein with a molecular mass 44 kDa, named DHN44, was detected in embryonic axes at 16 WAF and in cotyledons at 17 WAF, and its gradual accumulation was observed in mature seeds. With regard to the acquisition of DT, the strongest correlations were detected between electrolyte leakage, DHN44 accumulation, and the percentage of heat-stable proteins. These results suggest that developing beech seeds become tolerant to desiccation at 16 WAF. The effect of desiccation and ABA treatment on DHN44 synthesis was tested before (14 WAF) and after the DT acquisition (18 WAF). Depending on the maturation stage desiccation and ABA treatment can induce or enlarge DHN44 expression.     

The role of sugar, vitrification and membrane phase transition in seed desiccation tolerance

In a search for the mechanism of desiccation tolerance, a comparison was made between orthodox (desiccation-tolerant) soybean ( Glycine max [L.] Merrill) and recalcitrant (desiccation-intolerant) red oak ( Quercus rubra L.) seeds. During the maturation of soybean seeds, desiccation tolerance of seed axes is correlated with increases in sucrose, raffinose and stachyose. In cotyledons of mature oak seeds, sucrose levels are equal to those in mature soybeans, but oligosaccharides are absent. By using the thermally stimulated current method, we observed the glassy state in dry soybean seeds during maturation. Oak cotyledons showed the same phase diagram for the glass transition as did mature soybeans. By using X-ray diffraction, we found the maturation of soybeans to be associated with an increased ability of membranes to retain the liquid crystalline phase upon drying, whereas the mature oak cotyledonary tissue existed in the gel phase under similar dry conditions. These findings lead to the conclusion that the glassy state is not sufficient for desiccation tolerance, whereas the ability of membranes to retain the liquid crystalline phase does correlate with desiccation tolerance. An important role for soluble sugars in desiccation tolerance is confirmed, as well as their relevance to membrane phase changes. However, the presence of soluble sugars does not adequately explain the nature of desiccation tolerance in these seeds.     

Desiccation of the resurrection plant Craterostigma plantagineum induces dynamic changes in protein phosphorylation

Reversible phosphorylation of proteins is an important mechanism by which organisms regulate their reactions to external stimuli. To investigate the involvement of phosphorylation during acquisition of desiccation tolerance, we have analysed dehydration-induced protein phosphorylation in the desiccation tolerant resurrection plant Craterostigma plantagineum. Several dehydration-induced proteins were shown to be transiently phosphorylated during a dehydration and rehydration (RH) cycle. Two abundantly expressed phosphoproteins are the dehydration- and abscisic acid (ABA)-responsive protein CDeT11-24 and the group 2 late embryogenesis abundant (LEA) protein CDeT6-19. Although both proteins accumulate in leaves and roots with similar kinetics in response to dehydration, their phosphorylation patterns differ. Several phosphorylation sites were identified on the CDeT11-24 protein using liquid chromatography-tandem mass spectrometry (LCMS/MS). The coincidence of phosphorylation sites with predicted coiled-coil regions leads to the hypothesis that CDeT11-24 phosphorylations influence the stability of coiled-coil interactions with itself and possibly other proteins.     

Temporal profiling of the heat-stable proteome during late maturation of Medicago truncatula seeds identifies a restricted subset of late embryogenesis abundant proteins associated with longevity

Developing seeds accumulate late embryogenesis abundant (LEA) proteins, a family of intrinsically disordered and hydrophilic proteins that confer cellular protection upon stress. Many different LEA proteins exist in seeds, but their relative contribution to seed desiccation tolerance or longevity (duration of survival) is not yet investigated. To address this, a reference map of LEA proteins was established by proteomics on a hydrophilic protein fraction from mature Medicago truncatula seeds and identified 35 polypeptides encoded by 16 LEA genes. Spatial and temporal expression profiles of the LEA polypeptides were obtained during the long maturation phase during which desiccation tolerance and longevity are sequentially acquired until pod abscission and final maturation drying occurs. Five LEA polypeptides, representing 6% of the total LEA intensity, accumulated upon acquisition of desiccation tolerance. The gradual 30-fold increase in longevity correlated with the accumulation of four LEA polypeptides, representing 35% of LEA in mature seeds, and with two chaperone-related polypeptides. The majority of LEA polypeptides increased around pod abscission during final maturation drying. The differential accumulation profiles of the LEA polypeptides suggest different roles in seed physiology, with a small subset of LEA and other proteins with chaperone-like functions correlating with desiccation tolerance and longevity.     

板栗种子发育期间ABA等生理指标与脱水耐性的相关性研究

对板栗种子发育期间胚轴和子叶中部分生理指标的变化以及它们与板栗种子脱水耐性的相关性进行研究.结果表明:随着板栗种子不断发育,在花后80 d胚轴和子叶中ABA、淀粉、可溶性蛋白质量分数同步达到最大值,可溶性糖质量分数达到最低,此时板栗种子的脱水耐性最强,可确定为板栗的最佳采收期.另外,通过相关分析可知,板栗种子在发育期间...     

Desiccation tolerance of Sphagnum revisited: a puzzle resolved

As ecosystem engineers, Sphagnum mosses control their surroundings through water retention, acidification and peat accumulation. Because water retention avoids desiccation, sphagna are generally intolerant to drought; however, the literature on Sphagnum desiccation tolerance (DT) provides puzzling results, indicating the inducible nature of their DT. To test this, various Sphagnum species and other mesic bryophytes were hardened to drought by (i) slow drying; (ii) ABA application and (iii) chilling or frost. DT tolerance was assessed as recovery of chlorophyll fluorescence parameters after severe desiccation. We monitored the seasonal course of DT in bog bryophytes. Under laboratory conditions, following initial de‐hardening, untreated Sphagnum shoots lacked DT; however, DT was induced by all hardening treatments except chilling, notably by slow drying, and in Sphagnum species of the section Cuspidata. In the field, sphagna in hollows and lawns developed DT several times during the growing season, responding to reduced precipitation and a lowered water table. Hummock and aquatic species developed DT only in late autumn, probably as a response to frost. Sphagnum protonemata failed to develop DT; hence, desiccation may limit Sphagnum establishment in drier habitats with suitable substrate chemistry. Desiccation avoiders among sphagna form compact hummocks or live submerged; thus, they do not develop DT in the field, lacking the initial desiccation experience, which is frequent in hollow and lawn habitats. We confirmed the morpho‐physiological trade‐off: in contrast to typical hollow sphagna, hummock species invest more resources in water retention (desiccation avoidance), while they have a lower ability to develop physiological DT.     

The Maillard reaction and oxidative stress during aging of soybean seeds

The chemical reactions that may lead to the loss of seed viability were investigated both during the accelerated aging and natural aging of soybeans ( Glycine max Merrill cv. Chippewa 64). Under conditions of accelerated aging (36°C and 75% RH), fluorescence of soluble proteins accumulated, which was closely correlated with the loss of seed germinability and vigor. We were able to show this correlation by using partially purified proteins for the assay. Fluorescence also increased in seeds under good storage conditions (5°C for up to 21 years), although there was a less significant correlation between seed viability and the accumulation of fluorescent products during the time of natural aging. The rise in protein fluorescence is interpreted as an increase of Maillard products. The carbonyl content of soluble proteins (a measure of the oxidative damage) did not change significantly during either accelerated aging or natural aging: however the elimination of carbonyls during germination seemed to be hindered in seeds that had poor germination. The Maillard reaction may be a consequence of the formation of reducing sugars through a gradual hydrolysis of oligosaccharides during aging. Preliminary evidence from the natural aging study showed that, when seeds were in the glassy state, the sugar hydrolysis was inhibited. These results suggest that the Maillard reaction and oxidative reaction may play an important role in seed deterioration.     

Acquisition of desiccation tolerance by isolated maize embryos exposed to different conditions: the questionable role of endogenous abscisic acid

The effect of exogenous ABA on acquisition of desiccation tolerance has been well documented for the embryos of several species. including maize ( Zea mays L.). It has also been suggested that endogenous ABA plays a role in regulating the same phenomena. To test this hypothesis, endogenous ABA was quantified by radioimmunoassay. Our results show that: (1) during embryogenesis in maize, endogenous ABA increase-concomitantly with the acquisition of desiccation tolerance: (2) ABA deficient embryos of the vp 5 mutant are desiccation intolerant, but tolerance can he induced by exogenous ABA: and (3) desiccation tolerance is acquired if desiccation sensitive embryos undergo a slow drying treatment, during which ABA increases. However, when embryos were preincubated in fluridone to prevent ABA accumulation during slow drying, desiccation tolerance was induced in spite of the low level of endogenous ABA in the embryo. Our results cast doubts on an exclusive role of ABA in the acquisition of desiccation tolerance in maize embryo.     

Artificial seeds of alfalfa (Medicago sativa L.). Induction of desiccation tolerance in somatic embryos

Summary The use of somatic embryos from cell culture systems in the clonal propagation of plants would be greatly facilitated if the somatic embryos could be dried and stored in a dormant state similar to true seeds. A cell culture system was developed for alfalfa (Medicago sativa L.) line RL34 which gave high yields of somatic embryos in an approximately synchronized pattern. These somatic embryos were treated with abscisic acid (ABA) at the cotyledonary stage of development to induce desiccation tolerance. With no visual preselection, approximately 60% of the dried embryos converted into plants upon reimbibition. When high quality embryos were selected prior to drying, 90 to 100% conversion rates were observed. The timing of the application of ABA in terms of embryo development was critical with an optimum being at cotyledonary stage spanning approximately 4 days; thus, synchronized embryo development is required for optimal expression in bulk samples. The vigor of the seedlings from dried somatic embryos was greater than those from embryos which had not been dried, but remained substantially lower than those from true seeds.     

Nuclear and cytoplasmic "stay-green" mutations of soybean alter the loss of leaf soluble proteins during senescence

In soybean ( Glycine max [L.] Merr.) the homozygous combination of the recessive alleles dI and d2 (i.e., dldld2d2 ) at two different nuclear loci or the cytoplasmic gene cytG inhibit chlorophyll degradation during senescence; i.e. their leaves are green when they are shed. The main objectives of the present work were: (J) to determine whether these "stay-green" genes also interfere with the loss of the bulk of leaf soluble proteins and ribulose bisphospnate carboxylase/oxygensase (Rubisco; EC 4.1.1.39) during senescence and (2) to relate this to alterations in leaf proteolytic activity. Leaves of the normal. Yellowing cvs Clark and Harosoy lost about 90% of their soluble proteins before abscission. The abscising leaves of these cultivars contained no detectable Rubisco. By contrast, protein degradation was significantly less in leaves of near-isogenic lines of Clark and Harosoy carrying dIdId2d2 , with or without G (a dominant nuclear gene in a third locus causing green seed coats). These leaves still retained 50% of the soluble protein and large amounts of both subunits of Rubisco at the time of abscission. Alone, neither dl nor d2 had any effect. The cytoplasmic gene cytG slowed the loss of Rubisco. although eventually when leaves were shed they contained as little Rubisco as Clark. Despite inhibition (i.e. dIdId2d2 and GGdIdId2d2 ) or retardation (i.e. cytG ) of protein loss, these mutant genotypes did not differ from Clark in the breakdown of endogenous Rubisco by leaf extracts ("autodigestion"). The wild-type alleles in the dI and d2 loci may control a central regulatory process of the senescence syndrome.     

栽培大豆和野生大豆耐盐性及离子效应的比较

以国际上常用的耐盐大豆（Glycine max L.)品种Lee68为对照,在发芽期和苗期两个阶段,利用发芽指数、指害指数和耐盐系数等指标对一年生具盐腺野生大豆（Glycine soja L.)和部分栽培大豆（Glycine max L.)及某些野生大豆品系或品种的耐盐性进行了比较,讨论了耐盐指标的可行性。从离子效应方面比较了Na^ 和Cl^-对大豆发芽率的影响,并对具盐腺野生大豆的耐盐机理进行了初步分析。结果表明,大豆品种的耐盐性在发芽期和苗期无一致相关性。轻度等渗胁迫下,Na^ 对种子发芽率的抑制作用大于Cl^-,而重度等渗胁迫下则相反。通过减少由根系吸收的Na^ 、Cl^-向叶片的运输,维持叶片中较高含量的K^ ,减轻盐离子毒害,可能是具盐腺野生大事耐盐的主要生理机制之一。     

Abscisic acid and osmotic stress or slow drying independently induce desiccation tolerance in mutant seeds of Arabidopsis thaliana

Action of endogenous abscisic acid (ABA) is absent in the ABA-deficient and -insensitive double mutant ( aba-1abi3–1 ) seeds of Arabidopsis thaliana . Thus, responses to osmotic stress and dehydration can be studied without interference of endogenous ABA. Seeds of this double mutunt are viable hut desiceation-intolerant. However, desiccation tolerance can he induced by either (1) slow dehydration of immature seeds; (2) treatment of immature seeds with osmotica or; (31 due to the leakiness of the ABA-insensitivty mutation, by application of exogenous ABA. Consequently it is concluded that either ABA or osmotic- or dehydration-stress and related gene expression meets the minimal requirements for acquisition of desiccation tolerance in seeds of Arabidopsis thalianna .     

Gaba shunt in developing soybean seeds is associated with hypoxia

In the present study we investigated the proposal that the γ-aminobutyrate (Gaba) shunt in developing soybean (Glycine max [L.] Merr.) seeds is associated with hypoxia. The ontogeny and pH profile of enzymes associated with glutamate metabolism (glutamate decarboxylase [EC 4.1.1.15]. Gaba transaminase [EC 2.6.1.19], succinic semialdehyde dehydrogenase [EC 1.2.1.16], glutamate dehydrogenase [EC 1.4.1.2], glutamate:oxaloacetate transaminase [EC 2.6.1.1], glutamate:pyruvate transaminase [EC 2.6.1.2] and 2-oxoglutarate dehydrogenase complex [EC 1.2.4.2]) and hypoxia (alcohol dehydrogenase [ADH, EC 1.1.1.1] and pyruvate decarboxylase [PDC, EC 4.1.1.1]) were determined in cotyledons, nucellus and seed-coat tissues. Gaba-shunt enzymes were ubiquitous in the developing seed. Activities of enzymes catalyzing glutamate-C entry into the Krebs cycle via 2-oxoglutarate were generally greater than those of Gaba-shunt enzymes. In cotyledons, the activity of ADH increased throughout seed development (up to 72 days after anthesis [DAA]), whereas PDC was static during early development, then increased. In contrast, the activities of ADH and PDC in maternal tissues (nucellus and seed coat) were initially high, then declined dramatically after 37 DAA. The adenylate energy charge (AEC) = ([ATP]+0.5 [ADP])/ ([ATP] + [ADP] + [AMP]) of soybean seeds from fruits (37 DAA) frozen in situ was low (0.67±0.01) compared to the AEC of adjacent pod tissue (0.82 ± 0.04) and cotyledons exposed to air (0.84 ± 0.01). A 60-min time-course study showed that the rate of [U-¹⁴C]-glutamate catabolism by an intact excised cotyledon at 37 DAA was markedly lower at 8 and 0% O₂ than at 21%; the pool size of [¹⁴C]-Gaba was unaffected. The data indicated that: (1) Gaba-shunt activity is not a response to limited glutamate deamination/transamination: (2) the soybean seed is hypoxic; and (3) the relative partitioning of glutamate-C through glutamate decarboxylase is increased by hypoxia.     

Lipid peroxidation and peroxide-scavenging in soybean seeds during aging

The possible role of lipid peroxidation in seed deterioration was investigated during natural aging and accelerated aging of seeds of edible soybean ( Glycine max [L], Merr. cv. Kaohsiung Selection No. 1). Natural aging was achieved by sealing the seeds in aluminum foil bags coated with polyethylene and storing the seeds at room temperature for 3 to 12 months. Accelerated aging was obtained by incubating the seeds at 45°C and close to 100% relative humidity for 3 to 12 days, after which the seeds were air dried to their original moisture level (8%). The results indicate that both natural and accelerated aging enhanced lipid peroxidation, as germination was depressed. Aging also inhibited the activity of superoxide dismutase, catalase, ascorbate peroxidase and peroxidase. The changes in germination and physiological activities, expressed as a function of aging duration, were somewhat similar in the two aging treatments.     

Salt tolerance of soybean cultivars

Soybean (Glycine max) cv. Clark tolerated NaCl salinity up to osmotic potential in soil -1800 kPa, cv. Forest to -1500 kPa and cv. Kint to -1200 kPa. The tolerance of cvs. Clark and Forest was found to be related to the accumulation of soluble proteins, amino acids, proline, K and Ca. The sensitivity of cv. Kint was found to be associated with decrease in water, saccharide, protein, K and Ca contents. The leaf osmotic potential decreased in cvs. Clark and Forest while remained unchanged in cv. Kint. This revised version was published online in July 2006 with corrections to the Cover Date.     

Light-quality and irradiance effects on pigments, light-harvesting proteins and Rubisco activity in a chlorophyll- and light- harvesting-deficient soybean mutant

Soybean ( Glycine max [L.] Merrill) plants, normal green (Clark L1) and mutant yellow (Clark y₉y₉), were grown in (1) full-spectrum solar irradiation; (2) either red plus far-red or blue plus far-red; (3) either red or blue light with no far-red light. Young leaves harvested from first (1TF) trifoliolate or fifth (5TF) trifoliolate stages of development showed that the mutant plants express pigment and protein deficiencies as a direct function of irradiance. Response of the mutant to light quality indicates that blue light slightly enhances expression of the mutation at higher irradiances. Direct response of light-harvesting proteins of photosystem 2 (LHCP2) and light-harvesting protein of photosystem 1 (LHCP1) to light quality increases the ratio of LHCP1/LHCP2 in blue light compared to that in red or red/far-red light. Rubisco proteins and Rubisco activity (leaf area basis) are directly related to irradiance level but are enhanced in blue light over equal irradiance red. This enhancement is not shown in the presence of far-red light.     

The effect of methionine, ethylene and polyamine catabolic intermediates on polyamine accumulation in detached soybean leaves

In the present study we determined the effects of methionine, intermediates of polyamine catabolic pathways and inhibitors of either ethylene biosynthetic or polyamine catabolic pathways on polyamine accumulation in soybean leaves. Inhibitors to SAM decarboxylase and spermidine synthase, methylglyloxal-bis-(guanylhy-drazone) and cyclohexylamine, respectively, suggest that methionine may provide aminopropyl groups for the synthesis of polyamine via S-adenosylmethionine (SAM). Results from experiments that utilized a combination of compounds which altered either ethylene or polyamine biosynthesis, namely, aminoethoxyvinyl glycine, CoSO₄, 2,5-norbornadiene, and CuSO₄, suggest the two pathways compete for a common precursor. However, exogenous addition of ethylene (via ethephon treatments) had little or no effect on polyamine biosynthesis. Likewise, polyamine treatments had little or no effect on ethylene biosynthesis. These data suggest that there are few or no inhibitory effects from the end products of one pathway on the synthesis of the other. Data from leaves treated with metabolic intermediates in the catabolic pathway of polyamines and inhibitors of enzymes in the catabolic pathway, i.e. aminoguanidine, hydroxyethyldrazine and gabaculine, suggest that the observed increases in polyamine titers were not due to decreased catabolism of the polyamines. One catabolic intermediate, γ-aminobutyric acid (GABA), elevated putrescine, spermidine and spermine by 12-, 1.4-, and 2-fold, respectively, Ethylene levels decreased (25%) in GABA-treated leaves. This small decrease in ethylene could not account for such large increase in putrescine titers. Further analysis demonstrated that the GABA-mediated polyamine accumulation was inhibited by difluoromethylarginine, an inhibitor of arginine decarboxylase, but not by difluoromethylornithine, an inhibitor of ornithine decarboxylase. These data suggest that GABA directly or indirectly affects the biosynthesis of polyamines via arginine decarboxylase.     

Abscisic-acid-induced drought tolerance in Funaria hygrometrica Hedw.

Three-week-old protonemata of Funaria hygrometrica Hedw. cultivated in Petri dishes tolerate slow drying (24 h to complete dryness) but not rapid drying (1h to complete dryness). Slowly dried mosses show, on a dry-weight basis, a sixfold increase in abscisic-acid (ABA) contents during the drying process. Rehydrated, slowly dried protonemata have the ability to tolerate subsequent rapid drying. When ABA is added to three-week-old protonemata at a concentration of 10 M for 16 h, tolerance to rapid drying is induced. These data indicate that the induction of drought tolerance in Funaria hygrometrica is mediated by ABA. Mosses treated with ABA loose their water as fast as controls do; therefore, ABA does not act via reduced water loss. However, induction of synthesis of new proteins by ABA may form an important part of the drought tolerance because 10 M cycloheximide inhibits the ABA-mediated tolerance to rapid drying.Abbreviations ABA abscisic acid - CHI cycloheximide - DW dry weight - FW fresh weight - RWL relative water loss This work was supported by grants from the Deutsche Forschungs-gemeinschaft and by a NATO fellowship awarded to R.M. Ros Espin.