Sugar effects on membrane damage during desiccation of pea embryo protoplasts

Desiccation tolerance of protoplasts isolated from germinating pea (Pisum sativum L. cv. 'Alaska') embryonic axes depends, in part, on the osmotic strength and composition of the suspending medium. To determine the reason for this dependence and whether treatment with different solutions results in different types of damage, protoplast recovery and survival were assessed after dehydration to a range of water contents. Protoplasts were derived from germinating axes that had intermediate desiccation tolerance. Protoplasts were isolated and resuspended in buffers containing sucrose/raffinose (85:15, w/w) or sorbitol, which were isotonic or hypertonic to the cells of the embryonic axis, then were flash-dried to a range of water contents. Protoplasts were rehydrated and stained with fluorescein diacetate (FDA) to assess survival and to estimate two types of membrane injury: lysis and the loss of semipermeability. In all treatments, protoplast survival dropped sharply during the initial phase of dehydration due to lysis. Protoplast survival was greater in hypertonic sucrose/raffinose buffer than in isotonic sucrose/raffinose buffer, or in the latter made hypertonic by the addition of sorbitol. When sorbitol was substituted for sucrose/raffinose in either the isolation or desiccation buffer, or both, protoplast survival at intermediate and low hydrations decreased due to a loss of membrane semipermeability. The results indicate that additional sucrose/raffinose is beneficial for the desiccation tolerance of protoplasts, the benefit is not due to a simple osmotic effect, and the benefit is greatest at water contents less than 0.5 g g(-1) DW, where the presence of the sugars appears to protect membrane semipermeability.     

Onset of desiccation tolerance during development of the barley embryo

We have investigated events which take place in the developing barley (Hordeum vulgare L.) embryo during its acquisition of desiccation tolerance. Excised embryos are capable of precocious germination as early as 8 d after pollination (DAP). At this age, however, they are not capable of resisting a desiccation treatment which induces a loss of 96–98% of their initial water content. At 16 DAP the embryos germinate despite the drastic drying treatment. The pattern of in-vivo and in-vitro proteins synthesized by the developing embryos from 12 DAP (desiccation-intolerant) and 16 DAP (desiccation-tolerant) were compared. A set of 25–30 proteins was identified which is denovo synthesized or enhanced during the developmental period leading to desiccation tolerance. Abscisic acid (ABA; 100 M) applied in vitro for 5 d to 12-DAP embryos induces desiccation tolerance and represses a subset of polypeptides preferentially associated with 16-DAP embryos. During in vitro culture of barley embryos ABA stimulates the appearance of a set of proteins and prevents the precocious germination allowing embryogenesis to continue in vitro. It also suppresses a set of germination-related proteins which appear 4 h after the incubation of the dissected embryo on a germination medium without ABA. Almost all mRNAs remain functional for translation when isolated embryos are dried at the desiccation-intolerant and tolerant stages of embryo development.Abbreviations ABA abscisic acid - DAP days after pollination - GM germination medium - poly(A)RNA polyadenylated RNA - SDS sodium dodecyl sulfate     

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.     

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.     

The loss of desiccation tolerance during germination: An ultrastructural and biochemical approach

Summary Rye embryos of high viability and vigour can be imbibed for 1 hour, dehydrated and subsequently rehydrated without harm. However, extension of the imbibition period results in progressive structural damage to cells of both the embryonic root and the coleorhiza. Greatest sensitivity to this treatment is shown by the microtubule assembly system and the plasmalemma which loses its integrity permitting the egress of ribosomes and lipid towards the cell wall. Further stress results in fragmentation of the endoplasmic reticulum, disruption of plastid, mitochondrial and nuclear membranes and the dispersion of the contents of provacuoles. Damage is initiated during the drying of imbibed embryos but it is compounded by subsequent rehydration. Coleorhiza cells, particularly those distal to the root, which normally develop very rapidly during the early hours of germination are most sensitive to desiccation. The onset of sensitivity to desiccation (ca. 3 hours after imbibition) corresponds with a transitory halt in the increasing rate of protein synthesis and with the start of DNA replication. These results are discussed in relation to DNA repair and the hardening of seeds to stimulate rapid growth following rehydration.     

Patterns of protein synthesis during the germination of pea axes,and the effects of an interrupting desiccation period

As germination of axes of Pisum sativum L. seeds progressed, profound quantitative and qualitative changes occurred in the patterns of protein synthesis. This was shown by fluorography of gels following two-dimensional polyacrylamide gel electrophoresis separation of [³⁵S]methioninelabelled proteins. The effects of desiccation during germination on these in-vivo protein-synthesis patterns were followed. Desiccation differentially affected the synthesis of proteins. Usually, however, upon rehydration following desiccation the types of proteins being synthesized were recognizable as those synthesized earlier during imbibition of control, once-imbibed axes: seeds imbibed for 8 h, and then dried, did not recommence synthesis of proteins typical of 8-h-imbibed control seeds, but rather of 4-h-imbibed control seeds. Seeds imbibed for 12 h, and then dried and rehydrated, synthesized proteins typical of 4-h-and 8-h-control seeds. Thus drying of germinating pea axes caused the proteinsynthesizing mechanism to revert to producing proteins typical of earlier stages of imbibition. Drying during germination never caused the seed to revert to the metabolic status of the initial mature dry state, however.Abbreviation DR dried and rehydrated     

Effect of the pea embryo on formation and degeneration of the mitochondrial membrane in cotyledons during germination

The effect of removal of the embryo on the properties of mitochondriain pea cotyledons was investigated. During imbibition of theseeds, mitochondrial activity was enhanced in the cotyledons.In later stages of germination, respiratory activity of themitochondria decreased gradually, and no response of the mitochondriato exogenous ADP was observed. Moreover, considerable activityof cytochrome oxidase wasrecovered in the post-mitochondrialfraction. Mitochondrial fractions isolated from senescent cotyledonscontained only fragmented particles of mitochondria. On theother hand, in cotyledons excised from the seeds and cultivatedunder wet condition, the initial development of mitochondriademonstrated in the attached cotyledons was suppressed. However,respiratory activity of the mitochondria increased in the laterstages of cultivation. The mitochondria remained unfragmentedand responded to exogenous ADP during all stages of cultivation.Also, a change in the density of mitochondria which occurredin the germinating attached cotyledons was delayed in the cultivatedexcised cotyledons. (Received February 27, 1973; )     

Expression profiling of reciprocal maize hybrids divergent for cold germination and desiccation tolerance

Recombinant inbred lines (RILs) derived from B73 x M017 were screened for cold germination (CG) and desiccation tolerance (DT) phenotypes. Reciprocal F(1) hybrids were made between divergent RILs, and hybrids that showed differential phenotypes (parent-of-origin effect) for CG or DT were selected for profiling mRNA and protein expression. mRNA and proteins were extracted from embryo axes of seed germinated for 11 d at 12.5 degrees C in the dark and developing embryos at 40% seed moisture (R5 stage) for CG and DT, respectively. GeneCalling analysis, an open-ended mRNA profiling method, identified 336 of 32,496 and 656 of 32,940 cDNA fragments that showed >or=1.5-fold change in expression between the reciprocal F(1) hybrids for CG and DT, respectively. Protein expression map (PEM) analysis, an open-ended two-dimensional polyacrylamide gel electrophoresis, identified 117 of 2,641 and 205 of 1,876 detected proteins to be differentially expressed with >or=1.5-fold change between the reciprocal F(1) hybrids in CG and DT samples, respectively. A subset of these proteins was identified by tandem mass spectrometry followed by database query of the spectra. The differentially expressed genes/proteins were classified into various functional groups including carbohydrate and amino acid metabolism, ion transporters, stress and defense response, polyamine metabolism, chaperonins, cytoskeleton associated, etc. Phenotypic analysis of seed from self-pollinated ears of the reciprocal F(1) hybrids displayed small differences compared with the reciprocal hybrids themselves, suggesting a negligible effect of cytoplasmic factors on CG and DT traits. The results provide leads to improving our understanding of the genes involved in stress response during seed maturation and germination.     

假槟榔种子催芽技术和脱水耐性的研究

为提高假槟榔的人工种植技术,对其种子做了不同的化学催芽处理,以寻求种子的有效催芽方法,并对种子脱水耐性进行了探讨。结果表明:20%过氧化氢和98%浓硫酸浸泡5min,0.3%亚硝酸钠和0.2%硝酸钾溶液浸种24h后,发芽率显著升高,速度显著加快,尤以浓硫酸和硝酸钾处理效果为好;200～1000mg/L赤霉素和20～100mg/L激动素溶液浸泡24h也显著促进种子萌发,但催芽效果与溶液浓度有关。成熟种子轻度脱水,发芽率有所上升,但含水量下降至17%以下,发芽率急剧下降,当含水量下降10%以下,发芽力完全丧失。由此可见,种子很可能是中间型种子。     

The role of recovery of mitochondrial structure and function in desiccation tolerance of pea seeds

Mitochondrial repair is of fundamental importance for seed germination. When mature orthodox seeds are imbibed and germinated, they lose their desiccation tolerance in parallel. To gain a better understanding of this process, we studied the recovery of mitochondrial structure and function in pea (Pisum sativum cv. Jizhuang) seeds with different tolerance to desiccation. Mitochondria were isolated and purified from the embryo axes of control and imbibed-dehydrated pea seeds after (re-)imbibition for various times. Recovery of mitochondrial structure and function occurred both in control and imbibed-dehydrated seed embryo axes, but at different rates and to different maximum levels. The integrity of the outer mitochondrial membrane reached 96% in all treatments. However, only the seeds imbibed for 12 h and then dehydrated recovered the integrity of the inner mitochondrial membrane (IMM) and State 3 (respiratory state in which substrate and ADP are present) respiration (with NADH and succinate as substrate) to the control level after re-imbibition. With increasing imbibition time, the degree to which each parameter recovered decreased in parallel with the decrease in desiccation tolerance. The tolerance of imbibed seeds to desiccation increased and decreased when imbibed in CaCl(2) and methylviologen solution, respectively, and the recovery of the IMM integrity similarly improved and weakened in these two treatments, respectively. Survival of seeds after imbibition-dehydration linearly increased with the increase in ability to recover the integrity of IMM and State 3 respiration, which indicates that recovery of mitochondrial structure and function during germination has an important role in seed desiccation tolerance.     

Genetic basis of barley caryopsis dormancy and seedling desiccation tolerance at the germination stage

The genomic regions controlling caryopsis dormancy and seedling desiccation tolerance were identified using 152 F₄ lines derived from a cross between Mona, a Swedish cultivar, and an Israeli xeric wild barley Hordeum spontaneum genotype collected at Wadi Qilt, Israel. Dormancy, the inability of a viable seed to germinate, and desiccation tolerance, the ability of the desiccated seedlings to revive after rehydration, were characterized by fitting the germination and revival data with growth curves, using three parameters: minimum, maximum, and slope of germination or revival rate derived by the least square method. The genetic map was constructed with 85 genetic markers (SSRs, AFLPs, STSs, and Dhn genes) using the multipoint-mapping algorithm. Quantitative trait loci (QTLs) mapping was conducted with the multiqtl package. Ten genomic regions were detected that affected the target traits, seven of which affected both dormancy and desiccation tolerance traits. Both the wild barley genotype and the Swedish cultivar contributed the favorite alleles for caryopsis dormancy, whereas seedling desiccation tolerance was attributed to alleles descending from the cultivar. The results indicate that some barley dormancy genes are lost during domestication and that dormancy QTLs are associated with abiotic stress tolerance.     

Legumin and albumin of pea cotyledons during seed germination

During 6 days of pea seed germination the depletion of legumin with mol. m. 390 000 from protein bodies was observed. SDS-PAGE indicated that the legumin subunits with mol. m. 41 700 and 21 000 were prevailing. Only the former of these, probably corresponding to α-subunit, was degraded rapidly during 6 days of germination. Water-soluble proteins (albumins) prepared from pea cotyledons were separated by preparative IEF into proteins with pI 7.1, 6.5, 6.0, 5.4, 5.0, and 4.6. During 6 days the components of albumin with pI 7.1, and 6.5 were dramatically depleted. Major fractions with pI 6.5, 6.0, and 5.4 were subjected to SDS-PAGE and their subunit composition was determined. Moreover, albumin of pea cotyledons was resolved into 13 components by SDS-PAGE. Mobilization of albumin began from the degradation of components with higher mol. m. during germination.     

Sugar and starch changes in pea cotyledons during germination

Changes in starch and sugar contents in the cotyledons during germination have been compared in a smooth (cv. Alaska) and a wrinkled (cv. Progress) cultivar of the garden pea ( Pisum sativum L.). In both cultivars there was an initial accumulation of sucrose due to the hydrolysis of sucrosyl oligosaccharides, but galactose did not accumulate in the cotyledons. Starch mobilization in the Progress pea was linear with time and started before the rise in α-amylase (EC 3.2.1.1) activity in the cotyledons; sucrose was synthesized in the cotyledons, and their excision from the axis resulted in an additional accumulation of this sugar. In the Alaska pea, the onset of starch hydrolysis coincided with the rise in α-amylase activity; no accumulation of sucrose was found in excised cotyledons, whilst the sucrose content decreased continuously in attached cotyledons.
The same sugars were found in the cotyledons of both cultivars, suggesting a common pathway for starch breakdown. Maltose, maltotriose and linear malto-dextrins were not present and only trace amounts of glucose were detected, suggesting a degradation of starch by phosphorylase after an initial attack by α-amylase. α-Amylase activity in the cotyledons was higher in the presence of the axis, but was influenced by the water content of the cotyledons. Transient changes in α-amylase activity correlated well with changes in the rate of starch hydrolysis, but after 2–3 days starch mobilization was reduced in excised cotyledons probably due to the resynthesis of starch.     

Impact of culture age on conidial germination,desiccation and UV tolerance of entomopathogenic fungi

The impact of culture age on conidial yields, germination and tolerance to UV exposure of freshly harvested and dry conidia produced by five entomopathogenic fungal (EPF) isolates was studied. Beauveria bassiana, Metarhizium anisopliae, Lecanicillium lecanii and Lecanicillium muscarium were grown on potato dextrose agar (PDA) medium for 7 or 14 days at 25°C. While the age of cultures had a significant impact on the germination rate of conidia produced by isolates L. lecanii CBS 122.175 and B. bassiana LMSA 1.01.093, other EPF isolates germinated at the same rate regardless of the culture age. When exposed to UV radiation, conidia produced by all isolates germinated at a lower rate compared to the non-irradiated conidia, although this decrease in germination (20–80% decrease) was unaffected by the culture age. Air-drying had only a slight impact on conidial germination (0–60% decrease). Under the conditions of this study, the stability of irradiated conidia produced by M. anisopliae LMSA 1.01.197 and B. bassiana CBS 110.25 was significantly increased when conidia were dried prior to UV exposure. This increase in tolerance to stress of dried conidia might be caused, at least partially, by the low metabolic activity associated with dehydration.     

9种形态生理休眠的种子脱水对萌发和胚胎生长的影响

9种形态生理休眠的种子脱水对萌发和胚胎生长的影响 在具有形态生理种子休眠(MPD)的物种中，吸胀种子脱水对胚胎生长和萌发的影响鲜为人知。我们研究了9种不同MPD水平的种子对脱水的反应。对每个物种进行对照实验，使种子永久保持水化并暴露在最佳层积-培养顺序中以促进胚胎生长。同时也开展了室温条件下脱水中断种子层积处理1个月的实验。研究结果显示，具有非深度简单MPD的白藤铁线莲(Clematis vitalba)和高山茶藨子(Ribes alpinum)的胚生长 和种子活力均不受干燥影响，但干燥使高山茶藨子的萌发力下降了16%。具有深度简单上胚轴MPD的黄 水仙(Narcissus pseudonarcissus)种子在不同的胚生长阶段呈现脱水耐受性，但其萌发力略有下降。具有不同 MPD复杂水平的物种对脱水的反应更为多变：具有中度复杂MPD的Delphinium fissum亚种与具有深度复杂MPD的峨参(Anthriscus sylvestris)和熊根芹(Meum athamanticum)，具有脱水耐受性。与之相反，具有非深度复杂MPD的鹅莓(Ribes uva-crispa)、中度复杂MPD的Lonicera pyrenaica和深度复杂MPD的Chaerophyllum aureum，脱水后萌发力下降。虽然具有MPD简单水平的种子能够具备脱水耐受性，但一些具有复杂水平MPD的种子也具有很高的耐受性。因此，脱水不诱导胚生长后期的次生休眠。9种植物中大多数的吸胀种子的脱水耐受性可能表征其对地中海地区气候变化的适应性。     

Changes in chromatin structure associated with germination of maize and their relation with desiccation tolerance

Morphological and physicochemical measurements of chromatin condensation were made on germinating maize (Zea mays L.) radicles to determine whether the loss of genetic activities that occurs during the loss of desiccation tolerance is linked to irreversible changes in chromatin condensation. Chromatin samples were compared at different stages of germination (0, 24 and 72 h after imbibition), before (control) and after 24 h of desiccation. Morphological changes in chromatin structure and condensation were characterized by a qualitative and quantitative electron microscope study of chromatin which was allowed to spread in 0.2 mol m^?3 EDTA and then laid on coated microscope grids. The experiments showed similar levels of chromatin condensation in quiescent embryos and 24-h-old radicles (desiccation-tolerant material). After 72 h of imbibition, when radicle emergence and desiccation intolerance had ceased, the chromatin underwent a major decondensation towards various lower order folded structures. Regardless of the desiccation tolerance stage, an in vivo drying treatment of 24- and 72-h-old radicles before chromatin extraction did not induce significant changes in the extent of condensation compared to their respective controls. Similar conclusions were drawn from measurements of several spectroscopy properties (absorbance ratios: A₂₆₀/A₂₄₀, A₂₆₀/A4₀₀; thermal denaturation, and linear electric dichroism) of chromatin fragments that were obtained after nuclease digestion and then dissolved in 0-2 mol m^?3 EDTA. In quiescent and 24-h-old material, chromatin fragments were poorly soluble but highly stable during thermal denaturation. Chromatin fragments were 3-5-fold more soluble and less thermally stable in 72-h-old material than in 24-h-old material. In vivo desiccation had no significant effects on these properties compared to the respective controls. Collectively these data suggest that desiccation did not induce irreversible changes in the condensation properties of chromatin. The likelihood that the decondensation process occurring during germination is linked to the loss of desiccation tolerance is discussed.     

Ethanolamine incorporation into membranes of pea embryonic axes during germination

Changes in the ethanolamine pool of the embryonic axes of pea seeds exposed to different temperatures during imbibition and germination were followed. The ethanolamine pool decreased except during imbibition at 25°. Label from ethanolamine was incorporated almost entirely into phosphatidylethanolamine with incorporation into phosphatidylcholine being observed only after imbibition and germination at 25°. The incorporation of ethanolamine was apparently less sensitive to temperature than that of choline and glycerol, previously reported. Preliminary results also show an effect of the imbibition temperature on some of the membrane proteins, but most did not seem to be affected.     

Postgenomic analysis of desiccation tolerance

Desiccation tolerance is the capacity to survive complete drying. It is an ancient trait that can be found in prokaryotes, fungi, primitive animals (often at the larval stages), whole plants, pollens and seeds. In the dry state, metabolism is suspended and the duration that anhydrobiotes can survive ranges from years to centuries. Whereas genes induced by drought stress have been successfully enumerated in tissues that are sensitive to cellular desiccation, we have little knowledge as to the adaptive role of these genes in establishing desiccation tolerance at the cellular level. This paper reviews postgenomic approaches in a variety of desiccation tolerant organisms in which the genetic responses have been investigated when they acquire the capacity of tolerating extremes of dehydration or when they are dry. Accumulation of non-reducing sugars, LEA proteins and a coordinated repression of metabolism appear to be the essential and universal attributes that can confer desiccation tolerance. The protective mechanisms of these attributes are described. Furthermore, it is most likely that other mechanisms have evolved since the function of about 30% of the genes involved in desiccation tolerance remains to be elucidated. The question of the overlap between desiccation tolerance and drought tolerance is briefly addressed.