Desiccation of Phaseolus vulgaris Seeds During and Following Germination, and its Effect Upon the Translatable mRNA Population of the Seed Axes

Misra, S. and Bewley, J. D. 1986. Desiccation of Phaseolus vulgansseeds during and following germination, and its effect uponthe translatable mRNA population of the seed axes.—J.exp. BoL 37: 364–374. After imbibition and germination, seeds of P. vulgaris passfrom a stage where they are insensitive to desiccation to astage where they are sensitive. Desiccation of seeds duringthe sensitive stage results in an almost total impairment ofprotein synthesis upon subsequent rehydration. Seeds desiccatedduring the desiccation-tolerant stage, however, resume proteinsynthesis at almost control levels. The protein patterns obtained following in Vitro translationof bulk RNA from fresh imbibed, desiccated, and desiccated-rehydratedseed axes were qualitatively similar at 5 HAI (the desiccation-tolerant stage). The drying treatment resulted in increasedintensity of extant proteins at 5 and 12 HAI. At 12 HAI (thetransition stage between the desiccation-tolerant and desiccation-intolerantphases) desiccation and subsequent rehydration triggered synthesisof a unique set of proteins-the rehydration proteins. At 20HAI (the desiccation-intolerant stage) desiccation resultedin an overall decline in the intensity of proteins synthesizedin vitro. Also the rehydration proteins were not synthesizedin response to a drying and rehydration treatment at this time. Key words: Seed germination, desiccation, mRNA, in vitro translation, Phaseolus vulgaris     

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     

Desiccation of Axes of Phaseolus vulgaris during Development of a Switch from a Development Pattern of Protein Synthesis to a Germination Pattern

Immature seeds of Phaseolus vulgaris removed from the pod at 32 days of development do not germinate unless first subjected to a desiccation treatment. This change from development to germination caused by premature drying is mirrored in the pattern of protein synthesis by the axes. Rehydrated axes from 32-day-developed seeds cease to synthesize proteins that are uniquely associated with development, but instead synthesize some proteins that are similar to those made in the germinating axes from mature dry seeds. Desiccation of 22-day-developed seeds does not lead to their germination, nor does it cause a switch from a developmental to a germination mode of protein synthesis by the axes. It is proposed that desiccation plays a role in permanently suppressing developmental protein synthesis and in inducing germination protein synthesis.     

The Messenger RNA Population in the Embryonic Axes of Phaseolus vulgaris During Development and Following Germination

Misra, S. and Bewley, J. D. 1985. The messenger RNA populationin the embryonic axes of Phaseolus vulgaris during developmentand following germination.—J. exp. Bot. 36: 1644–1652. Messenger RNAs were extracted from young, mid-maturation, late-maturation,mature-dry, and 20-h-germinated embryonic axes of Phaseolusvulgaris cv. Taylor's Horticultural. They were translated invitro in a rabbit reticulocyte lysate protein synthesizing system.Analysis of the translation products using two-dimensional polyacrylamidegel electrophoresis indicated that there were substantial changesin the messenger RNA populations of developing and germinatingaxes. The number of polypeptides synthesized increased sharplyat 20–22 d after pollination and then declined. Therewas a parallel increase and decrease in the Poly(A)⁺ contentof the seed axis. The analysis showed that certain messageswere present throughout development and were stored in maturedry seed. These messages were degraded upon subsequent rehydration.Some messages appeared during mid-maturation but declined duringlater stages of development and were absent from the matureseed. In the germinating seed a set of messages unique to germinationappeared. Key words: —Seed development, germination, mRNA, in vitro translation, Phaseolus vulgaris     

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     

Relation Between Desiccation Sensitivity and Membrane Permeability of Developing Clausena lansium Seeds

Desiccation sensitivity and its relation to membrane permeability of the embryonic axes of the developing wampee (Clausena lansium (Lour.) Skeels) seeds were studied by measuring the changes in electrolyte leakage, germination and vigor index after the embryonic axes were rapidly air-dried to various water contents. During development, the fresh and dry weight per seed reached nearly maximum value at 72 d after anthesis, but the dry weight per embryonic axis continuously increased until 85 d after anthesis. The embryonic axes acquired the full capacity for germination at 58 d after anthesis and their vigor index continuously rose up from 51 to 92 d after anthesis. The electrolyte leakage of the developing the embryonic axes linearly declined to the minimum value at 72 d after anthesis and then went up again. The electrolyte leakage of the embryonic axes was higher than that of the whole seeds at the same time. The immature embryonic axes did not germinate completely, while mild desiccation could improve their viability. Any degree of desiccation decreased the vigor index of the embryonic axes which have reached physiological maturation and the decline of vigor index was corresponded to the increase of electrolyte leakage. According to this experiment, the authors concluded that wampee seeds did not gain desiccation-tolerance which was a characteristic of orthodox seeds during development. High water content was essential for maintaining membrane integrity and stabiligy of matured wampee seeds. The injury of seed viability during dehydration could be estimated by using the electro-conductivity method.     

The recalcitrant plant species, Castanospermum australe and Trichilia dregeana, differ in their ability to produce dehydrin-related polypeptides during seed maturation and in response to ABA or water-deficit-related stresses

In constrast to seeds of orthodox species, those of recalcitrantspecies do not acquire desiccation tolerance during their developmentand are shed from the parent plant at high water contents. Dehydrinproduction in seeds of recalcitrant species was examined duringdevelopment and germination, in response to abscisic acid (ABA),and following the imposition of various water-deficit-relatedstresses, including desiccation, water stress, high salt, highosmolarity, and low temperature. Two tropical species exhibiteda differential capacity to produce dehydrin-related proteinsduring seed maturation. Dehydrins were present in axes and cotyledonsof Castanospermum australe seeds during mid-maturation and atmaturity. In Trichilia dregeana, no dehydrin-related polypeptideswere detected in the mature seed. During the development ofC. australe seeds, the nature of the dehydrin related polypeptidesaccumulated in the cotyledons and axis changed and new polypeptideswere detected in the mature seeds that were not present duringmid-maturation. The dehydrins present in cotyledons of matureseeds (31, 37 and 40 kDa) were still detectable after germination(i.e. in untreated seedlings). These dehydrins became less abundantin the cotyledons of C. australe seedlings following ABA andall stress treatments except cold, although most of the dehydrinswere still detectable. An exception was the desiccation-treatedseedlings, in which no dehydrins were detected. In the rootsof C. australe seedlings, no dehydrins were found after germinationnor were they induced in the root by ABA or any of the stresstreatments imposed on seedlings. Seedlings of Trichilia dregeanadid not produce dehydrins in the roots or cotyledons when exposedto ABA or water-deficit-related stresses. Key words: Dehydrin, ABA, desiccation, recalcitrant, seed     

Desiccation and Freezing Sensitivity in Recalcitrant Seeds of Tea, Cocoa and Jackfruit

Investigations were undertaken on the desiccation and freezingsensitivity of recalcitrant seeds of three species: tea [Camelliasinensis (L.) O. Kuntze], cocoa (Theobroma cacao L.) and jackfruit(Artocarpus heterophyllus Lamk.). All species showed changesin the physiological characteristics, desiccation and freezingsensitivity of both the seed and the embryonic axes with increasingseed maturity. Fully mature seeds of tea, cocoa and jackfruitsurvived desiccation to 24, 35 and 31% moisture content, respectively,but at these moisture levels seeds were not able to toleratefreezing in liquid nitrogen (-196 °C). Some survival ofcryopreservation was, however, achieved for excised embryonicaxes of partially and fully-mature tea and jackfruit seeds afterdrying to 14% moisture content; cocoa axes were totally freezingsensitive at all three stages of physiological maturity studied.Biochemical investigations on fully mature axes after desiccationand freezing showed that the decline in viability with moisturelevel was associated with increased leachate conductivity, lipidperoxidation products and/or soluble carbohydrates. Evidencefor disruption of cell membranes during desiccation and freezingwas supported by ultrastructural studies.Copyright 1995, 1999Academic Press Camellia sinensis (L.) O. Kuntze, Theobroma cacao L., Artocarpus heterophyllus Lamk., Phaseolus vulgaris L., tea, cocoa, jackfruit, french bean, seeds, embryonic axes, desiccation, freezing, cryopreservation     

Desiccation-Tolerant and Desiccation-Intolerant Stages during the Development and Germination of Phaseolus vulgaris Seeds

Embryonic axes of Phaseolus vulgaris undergo a transition fromdesiccation-intolerance to desiccation-tolerance in the courseof their development. Biochemical and ultrastructural observationsindicate that desiccation and rehydration during the early intolerantdevelopmental stages drastically reduces the metabolic and cellularintegrity of the axis. During the desiccation-tolerant stagesuch perturbations do not occur. Coincident with the acquisitionof desiccation-tolerance during development the seeds gain thecapacity to germinate upon subsequent rehydration. Drying presumablyacts to terminate developmental processes and to initiate themetabolic processes essential for the completion of germination.During germination of the mature axis, desiccation and rehydration,up to 12 h from the start of imbibition, does not affect subsequentseedling growth and development. But gradually desiccation-tolerancedecreases and metabolism is severely and irreversibly reducedby drying.     

Long-lived Messenger RNA and its Relationship to Protein Synthesis During Germination of Pea (Pisum sativum L.) Seeds

Synthesis of both protein and RNA is initiated very early ingermination in the embryo axes of pea seeds. The early RNA synthesisinvolves all three types, although there is some evidence forpreferential synthesis of mRNA in the first few hours afterthe onset of imbibition. In addition to newly synthesized mRNA,the embryo axis also contains long-lived mRNA. The amount ofthis long-lived mRNA declines markedly during the first 20 hof germination. Synthesis of both protein and RNA is initiated very early ingermination in the embryo axes of pea seeds. The early RNA synthesisinvolves all three types, although there is some evidence forpreferential synthesis of mRNA in the first few hours afterthe onset of imbibition. In addition to newly synthesized mRNA,the embryo axis also contains long-lived mRNA. The amount ofthis long-lived mRNA declines markedly during the first 20 hof germination. Results from in vitro and in vivo protein synthesis experimentsand from studies of polysome formation suggest that much ofthe long-lived mRNA present in the embryo axis does not directprotein synthesis. The increase in the rate of protein synthesisduring germination is thus dependent on recruitment of newlysynthesized mRNA molecules. Pea, Pisum sativum L., germination, mRNA, protein synthesis     

Embryo Water Status and Survival in the Recalcitrant Species Quercus robur L: Evidence for a Critical Moisture Content

The responses of Q. robur L. fruits, seeds and embryonic axesto desiccation are characterized and discussed in relation tocurrent knowledge of recalcitrant seed behaviour. A relationshipbetween viability and seed moisture content is described. Thisrelationship was unaffected by rate of drying, year of harvestor presence of the pericarp. Desiccation sensitivity did notincrease with storage. Excised embryonic axes survived to lower moisture contents thanintact seeds. However, in the intact seed, loss of viabilityappeared to be determined by a critical moisture content inthe cotyledons. Consequently, the level of desiccation tolerancewithin the axis attached to cotyledons was not determined byaxis drying rate. A link is drawn between the difference in the desiccation toleranceof embryonic axes and of cotyledons, and estimates of theirdifferent levels of matrix-bound water. The results presentedare consistent with a critical moisture content for survivalwhich is determined by the loss of all free cellular water.This hypothesis takes account of the differential desiccationsensitivity of seed tissues and differences in desiccation tolerancebetween species.     

Characterization of RNA synthesized during germination of Blastocladia ramosa zoospores

The synthesis of RNA was studied during the synchronous germination of Blastocladia ramosa zoospores. Comparison of RNA synthesis during germination of B. ramosa and Blastocladiella emersonii zoospores revealed that B. ramosa has a longer lag time before RNA synthesis is initiated and, in addition, the rate of RNA synthesis is ten-fold lower in B. ramosa. Zoospores of B. ramosa were shown to contain pre-formed messenger RNA but this messenger RNA directs only a portion of the protein synthesis which occurs during early germination. The conclusion that the remainder of the protein synthetic activity of the germinating spores is due to new message synthesis was supported by demonstrating that the timing of the initation of protein synthesis on new messages correlates with the time RNA synthesis is initiated. New message synthesis was also demonstrated by the incorporation of label into RNA which contains a poly (A) fragment. Synthesis of all classes of RNA including ribosomal, messenger, and transfer RNA was shown to be initiated at the same time. The implications of this observation are discussed.     

The Role of Maturation Drying in the Transition from Seed Development to Germination: V. RESPONSES OF THE IMMATURE CASTOR BEAN EMBRYO TO ISOLATION FROM THE WHOLE SEED: A COMPARISON WITH PREMATURE DESICCATION

Kennode, A. R, and Bewley, J. D. 1988. The role of maturationdrying in the transition from seed development to germination.V. Responses of the immature castor bean embryo to isolationfrom the whole seed; a comparison with premature desiccation.—J.exp. Bot. 39: 487–497. Desiccation is an absolute requirement for germination and post-germinativegrowth of whole seeds of the castor bean, whether desiccationis imposed prematurely during development, at 35 d after pollination(DAP) or occurs naturally during late maturation (50–60DAP). Desiccation also plays a direct role in the inductionof post-germinative enzyme synthesis in the cotyledons of embryosin the intact seed; this event is not simply due to the presenceof a growing axis. Isolation of embryos from the developingcastor bean seed at 35 DAP results in both germination and growth,despite the absence of a desiccation event. We have comparedthe metabolic consequences of premature drying of whole seeds(35 DAP) and isolation of the developing 35 DAP embryos. Inboth cases, hydrolytic events involved in the mobilization ofstored protein reserves proceed in a similar manner and mirrorthose events occurring within germinated mature seeds. Thereare differences, however, for post-germinative enzyme (LeuNAaseand isocitrate lyase) production occurs to a lesser extent innon-dried isolated embryos than in those from prematurely dried(35 DAP) whole seeds, or from mature dry (whole) seeds. Desiccationof the 35 DAP whole seed does not alter the subsequent responseof the embryo upon isolation. Thus, while drying does not affectthe metabolism of isolated embryos, it has a profound effecton that of embryos within the intact seed. Tissues surroundingthe embryo in the developing intact seed (viz. the endosperm)maintain its metabolism in a developmental mode and inhibitgermination. This effect of the surrounding tissues can onlybe overcome by drying or by their removal. Key words: Metabolism, isolation, desiccation, embryo, endosperm, castor bean, development, germination     

Enhanced Maturation and Desiccation Tolerance of White Spruce [Picea glauca (Moench) Voss] Somatic Embryos: Effects of a Non-plasmolysing Water Stress and Abscisic Acid

A non-plasmolysing moisture stress effected by polyethyleneglycol (PEG) was beneficial when applied to maturing white spruce(Picea glauca) somatic embryos for the following reasons. Anosmotic treatment of 5.0–7.5% PEG stimulated a threefoldincrease in the maturation frequency. The osmotically treatedsomatic embryos displayed higher dry weights and lower moisturecontents than the controls, indicating a greater accumulationof storage reserves. Moisture contents of mature, osmotically-treated,hydrated somatic embryos were 40–45%, in contrast to 57%for the non-osmotically treated controls. Desiccation was achievedby placing the somatic embryos in a range of relative-humidityenvironments. No clear trend for the effect of PEG on survivalof desiccated somatic embryos was observed; mean survival valuesranged from 34 to 62% when somatic embryos from all osmotictreatments were desiccated for 14 d at 81% relative humidity.Following this desiccation treatment, somatic embryos from allosmotic concentrations had moisture contents of 26–31%,similar to the 32% recorded for unimbibed zygotic embryos. Afterimbibition, moisture contents for these zygotic and somaticembryos were in the order of 60%. Somatic embryos matured withPEG remained quiescent during desiccation due to their low initialmoisture contents, and gave rise to plantlets of normal appearance.Gradual desiccation of the somatic embryos directly followingmaturation with abscisic acid (ABA) was crucial to survivalduring desiccation. A plasmolysing water stress effected bysucrose at osmotic potentials similar to PEG was detrimentalto somatic embryo maturation, thereby emphasizing the importanceof the choice of osmoticum. Desiccation, maturation, osmotic potential, Picea glauca, polyethylene glycol, somatic embryo, water stress, white spruce     

Free proline content and sensitivity to desiccation and heat during yeast sporulation and spore germination.

Ascospores of a strain of Saccharomyces cerevisiae Hansen were less sensitive to desiccation and heat than vegetative cells. Desiccation resistance was acquired earlier during sporulation and lost later during spore germination than heat resistance. As spores matured, resistance to both stresses increased. With the exception of the first few hours in sporulation medium, when proline appeared to be utilized, the intracellular free proline content increased during sporulation and decreased during spore germination. Not all the proline lost could be detected in the germination medium, indicating that some was metabolically utilized by the germinating spores. Since exogenous proline supplied to vegetative or sporulating cells before desiccation increased their survival, it is suggested that the high level of free proline in mature spores may protect against desiccation stress.     

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.     

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.     

Studies on Protein Synthesis in Tortula ruralis: POLYRIBOSOME REFORMATION FOLLOWING DESICCATION

Desiccation of Tortula ruralis was achieved rapidly by placingthe moss on the laboratory bench, or more slowly by placingit in desiccators with atmospheres of high relative humidities.Unlike the rapidly desiccated moss, the slower desiccated mossretained no polyribosomes in the dehydrated state, althoughpolyribosome reformation and protein synthesis resumed on reintroductionof the moss to water. Protein synthesis commenced on rehydrationof the slower desiccated moss at a greater rate than on rehydrationof the faster desiccated moss. A lack of correlation betweenendogenous ribonuclease activity and polyribosome levels extractedfrom the moss suggests that the observed reduction in polyribosomesduring desiccation was not due to their degradation but wasmore likely a consequence of stress-induced restriction on reinitiationof existing messenger RNA. The observed protein synthesis onrehydration of the moss was largely independent of any priorRNA synthesis.