Reprogramming of Protein Synthesis from a Developmental to a Germinative Mode Induced by Desiccation of the Axes of Phaseolus vulgaris

Immature seeds of Phaseolus vulgaris cv Taylor's Horticultural removed from the pod at 32 days of development do not germinate unless first subjected to desiccation. Our results show that premature drying not only redirects metabolism from a developmental to a germination program but it does so permanently, thus effecting an irreversible switch. This is shown by in vitro protein synthesis, and analysis of poly(A)⁺ mRNA with a cDNA probe specific for phaseolin message. For example, the pattern of proteins synthesized in vitro by the mRNA fraction from fresh and prematurely dried axes show strong similarities; on the other hand, the mRNA population from rehydrated axes code for a different set of proteins. Also, the message for phaseolin is preserved following the normal maturation process and premature desiccation of seeds. Following rehydration of immature seeds at the desiccation-tolerant stage, this message is no longer detectable in the axes.     

Proteins in development and germination of a desiccation sensitive (recalcitrant) seed species

In the recalcitrant seeds of Avicennia marina, protein content and the rates of protein synthesis increase during histodifferentiation. This is similar to the situation in desiccation tolerant seeds. During the stage of reserve accumulation the protein content and rates of synthesis remain constant and there is no de novo synthesis of proteins which might qualify as storage proteins. There is also no change in the nature of proteins present in either axis or cotyledonary tissues during development or germination. Similarly, fluorographs of axis proteins show only very limited changes in the patterns of protein synthesis during development and germination, at least until the onset of root growth. Heat-stable proteins are present from an early developmental stage. However, no late embryogenic abundant (LEA) proteins are synthesised during the late stages of development, indicating that seedling establishment is independent of such maturation proteins. It is suggested that the lack of desiccation tolerance of A. marina seeds might be related to the absence of desiccation-related LEAs. Although the rate of protein synthesis increases during germination, protein metabolism appears to remain qualitatively the same as that occurring during development. The present results suggest that in these desiccation sensitive seeds, protein metabolism characterising development changes imperceptibly into that of germination.     

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-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.     

Development of Desiccation Tolerance during Embryogenesis in Rice (Oryza sativa) and Wild Rice (Zizania palustris) (Dehydrin Expression,Abscisic Acid Content,and Sucrose Accumulation)

The ability of seeds to withstand desiccation develops during embryogenesis and differs considerably among species. Paddy rice (Oryza sativa L.) grains readily survive dehydration to as low as 2% water content, whereas North American wild rice (Zizania palustris var interior [Fasset] Dore) grains are not tolerant of water contents below 6% and are sensitive to drying and imbibition conditions. During embryogenesis, dehydrin proteins, abscisic acid (ABA), and saccharides are synthesized, and all have been implicated in the development of desiccation tolerance. We examined the accumulation patterns of dehydrin protein, ABA, and soluble saccharides (sucrose and oligosaccharides) of rice embryos and wild rice axes in relation to the development of desiccation tolerance during embryogenesis. Dehydrin protein was detected immunologically with an antibody raised against a conserved dehydrin amino acid sequence. Both rice and wild rice embryos accumulated a 21-kD dehydrin protein during development, and an immunologically related 38-kD protein accumulated similarly in rice. Dehydrin protein synthesis was detected before desiccation tolerance had developed in both rice embryos and wild rice axes. However, the major accumulation of dehydrin occurred after most seeds of both species had become desiccation tolerant. ABA accumulated in wild rice axes to about twice the amount present in rice embryos. There were no obvious relationships between ABA and the temporal expression patterns of dehydrin protein in either rice or wild rice. Wild rice axes accumulated about twice as much sucrose as rice embryos. Oligosaccharides were present at only about one-tenth of the maximum sucrose concentrations in both rice and wild rice. We conclude that the desiccation sensitivity displayed by wild rice grains is not due to an inability to synthesize dehydrin proteins, ABA, or soluble carbohydrates.     

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。     

Analyses to determine the role of embryo immaturity in dormancy maintenance of yellow cedar (Chamaecyparis nootkatensis) seeds: synthesis and accumulation of storage proteins and proteins implicated in desiccation tolerance

Development of yellow cedar seeds is completed by about 17-21 months after pollination. Following dispersal from the parent plant, the seeds exhibit a low capacity for germination and typically require an additional year to meet their moist chilling requirements and break dormancy. Biochemical analyses were undertaken in order to address whether seed dormancy is imposed and maintained because the embryo or megagametophyte is immature at the time of seed shedding and hence requires time to complete developmental events before dormancy can be terminated. Major protein reserves of the embryo and megagametophyte are the buffer-insoluble crystalloid (legumin) storage proteins and the water-soluble albumin proteins. SDS-PAGE, fluorography of in vivo synthesized proteins and Western blot analyses showed that the greatest increase in protein reserve synthesis and accumulation occurred between the first and second years of development; deposition of soluble and insoluble storage protein was largely completed in seeds of second-year cones by August, 2-3 months prior to seed dispersal. The period associated with greatest accumulation of storage proteins was accompanied by an increased accumulation of two ER-resident proteins associated with post-translational maturation of storage proteins (binding protein and protein disulphide isomerase). Accumulation of proteins implicated in the acquisition of desiccation tolerance (dehydrins and the tonoplast intrinsic protein, -TiP) occurred between the first and second years of development. Several heat-stable proteins and some of the proteins associated with late development continued to be synthesized after seed shedding and in 13 d moist-chilled mature seeds. However, this did not include the major dehydrin-like protein of yellow cedar seeds. Further, the continued synthesis of heat-stable proteins does not appear to be a factor preventing the germination of yellow cedar seeds following dispersal from the parent plant; rather, the mechanism of dormancy is primarily coat-imposed.     

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     

Premature Drying, Fluridone-Treatment, and Embryo Isolation During Development of Maize Kernels (Zea mays L.) Induce Germination, but the Protein Synthetic Responses are Different. Potential Regulation of Germination and Protein Synthesis by Abscisic Acid

Freshly harvested, developing kernels of maize (Zea mays L.)do not germinate up to 77 d after pollination, but can be inducedto do so by fluridone, premature desiccation, and isolationof the developing embryo. The pattern of protein synthesis indeveloping maize embryos is distinct from that during germinationand subsequent seedling growth. Premature desiccation at 35DAP elicits a pattern of protein synthesis upon rehydrationwhich is similar to that in germinated embryos from mature drykernels. Fluridone-induced viviparous germination is accompaniedby changes in the synthesis of some proteins to a post-germinativepattern, but some developmental proteins continue to be synthesized.Embryos isolated from developing kernels at 35 DAP germinatewhen incubated on water; they also produce some developmentalproteins during germination. Kernels from developing cobs at35 DAP which are detached from the mother plant and maintainedin an atmosphere of high relative humidity (moist controls)do not germinate, but neither do they continue a clearly definedpattern of either developmental or germinative protein synthesis.Drying is thus critical to effect a clear transition of proteinsynthesis from a developmental to a germinative mode in maizeembryos. Abscisic acid within the developing embryos is reduced by fluridone,but to a lesser extent by premature drying or maturation drying.Changes in sensitivity to abscisic acid by the developing embryomay be as, or more, important in permitting germination, andthe attendant synthesis of proteins, than changes in abscisicacid content. Key words: Maize (Zea mays L.), germination, vivipary, desiccation, abscisic acid     

Protein Synthesis in the Axes of Polyethylene Glycol-Treated Pea Seed and during Subsequent Germination

Germination of Alaska pea seeds is inhibited by –0.3 MPapolyethylene glycol but upon subsequent transfer to water, germinationis completed rapidly and radicle emergence occurs more quicklythan in water-imbibed seeds. Protein synthesis is reduced inthe axes of seeds imbibed on PEG but increases upon their returnto water, though not to the level exhibited by axes germinatedon water. Mobilization of proteins in the axes is retarded bytheir failure to complete germination on PEG, although somedoes occur. The quantitative reduction in protein synthesisresulting from incubation in osmoticum is not accompanied bymarked qualitative changes. The block to germination is notobviously associated with a restriction in synthesis of anyparticular protein or set of proteins; conversely, no ‘water-stress’proteins are synthesized in the presence of PEG. The synthesisof growth-specific proteins is prevented by PEG, but these increaseupon relief from the osmoconditioning treatments. These observationsdispute earlier claims for accelerated protein synthesis resultingfrom PEG treatments. Key words: Osmotic priming, Pisum sativum, germination, protein synthesis     

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     

Maturation proteins associated with desiccation tolerance in soybean

A set of proteins that accumulates late in embryogenesis (Lea proteins) has been hypothesized to have a role in protecting the mature seed against desiccation damage. A possible correlation between their presence and the desiccation tolerant state in soybean seeds (Glycine max L. Chippewa) was tested. Proteins that showed the same temporal pattern of expression as that reported for Lea proteins were identified in the axes of soybean. They were distinct from the known storage proteins and were resistant to heat coagulation. The level of these “maturation” proteins was closely correlated with desiccation tolerance both in the naturally developing and in the germinating seed: increasing at 44 days after flowering, when desiccation tolerance was achieved, and decreasing after 18 hours of imbibition, when desiccation tolerance was lost. During imbibition, 100 micromolar abscisic acid or Polyethylene glycol-6000 (−0.6 megapascals) delayed disappearance of the maturation proteins, loss of desiccation tolerance, and germination. During maturation, desiccation tolerance was prematurely induced when excised seeds were dried slowly but not when seeds were held for an equivalent time at high relative humidity. In contrast, maturation proteins were induced under both conditions. We conclude that maturation proteins may contribute to desiccation tolerance of soybean seeds, though they may not be sufficient to induce tolerance by themselves.     

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。     

Role of Abscisic Acid in the Induction of Desiccation Tolerance in Developing Seeds of Arabidopsis thaliana

In contrast to wild-type seeds of Arabidopsis thaliana and to seeds deficient in (aba) or insensitive to (abi3) abscisic acid (ABA), maturing seeds of recombinant (aba,abi3) plants fail to desiccate, remain green, and lose viability upon drying. These double-mutant seeds acquire only low levels of the major storage proteins and are deficient in several low mol wt polypeptides, both soluble and bound, and some of which are heat stable. A major heat-stable glycoprotein of more than 100 kilodaltons behaves similarly; during seed development, it shows a decrease in size associated with the abi3 mutation. In seeds of the double mutant from 14 to 20 days after pollination, the low amounts of various maturation-specific proteins disappear and many higher mol wt proteins similar to those occurring during germination are induced, but no visible germination is apparent. It appears that in the aba,abi3 double mutant seed development is not completed and the program for seed germination is initiated prematurely in the absence of substances protective against dehydration. Seeds may be made desiccation tolerant by watering the plants with the ABA analog LAB 173711 or by imbibition of isolated immature seeds, 11 to 15 days after pollination, with ABA and sucrose. Whereas sucrose stimulates germination and may protect dehydration-sensitive structures from desiccation damage, ABA inhibits precocious germination and is required to complete the program for seed maturation and the associated development of desiccation tolerance.     

A mutant of Arabidopsis which is defective in seed development and storage protein accumulation is a new abi3 allele

In order to investigate the role of the plant hormones gibberellin (GA) and abscisic acid (ABA) in seed development and germination the GA biosynthetic inhibitor, Uniconazol, was used to isolate mutants with abnormal germination profiles. In one of these mutants, the ability to germinate on Uniconazol is due to a mutation in the ABI3 gene. However, unlike the previously reported abi3 mutant, this line displays an array of seed-specific developmental defects. The accumulation of seed reserve proteins is dramatically reduced due to reduced levels of the storage protein mRNA. The embryos remain green throughout development and are desiccation intolerant. However, immature seeds are completely non-dormant and grow normally. These results suggest the ABI3 gene is essential for the synthesis of seed storage proteins and for the protection of the embryo during desiccation.     

The Role of Maturation Drying in the Transition from Seed Development to Germination: II. POST-GERMINATIVE ENZYME PRODUCTION AND SOLUBLE PROTEIN SYNTHETIC PATTERN CHANGES WITHIN THE ENDOSPERM OF Ricinus communis L. SEEDS

Kermode, A. R. and Bewley, J. D. 1985. The role of maturationdrying in the transition from seed development to germination.II. Post–germinative enzyme production and soluble proteinsynthetic pattern changes within the endosperm of Ricinus communisL. seeds.—J. exp. Bot. 36: 1916–1927. Immature seedsof Ricinus communis L. cv. Hale (castor bean) removed from thecapsule at 30 or 40 d after pollination (DAP) do not germinateunless first subjected to a desiccation treatment. This changefrom development to germination elicited by premature desiccationis also mirrored by a change, upon subsequent rehydration, inthe pattern of soluble protein synthesis within the endospermstorage tissue. Following rehydration of prematurely dried 30or 40 DAP seeds, soluble proteins characteristic of developmentcease to be synthesized after 5 h of imbibition, and those uniquelyassociated with germination and growth are then produced. Apattern of soluble storage protein breakdown comparable to thatfound in endosperms from mature seeds following imbibition isalso observed. In contrast, hydration of 40 DAP seeds immediatelyfollowing detachment from the mother plant results in a continuationof the developmental pattern of protein synthesis. Prematuredesiccation at 40 DAP elicits the production within the endospermof enzymes involved in protein reserve breakdown (leucyl ß–naphthylamidase;LeuNAase) and lipid utilization (isocitrate lyase; ICL) to levelscomparable to those observed in mature–hydrated endosperms.It is proposed that drying plays a role in redirecting metabolismfrom a developmental to a germinative mode; it also appearsto be a prerequisite for the induction of hydrolytic enzymesessential to the post–germinative (growth) phase of seedlingdevelopment. Key words: Desiccation-tolerance, germinability, seed development, castor bean