The Role of Maturation Drying in the Transition from Seed Development to Germination: III. INSOLUBLE PROTEIN SYNTHETIC PATTERN CHANGES WITHIN THE ENDOSPERM OF Ricinus communis L. SEEDS

Kermode, A. R., Gifford, D. J. and Bewley, J. D. 1985. The roleof maturation drying in the transition from seed developmentto germination. III. Insoluble protein synthetic pattern changeswithin the endosperm of Ricinus communis L. seeds.—J.exp. Bot. 36: 1928–1936. Immature seeds of Ricinus communisL. cv. Hale (castor bean) removed from the capsule at 30 or40 days after pollination (DAP) can be induced to germinateby being subjected to drying. This desiccation–inducedswitch from development to germination is mirrored by a change,upon subsequent rehydration, in the pattern of insoluble proteinsynthesis within the endosperm storage tissue. During normaldevelopment from 25–40 DAP there is rapid synthesis ofthe insoluble (11S) crystalloid storage protein. At later stagesof development (45 and 50 DAP), crystalloid protein synthesisdeclines markedly and synthesis of new insoluble proteins commences.Following premature drying at 30 or 40 DAP, the pattern of insolubleprotein synthesis upon rehydration is virtually identical tothat following imbibition of the mature seed. Proteins synthesizedduring normal late development (at 45 and 50 DAP) are producedup to 48 h after imbibition; a subsequent change in the patternof insoluble protein synthesis occurs between 48 and 72 h. Thus,in contrast to the rapid switch in the pattern of soluble proteinsynthesis induced by drying, insoluble protein syntheses withinthe endosperm are redirected towards those uniquely associatedwith a germination/growth programme only after a considerabledelay following mature seed imbibition, or following rehydrationof the prematurely dried seed. Nevertheless, these results supportour contention that drying plays a role in the suppression ofthe developmental metabolic programme and in the permanent inductionof a germination/growth programme. Key words: Desiccation, crystalloid storage proteins, castor bean, seed development, seed germination     

The Role of Maturation Drying in the Transition from Seed Development to Germination: IV. PROTEIN SYNTHESIS AND ENZYME ACTIVITY CHANGES WITHIN THE COTYLEDONS OF RICINUS COMMUNIS L. SEEDS

Drying of immature seeds of Ricinus communis L. cv. Hale (castorbean) during the desiccation-tolerant phase of development causesthem to germinate upon subsequent rehydration. This desiccation-inducedswitch from development to germination is also mirrored by achange in the pattern of soluble and insoluble protein synthesiswithin the cotyledons of the castor bean. Following rehydrationof seeds prematurely dried at 40 d after pollination (DAP),cotyledonary proteins characteristic of development (e.g. storageproteins) are no longer synthesized; hydrolytic processes resultingin their degradation commence (after 12 h) in a manner similarto that observed following imbibition of the mature seed. Apattern of protein synthesis recognizable as germination/growth-associatedoccurs; premature drying has elicited a redirection in metabolismfrom a developmental to a germinative mode. Desiccation is alsorequired for the induction (within cotyledons of 35 DAP seeds)of enzymes involved in protein reserve breakdown (leucyl ß-naphthylamidase;LeuNAase) and lipid utilization (isocitrate lyase; ICL), anevent intimately associated with the post-germinative (growth)phase of seedling development. Thus, at a desiccation-tolerantstage of development, premature drying results in the suppressionof the developmental metabolic programme and a permanent switching-onof the germination/growth metabolic programme. Key words: Desiccation, metabolism, seed development, seed germination, castor bean, cotyledons     

The Role of Maturation Drying in the Transition from Seed Development to Germination: I. ACQUISITION OF DESICCATION-TOLERANCE AND GERMINABILITY DURING DEVELOPMENT 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.1. Acquisition of desiccation–tolerance and germinabilityduring development of Ricinus communis L. seeds.—J. exp.Bot. 36: 1906–1915. Seeds of Ricinus communis L. cv. Hale(castor bean) undergo a transition from desiccation–intoleranceto desiccation–tolerance approximately midway throughtheir development. Tolerance of slow desiccation is gained overonly a few days of development (between 20 and 25 d) and isachieved well before the completion of major developmental events,such as reserve deposition and the onset of normal maturationdrying. A tolerance of very rapid water loss brought about bydrying over silica gel is not acquired by this seed until nearmaturity. Coincident with the acquisition of tolerance to slowdesiccation the seeds gain the capacity to germinate upon subsequentrehydration. Germinability and capacity for normal post–germinativegrowth during the tolerant phase are not fully expressed unlessthe seed is dried at an optimal rate, which is dependent uponthe developmental stage of the seed. Drying presumably actsto terminate developmental processes and to initiate those metabolicprocesses necessary to prepare the seed for germination andgrowth. Key words: Desiccation-tolerance, germinability, seed development, castor bean     

On the Composition, Deposition and Mobilization of Proteins in the Cotyledons of Castor Bean (Ricinus communis L. cv. Hale) Seeds: Their Role as Storage Proteins

Proteins in the soluble and insoluble fractions, extracted frommature castor bean cv. Hale seed cotyledons, differ quantitativelyand qualitatively from their counterparts extracted from theendosperm. The soluble fraction contains no glycoproteins, andthe lectins RCA₁ and ricin D are absent. While the insolubleproteins are electrophoretically and immunologically similarto those in the endosperm, they do not form the 100 kD subunitdimers which characterize some of the endosperm insoluble crystalloidproteins. Rapid rates of deposition of all of the soluble andinsoluble proteins present in the mature seed cotyledons commences30–35 d after pollination (DAP) and continues until 45DAP. These proteins are mobilized rapidly beginning 1–2d after seed imbibition and this coincides with an increasein specific activity, in the cotyledons, of two aminopeptidasesand a carboxypeptidase. The soluble and insoluble proteins inthe cotyledons of the mature seed probably function as storageproteins and support the growth of the germinated seed priorto the mobilization of the major protein storage reserves ofthe endosperm. Key words: Ricinus communis, Castor bean, Hale cultivar, Cotyledon, Storage protein, Seed development, Seed germination     

The Role of Maturation Drying in the Transition from Seed Development to Germination: VII. EFFECTS OF PARTIAL AND COMPLETE DESICCATION ON ABSCISIC ACID LEVELS AND SENSITIVITY IN RICINUS COMMUNIS L. SEEDS

During mid-development (25–40 d after pollination: DAP)of the castor bean seed the amount of abscisic acid (ABA) increasesin both the endosperm and the embryo, declining substantiallythereafter until there is little present in the mature dry (60DAP) seed. Premature desiccation of the seed at 35 DAP alsoleads to a major decline in ABA within the embryo and endosperm.Partial water loss from the seed at 35 DAP which, like naturaland premature desiccation, leads to subsequent germination uponreturn of the seed to full hydration, causes a much smallerdecline in ABA levels. In contrast, ABA declines substantiallyin the non-dried (hydrated) control at 35 DAP, but the seedsdo not germinate. Hence, a clear negative correlation betweenABA content and germinability is not observed. Both drying,whether natural or imposed prematurely, and partial drying decreasethe sensitivity of the isolated embryo to exogenous ABA by about10-fold. The protein synthetic response of the castor bean embryo exposedto 0.1 mol m^–3 ABA following premature desiccation exhibitssome similarity to the response of the non-dried developingembryo—in both cases the synthesis of some developmentalproteins is enhanced by ABA, and germination is suppressed.Germination of mature seeds is also suppressed by 0.1 mol m^–3ABA, but the same developmental proteins are not synthesized.In the cotyledons of prematurely-desiccated seed, some proteinsare hydrolysed upon imbibition in 0.1 mol m^–3 ABA, a phenomenonthat occurs also in the cotyledons of similarly treated matureembryos, but not in developing non-dried embryos. Hence theembryo exhibits an ‘intermediate’ response uponrehydration in 0.1 mol m^–3 ABA following premature desiccation;viz. some of the responses are developmental and some germinative.Following natural or imposed drying, the isolated embryo becomesrelatively insensitive to 0.01 mol m^–3 ABA: germinationis elicited and post-germinative reserve breakdown occurs inthe radicle and cotyledons. The reduced sensitivity of the embryoto ABA as a consequence of desiccation may be an important factorin eliciting the switch to germination and growth within thewhole seed. Key words: Abscisic acid, desiccation, astor bean endosperm, seed development, germination, protein synthesis, isolated embryos, hormone sensitivity     

Gluconeogenesis in the castor bean endosperm: I. Changes in glycolytic intermediates

The control points of the Embden-Meyerhof-Parnas pathway in germinating castor bean (Ricinus communis) endosperms are sought in two ways: (a) by measuring the amounts of various glycolytic intermediates at intervals during the germination; (b) by determining the crossover points appearing during anoxia.     

Protein Synthesis During Rehydration, Germination and Seedling Growth of Naturally and Precociously Matured Soybean Seeds (Glycine max)

Soybean seeds [Glycine max (L.) Merr.] synthesize de novo andaccumulate several non-storage, soluble polypeptides duringnatural and precocious seed maturation. These polypeptides havepreviously been coined ‘maturation polypeptides’.The objective of this study was to determine the fate of maturationpolypeptides in naturally and precociously matured soybean seedsduring rehydration, germination, and seedling growth. Developingsoybean seeds harvested 35 d after flowering (mid-development)were precociously matured through controlled dehydration, whereasnaturally matured soybean seeds were harvested directly fromthe plant. Seeds were rehydrated with water for various timesbetween 5 and 120 h. Total soluble proteins and proteins radio-labelledin vivo were extracted from the cotyledons and embryonic axesof precociously and naturally matured and rehydrated seed tissuesand analyzed by one-dimensional PAGE and fluorography. The resultsindicated that three of the maturation polypeptides (21, 31and 128 kDa) that had accumulated in the maturing seeds (maturationpolypeptides) continued to be synthesized during early stagesof seed rehydration and germination (5–30 h after imbibition).However, the progression from seed germination into seedlinggrowth (between 30 and 72 h after imbibition) was marked bythe cessation of synthesis of the maturation polypeptides followedby the hydrolysis of storage polypeptides that had been synthesizedand accumulated during seed development. This implied a drasticredirection in seed metabolism for the precociously maturedseeds as these seeds, if not matured early, would have continuedto synthesize storage protein reserves. Glycine max (L.) Merr, soybean, cotyledons, maturation, germination/seedling growth     

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     

Vacuolation and Storage Protein Breakdown in the Castor Bean Endosperm following Imbibition

Cytochemical staining of sections prepared for light microscopy,electron microscope sections, and sodium dodecyl sulphate-polyacrylamidegel electrophoresis reveal that, following imbibition, storageproteins are mobilized from the protein bodies of the endospermof castor bean (Ricinus communis L. cv. Hale). This is accompaniedby fusion of protein bodies to form a central vacuole, beforeall the protein is hydrolyzed. Mobilization of the US crystalloidprotein complex and of the 2S albumin fraction commences 2 dafter imbibition and is completed within 2 d. This loss of proteinis accompanied by an increase in activity of three proteolyticenzymes, one carboxypeptidase and two -SH-dependent aminopeptidases.In contrast to the 11S and 2S protein fractions the lectins,located within the protein body, are mobilized only slowly andare present after the other proteins have been completely brokendown. Hence lectins may have a role other than as storage proteins. Key words: Castor bean, Protein breakdown, Storage protein, Lectin, Vacuolation, Seed germination     

Studies on the Development and Localization of Catalase and H(2)O(2)-generating Oxidases in the Endosperm of Germinating Castor Beans

During germination of castor bean seeds (Ricinus communis var. Hale), the changes of activity of catalase, uricase, and α-hydroxyacid oxidase of the endosperm follow a rise and fall pattern with a peak between day 4 and 5 similar to that observed for the glyoxylate cycle enzymes. After 3 days of germination, most of the activities of these enzymes are recovered from the glyoxysomal fraction separated by isopycnic sucrose density gradient centrifugation.     

Accumulation and Distribution of Nutrients in Fruits of Castor Bean (Ricinus communis L.)

The nutrition of developing fruits of Ricinus communis was studiednear Perth, Western Australia, where the species grows as aweed on poor sandy soil. Fruits required 60 days to mature anddehydration of the capsule began 20 days before the seeds ripened.Mature seeds accumulated 49 per cent of the fruit dry matterand over 80 per cent of its P, Zn and Cu, 50–80 per centof its Mg, N, Fe and Mn, 41–46 per cent of its S and Caand 11–21 per cent of its K and Na. Losses of nutrientsfrom capsules during fruit ripening were: Zn, 73 per cent, P,42 per cent, Cu, 23 per cent and Mn, 8 per cent. Dry matter,N, K, S, Ca, Mg, Na and Fe were not withdrawn from capsules.Apparent retranslocation from capsules could have provided from6–28 per cent of the Zn, Mn, P and Cu in mature seeds.Seeds from plants on poor sandy soil were small but had adequatelevels of nutrients when compared with those from plants growingon a fertile loam. Concentrations of all nutrients except P were higher in youngcapsules than in young seeds, but levels of N, P, Mg, Fe, Znand Cu were higher in mature seeds than in mature capsules.The intake of most nutrients by fruits was out of phase withdry matter accumulation, especially in capsules, and the elementsappeared to accumulate in fruit parts independently of eachother. Glutamine accounted for over 85 per cent of the amino-Nin phloem sap destined for fruits. Potassium made up over 90per cent of the inorganic cations in phloem exudate. Of theminor elements in the exudate, Fe was present at highest concentrationand Cu at the lowest. The results showed that retranslocation from the capsule madea very small contribution to the nutrition of seeds. It is suggestedthat R. communis would require a sustained supply of soil nutrientsto ensure maximum seed yield, partly due to the restricted retranslocationof most nutrients from capsules. Ricinus communis L., castor bean, mineral nutrition, translocation, retranslocation     

Glucose-6-Phosphate Dehydrogenase in Plastids from Developing Castor Bean Seeds

Glucose-6-phosphate dehydrogenase, together with the other enzymesof pentose phosphate pathway, was found in the cytosol as wellas in the plastid from developing castor bean (Ricinus communisL.) seeds. The plastid enzyme was found in both the matrix andthe membrane. The plastid enzyme has a sharp pH profile withthe optimum at 8.5, while the cytosolic enzyme has a broad pHprofile, optimum at 7.5. The plastid enzyme was inactivatedby storage at 0°C and by detergents such as Triton X-100,Brij and Nonidet, but the cytosolic enzyme was not. Slab geldisc electrophoresis indicated that three isoenzymes of glucose-6-phosphatedehydrogenase were found in the plastid but one enzyme in thecytosol of developing castor bean seed. From the presence ofglucose-6-phosphate dehydrogenase in the plastid, the operationof whole pentose phosphate pathway in this organelle of developingcastor bean seeds is suggested. (Received September 21, 1982; Accepted January 17, 1983)     

Seeds of tomato (Lycopersicon esculentum Mill.) which develop in a fully hydrated environment in the fruit switch from a developmental to a germinative mode without a requirement for desiccation

Seed water content is high during early development of tomato seeds (10–30 d after pollination (DAP)), declines at 35 DAP, then increases slightly during fruit ripening (following 50 DAP). The seed does not undergo maturation drying. Protein content during seed development peaks at 35 DAP in the embryo, while in the endosperm it exhibits a triphasic accumulation pattern. Peaks in endosperm protein deposition correspond to changes in endosperm morphology (i.e. formation of the hard endosperm) and are largely the consequence of increases in storage proteins. Storage-protein deposition commences at 20 DAP in the embryo and endosperm; both tissues accumulate identical proteins. Embryo maturation is complete by 40 DAP, when maximum embryo protein content, size and seed dry weight are attained. Seeds are tolerant of premature drying (fast and slow drying) from 40 DAP.Thirty-and 35-DAP seeds when removed from the fruit tissue and imbibed on water, complete germination by 120 h after isolation. Only seeds which have developed to 35 DAP produce viable seedlings. The inability of isolated 30-DAP seed to form viable seedlings appears to be related to a lack of stored nutrients, since the germinability of excised embryos (20 DAP and onwards) placed on Murashige and Skoog (1962, Physiol. Plant. 15, 473–497) medium is high. The switch from a developmental to germinative mode in the excised 30- and 35-DAP imbibed seeds is reflected in the pattern of in-vivo protein synthesis. Developmental and germinative proteins are present in the embryo and endosperm of the 30- and 35-DAP seeds 12 h after their isolation from the fruit. The mature seed (60 DAP) exhibits germinative protein synthesis from the earliest time of imbibition. The fruit environment prevents precocious germination of developing seeds, since the switch from development to germination requires only their removal from the fruit tissue.Abbreviations DAP days after pollination - kDa kilodaltons - SP1-4 storage proteins 1–4 - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - HASI hours after seed isolation - MS medium Murashige and Skoog (1962) medium This work is supported by National Science and Engineering Research Council of Canada grant A2210 to J.D.B.     

Electron transport dependent adenosine triphosphatase activity in castor bean endosperm mitochondria

The release of inorganic phosphate from ATP by mitochondriaisolated from endosperms of castor bean (Ricinus communis) wasstimulated by Mg⁺⁺, but not by Ca⁺⁺. EDTA, succinate, NADH₂or oligomycin depressed the reaction. The depression by succinatewas removed by KCN, antimycin A or anoxia. DNP alone did notaffect activity but did stimulate the Pi release in the presenceof succinate under aerobic conditions. Enhanced Pi release inthe presence of succinate and DNP was cancelled by KCN, antimycinA, oligomycin or anoxia. On the basis of these results, themechanism of ATPase action in castor bean endosperm mitochondriais discussed. (Received January 27, 1969; )     

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     

Albumin storage proteins in the protein bodies of castor bean

Of the total protein in the protein bodies of castor bean (Ricinus communis L.), approximately 40% is represented by a group of closely related albumins localized in the matrix of the organelle. This group of albumins has a sedimentation value of 2S and is resolved into several proteins of molecular weight around 12,000 daltons by sodium dodecyl sulfate-acrylamide gel electrophoresis. It has a high content of glutamate/glutamine and undergoes rapid degradation during the early stage of germination. In view of the abundance and ubiquitous occurrence of albumins in various seeds, we suggest that albumins, in addition to globulins, glutelins, and prolamines, are important storage proteins in seeds.     

The presence of ribulose 1,5-diphosphate carboxylase in the nonphotosynthetic endosperm of germinating castor beans

Ribulose 1,5-diphosphate carboxylase was detected in extracts of germinating castor bean (Ricinus communis var. Hale) endosperms. This is the first report of this enzyme in a nonphotosynthetic (no chlorophyll) plant tissue. Radioactive 3-phosphoglyceric acid has been identified as the principle product resulting from the enzymatic condensation of ¹⁴C-bicarbonate and ribulose-1,5-diP in endosperm extracts. The Km values of bicarbonate and ribulose-1,5-diP for the endosperm carboxylase are 1.14 × 10⁻²m and 7.5 × 10⁻⁵m, respectively. The carboxylase activity peaks at 4 days in endosperms of castor beans germinated in the dark. The specific activity of the carboxylase at this stage of germination is 4.3 μmoles of 3-phosphoglycerate formed/mg protein·hr. The presence of ribulose-1,5-diP carboxylase and other enzymes of the reductive pentose phosphate pathway show the potential of this pathway in castor bean endosperms.