Isoenzyme Activities during the Early Stages of Allium Radicle Germination

As a continuation of a study of the molecular events surrounding the establishment of a mature root apex, changes in the pattern and specific activity of the isoenzymes of aminopeptidase (AmP), esterase (EST), glucose-6-phosphate de-hydrogenase (G6PD) and glutamic-oxaloacetic transaminase (GOT) were investigated during the early germinative growth of the Allium radicle tip. Polyacrylamide disc gel electrophoresis revealed a total of 9 EST fractions. A shift in pattern and reactivity characteristic of pre-emergence to those characteristic of post-emergence was demonstrated. G6PD electropherograms displayed 5 isoenzyme bands. Relative specific activity analyses indicated that this enzyme may be more important during the very early stages of germination. Only quantitative differences were noted in the 3 AmP isoenzymes detected. The progressive increase in AmP isoenzyme activity during the period of maximal protein body degradation suggested a role for these exopeptidases in the degradation of reserve proteins. The 3 isoenzyme bands of GOT exhibited only quantitative changes during germination. Parallel increases in the quantity of soluble protein and GOT specific activity suggest that this isoenzyme is involved in the onion's nitrogen metabolism during germination.     

Hydrolysis of lipid and protein reserves in loblolly pine seeds in relation to protein electrophoretic patterns following imbibition

The correlation between changes in seed protein electrophoretic patterns and the hydrolysis of lipid and protein reserves of loblolly pine ( Pinus taeda L.) seed was studied. Seeds were incubated at 30°C for up to 12 days following stratification, then megagametophytes and embryos were assayed for lipid and protein content after each day of imbibition. The megagametophyte of mature seed was found to contain 20% lipid and 12% storage protein on a fresh weight basis. The embryo contained 26% lipid and 15% protein. Both lipid and protein reserves were depleted constantly following imbibition. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of soluble and insoluble protein fractions showed a 60 kDa protein that was representative of crystalloid-like proteins. These crystalloid-like proteins comprised 85% of the insoluble protein storage reserves. A small number of insoluble storage proteins, including a 47 kDa protein, were distinct in that they were unaffected by 2-mercaptoethanol treatment. The soluble fractions from both tissues were labelled with [³⁵S]-methionine, and incorporation was visualized by two-dimensional electrophoresis. Proteins were found to belong to one of three categories, those synthesized constitutively (comprising the bulk of newly synthesized proteins), those synthesized during germination or those synthesized after radicle emergence. Accompanying seed reserve hydrolysis were developmental shifts in protein pattern and synthesis, suggesting the possibility that control of hydrolysis is at the level of enzyme accumulation.     

Natural priming as an important metabolic event in the life history of Wigandia urens (Hydrophyllaceae) seeds

Natural priming promotes fast and synchronic seed germination and enhances the establishment of seedlings from Wigandia urens seeds. In this study, seeds of this species were buried in the field in three different sites, and each site was divided into three different microsites (natural priming). They were then exhumed and air-dried at the beginning of the rainy season. The control and exhumed W. urens seeds were tested for differences in germination, and in the protein patterns derived from heat-stable and phospho-protein enriched fractions, sucrose concentrations, amylase isoenzyme patterns, as well as catalase activity and H₂O₂ levels. All the exhumed seeds germinated faster and more synchronically than the control seeds. Nevertheless, the germinative pattern showed a microsite effect related to the microenvironmental conditions. Most exhumed samples kept the germinative advantages acquired from the soil during natural priming for 2 years but seeds from the most heterogeneous site (a forest gap) did not. Natural priming also promoted mobilization of 7-S globulin-like proteins, solubilization of the 11-S globulin-like proteins as well as sucrose consumption before radicle protrusion. Although there were no changes in starch concentration during the germination of either the control or buried seeds, a different isoenzyme amylase pattern was observed. Catalase activity decreased and hydrogen peroxide levels were lower in exhumed seeds during germination. The changes in the protein and sucrose patterns were related to advances in the germinative process acquired during burial. Metabolic advantages were maintained systematically in all seed samples along the 2 years. The ecological significance of natural priming is discussed.     

An evaluation of the potential of low temperature pre-sowing treatments of tomato seeds as a means of improving germination performance

Low temperature pre-sowing treatment of tomato seeds substantially enhanced germination rates and was much more effective at improving uniformity of germination than equivalent osmotic treatments. Although radicle emergence was inhibited by the low temperature, imbibition studies showed that seeds were maintained at lag-phase moisture contents during treatment and some aspects of germinative metabolism began. No evidence was found for improved seedling growth rates per se as a result of pretreatment: in fact, initial axis growth may be temporarily reduced, probably as a consequence of depletion of reserves during the treatment period.     

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     

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     

Early carbon mobilization and radicle protrusion in maize germination

Considerable amounts of information is available on the complex carbohydrates that are mobilized and utilized by the seed to support early seedling development. These events occur after radicle has protruded from the seed. However, scarce information is available on the role of the endogenous soluble carbohydrates from the embryo in the first hours of germination. The present work analysed how the soluble carbohydrate reserves in isolated maize embryos are mobilized during 6-24 h of water imbibition, an interval that exclusively embraces the first two phases of the germination process. It was found that sucrose constitutes a very significant reserve in the scutellum and that it is efficiently consumed during the time in which the adjacent embryo axis is engaged in an active metabolism. Sucrose transporter was immunolocalized in the scutellum and in vascular elements. In parallel, a cell-wall invertase activity, which hydrolyses sucrose, developed in the embryo axis, which favoured higher glucose uptake. Sucrose and hexose transporters were active in the embryo tissues, together with the plasma membrane H(+)-ATPase, which was localized in all embryo regions involved in both nutrient transport and active cell elongation to support radicle extension. It is proposed that, during the initial maize germination phases, a net flow of sucrose takes place from the scutellum towards the embryo axis and regions that undergo elongation. During radicle extension, sucrose and hexose transporters, as well as H(+)-ATPase, become the fundamental proteins that orchestrate the transport of nutrients required for successful germination.     

Immunochemical study of changes in reserve proteins of germinating soybean seeds

Changes in the reserve proteins of soybean seeds (Glycine max) were investigated by the techniques of disc electrophoresis and disc immunoelectrophoresis. Three different antisera were used in these studies, an anti-whole soybean extract serum 129, an anti-11S soybean protein monospecific serum 102, and an anti-7S soybean protein monospecific serum 132. At least 6 antigenically distinct components were found to be present in the proteins of the isolated soybean protein bodies. These components are metabolized at different rates during germination. The major soybean protein (11S component) is found to be present even after 16 days of germination, whereas the 7S component disappears after the ninth day. Histochemical observations of cotyledon sections during germination are also reported.     

Importance of methionine biosynthesis for Arabidopsis seed germination and seedling growth

Proteomics of Arabidopsis seeds revealed the differential accumulation during germination of two housekeeping enzymes. The first corresponded to methionine synthase that catalyses the last step in the plant methionine biosynthetic pathway. This protein was present at low level in dry mature seeds, and its level was increased strongly at 1-day imbibition, prior to radicle emergence. Its level was not increased further at 2-day imbibition, coincident with radicle emergence. However, its level in 1-day imbibed seeds strongly decreased upon subsequent drying of the imbibed seeds back to the original water content of the dry mature seeds. The second enzyme corresponded to S -adenosylmethionine synthetase that catalyses the synthesis of S -adenosylmethionine from methionine and ATP. In this case, this enzyme was detected in the form of two isozymes with different p I and M r. Both proteins were absent in dry mature seeds and in 1-day imbibed seeds, but specifically accumulated at the moment of radicle protrusion. Arabidopsis seed germination was strongly delayed in the presence of dl -propargylglycine, a specific inhibitor of methionine synthesis. Furthermore, this compound totally inhibited seedling growth. These phenotypic effects were largely alleviated upon methionine supplementation in the germination medium. The results indicated that methionine synthase and S -adenosylmethionine synthetase are fundamental components controlling metabolism in the transition from a quiescent to a highly active state during seed germination. Moreover, the observed temporal patterns of accumulation of these proteins are consistent with an essential role of endogenous ethylene in Arabidopsis only after radicle protrusion.     

Changes in Protein Synthesis upon Cytokinin-Mediated Adventitious Bud Induction and during Seedling Development in Norway Spruce, Picea abies

A pulse-treatment of embryos of Picea abies (L.) Karst with cytokinin efficiently and reproducibly induces the coordinate de novo formation of bud primordia from subepidermal cells. The cytokinin treatment also affects the germinative development of the embryo; chloroplast maturation is delayed, and cell elongation is completely suppressed. We have analyzed the protein patterns in developing spruce embryos with the aim of identifying proteins which are differentially synthesized during early bud-differentiation and germination. In addition to a set of major seed storage proteins and a large set of constitutively synthesized proteins, we distinguish two sets of proteins that showed different patterns of synthesis in relation to germination. One was synthesized at high rates during germination, and the second set during post-germinative seedling development. Twenty-two proteins were differentially synthesized in the bud-induced versus the germinating embryos. Interestingly, all 22 belonged to either the germination phase-abundant or the seedling protein sets, whereas the constitutively synthesized proteins were unaffected by the treatment. Proteins synthesized exclusively in bud-induced embryos were not found. In total, the bud-induction treatment caused a maintenance of a protein synthesis pattern typical for the germination phase in the nontreated embryos, and the de novo formation of buds was not preceded by a major change in gene expression in the tissue.     

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     

Lack of correlation between overall protein synthesis and the onset of cell elongation in germinating embryos of Haplopappus gracilis

10⁻⁵M abscisic acid (ABA) completely inhibits germination or (if seeds deprived of integuments are used) embryo elongation in Haplopappus gracilis (Nutt.) Gray. Nevertheless, considerable rates of protein and RNA synthesis were found in embryos grown in abscisic acid, at least during the early hours after sowing. On the contrary, seeds grown in cycloheximide + fusicoccin (a powerful promoter of cell expansion), where protein synthesis is almost completely inhibited, show full protrusion of radicle, thus simulating a "germination" process. These results suggest that some of the most important events involved in seed germination, i.e. protein and RNA synthesis, and cell elongation which leads to radicle protrusion, may not necessarily be linked together and are possibly regulated by different control mechanisms. Moreover, when seeds or embryos are grown in abscisic acid + fusicoccin, protein synthesis is considerable, cell elongation is greater than in water controls at least for 12 h, and germination in its early stages appears to be normal; but DNA synthesis and cell division are not resumed, possibly since some other factor is required. All these findings propose a reevaluation of criteria for defining successful germination.     

长喙毛茛泽泻胚中营养物质的积累与消耗

长喙毛茛泽泻是一种水生濒危植物。它的种子中没有胚乳,营养物质以淀粉和帽白体的形式贮藏在胚中。胚不同部位物质积累情况差异较大,下胚轴和子地细胞中的淀粉,蛋白体数目多,体积大,胚芽和胚根分生细胞中只贮藏有少量的淀粉粒和蛋白体。     

Relationships among cytokinins, ethylene and poly amines during the stratification-germination process in seeds of Acer saccharum

Relatively high levels of dihydrozeatin and trans-zeatin were detected in unstratified seeds of Acer saccharum Marsh. Both cytokinins increased substantially over the first 20 days of stratification at 5°C and then fell rapidly to values well below original levels by first germination on day 55. In seeds held at 20°C, a non-afterripening temperature, cytokinin levels remained constant for the first 10 days and then declined to their lowest levels by day 20. Levels of putrescine, spermidine and spermine in the radicles and cotyledons did not change during the full course of the afterripening process, but large increments were noted during radicle emergence. A large increase in ethylene production at germination suggests that competition for S-adenosyl-methionine by the ethylene and polyamine biosynthetic pathways did not inhibit synthesis of ethylene or polyamines during seedling emergence and establishment. In seeds stratified at 20°C, ethylene showed an exceptionally large peak early in the stratification period, but polyamine contents remained low throughout the test. The present results are consistent with the hypothesis that cytokinins play a significant role in overcoming the metabolic block present in dormant seeds. This conclusion is supported by data showing that high levels of cytokinins develop concurrently with the start of tissue differentiation and at the time when abscisic acid and phenolic inhibitors decline in stratifying seeds. Changes in ethylene and polyamine contents did not correlate with any events in the afterripening process; however, large increases in levels of these substances were closely associated with the germinative process and, in the case of polyamines, specifically with the start of cell division.     

Lipid-body lipoxygenase is expressed in cotyledons during germination prior to other lipoxygenase forms

Lipid bodies are degraded during germination. Whereas some proteins, e.g. oleosins, are synthesized during the formation of lipid bodies of maturating seeds, a new set of proteins, including a specific form of lipoxygenase (LOX; EC 1.13.11.12), is detectable in lipid bodies during the stage of fat degradation in seed germination. In cotyledons of cucumber (Cucumis sativus L.) seedlings at day 4 of germination, the most conspicuous staining with anti-LOX antibodies was observed in the cytosol. At very early stages of germination, however, the LOX form present in large amounts and synthesized preferentially was the lipid-body LOX. This was demonstrated by immunocytochemical staining of cotyledons from 1-h and 24-h-old seedlings: the immunodecoration of sections of 24-h-old seedlings with anti-LOX antiserum showed label exclusively correlated with lipid bodies of around 3 m in diameter. In accordance, the profile of LOX protein isolated from lipid bodies during various stages of germination showed a maximum at day 1. By measuring biosynthesis of the protein in vivo we demonstrated that the highest rates of synthesis of lipid-body LOX occurred at day 1 of germination. The early and selective appearance of a LOX form associated with lipid bodies at this stage of development is discussed.Abbreviations DAPI 4,6-diamidino-2-phenylindole - DIC differential interference contrast - FITC fluorescein-5-isothiocyanate - LOX lipoxygenase This work was financially supported by the Deutsche Forschungsgemeinschaft, SFB 286 (I.F., A.N., H.K.) and SFB 363 (B.H., C.W.).     

Chromosomal proteins of conifers

During imbibition and germination of jack pine seeds, the composition of the total extractable chromatin varied. Relative to DNA, the histone levels decreased as the nonhistone chromosomal proteins (NHCP) increased. New chromosomal proteins were synthesized after 2 days of imbibition as judged by recovery of ¹⁴C-amino acids from the major protein fractions. Phosphorylation of histones from ³²P-phosphoric acid was detected before the incorporation of ¹⁴C-amino acids. In the seed the synthesis and relative changes of chromatin coincided with a fall in total soluble protein and free arginine N. By contrast, adenylate energy charge, free glutamine N and in vitro template activity of chromatin increased during chromatin protein synthesis. When seeds had germinated for 4 days after the start of imbibition more radioactivity, derived from free ¹⁴C-amino acids, was recovered from the NHCP than from the histones. The percentage amino acid composition of most histone fractions remained stable, whereas the composition of NHCP changed more with time. The phosphorylation of NHCP was 8- to 41-fold greater than that of the histones. Phosphorylation of histone H4 was not detected at any stage of germination. Correlations between recovery of radioactivity (³²P and ¹⁴C) from chromosomal proteins and higher adenylate energy charge were positive.     

A proteomic analysis of rice seed germination as affected by high temperature and ABA treatment

Seed germination is a critical phase in the plant life cycle, but the specific events associated with seed germination are still not fully understood. In this study, we used two‐dimensional gel electrophoresis followed by mass spectrometry to investigate the changes in the proteome during imbibition of Oryza sativa seeds at optimal temperature with or without abscisic acid (ABA) and high temperature (germination thermoinhibition) to further identify and quantify key proteins required for seed germination. A total of 121 protein spots showed a significant change in abundance (1.5‐fold increase/decrease) during germination under all conditions. Among these proteins, we found seven proteins specifically associated with seed germination including glycosyl hydrolases family 38 protein, granule‐bound starch synthase 1, Os03g0842900 (putative steroleosin‐B), N‐carbamoylputrescine amidase, spermidine synthase 1, tubulin α‐1 chain and glutelin type‐A; and a total of 20 imbibition response proteins involved in energy metabolism, cell growth, cell defense and storage proteins. High temperature inhibited seed germination by decreasing the abundance of proteins involved in methionine metabolism, amino acid biosynthesis, energy metabolism, reserve degradation, protein folding and stress responses. ABA treatment inhibited germination and decreased the abundance of proteins associated with methionine metabolism, energy production and cell division. Our results show that changes in many biological processes including energy metabolism, protein synthesis and cell defense and rescue occurred as a result of all treatments, while enzymes involved in methionine metabolism and weakening of cell wall specifically accumulated when the seeds germinated at the optimal temperature.     

Developmental and germinative events can occur concurrently in precociously germinating Chinese cabbage (Brassica rapa ssp. Pekinensis) seeds

During germination in the siliquae of developing seeds of a precociously germinating (PG) line of Chinese cabbage (Brassica rapa spp. Pekinensis), the synthesis of cruciferin and oleosins was maintained and the activities of isocitrate lyase and malate synthase increased. Thus, both developmental and germinative/post-germinative events coexisted within the same PG seeds. Drying of the seeds resulted in the developmental events being switched off and germinative/post-germinative ones being initiated. Only about 18% of the seeds of the PG line completed germination in the siliquae, so the potential for germination of the remaining non-germinating seeds was tested during their development. Isolation of these developing seeds from the siliquae and imbibition in water induced PG, but developmental events were terminated and the seeds entered a germinative/post-germinative programme. This termination of developmental events in the induced PG seeds was not permanent, however, and they could be re-induced by incubating the seeds in ABA on Murashige and Skoog (MS) medium. If incubated on MS medium plus a low concentration of ABA, both developmental and germinative/post-germinative events could be induced to coexist in the PG seeds up to the time of establishment of the seedlings. Control of the developmental and germinative/post-germinative events in the seeds was influenced by the environment in which they were developing, which could be mimicked by manipulating the nutritional status and ABA content in which the seeds were in contact.Keywords: Abscisic acid, ABA, Brassica rapa, Chinese cabbage, stored reserves, precocious germination, seed development      

Molecular characterization of proteins in protein-body membrane that disappear most rapidly during transformation of protein bodies into vacuoles

During the post-germination growth of seeds, protein bodies fuse with one another and are converted to a central vacuole. To investigate this transition, protein-body membranes from dry seeds of pumpkin (Cucurbita sp.) were prepared and their protein components characterized. Five major proteins (designated MP23, MP27, MP28, MP32 and MP73) were detected in the protein-body membranes. A cDNA clone encoding both MP27 and MP32 has been isolated. The deduced precursor polypeptide was composed of a hydrophobic signal sequence, MP27 and MP32, in that order. A putative site of cleavage between MP27 and MP32 was located on the COOH-terminal side of asparagine 278, an indication that the post-translational cleavage may occur by the action of a vacuolar processing enzyme that converts proprotein precursors of seed proteins into the mature forms. Immunoelectron microscopic analysis showed that MP27 and MP32 were associated with protein-body membrane of dry pumpkin seeds. Among the five membrane proteins, MP27 and MP32 disappeared most rapidly during seedling growth. The degradation of MP27 and MP32 starts just after seed germination and proceeds in parallel with the transformation of the protein bodies into a vacuole.