Protein and nucleic acid synthesis during imbibition of dormant and after-ripened Vaccaria pyramidata seeds.

The regulation of nucleic acid and protein synthesis in dormant, thermodormant, and after-ripened embryos of Vaccaria pyramidata (Caryophyllaceae) has been studied. Germination of after-ripened V. pyramidata seeds is prevented by inhibitors of protein, RNA, and DNA synthesis. The synthesis of both protein and RNA is activated at the beginning of imbibition, whereas [³H]thymidine incorporation does not start until the second period of the imbibition phase. [³H]Thymidine incorporation is greatly reduced in embryos treated with cycloheximide or 6-methylpurine. There is no correlation between the level of [³H]uracil and l-[¹⁴C]leucine incorporation into macromolecules and the physiological state of the seeds: tRNA, ribosomal RNA, and poly(A)-containing RNA (probably mRNA) as well as proteins are synthesized at the same rate in both dormant and thermodormant embryos as in after-ripened embryos. The protein patterns of dormant and after-ripened embryos are similar, as shown by electrophoresis and electrofocusing of double-labeled proteins. The level of DNA synthesis, measured as [³H]thymidine incorporation, may, on the other hand, indicate the physiological activity of the seeds: [³H]Thymidine is incorporated at a high rate in after-ripened embryos only and remains at a low level in dormant or thermodormant embryos. This correlation is, however, observed only in the axes. DNA synthesis in the cotyledons does not show any relation to the developmental stage of the seeds. These results are discussed in relation to the regulation of dormancy and after-ripening of seeds.     

Differences in Patterns of Newly Synthesized Proteins in Imbibing Embryos of After-ripened and Aged Seeds of Agrostemma githago and Comparison of Protein Synthesis In Vivo and In Vitro

Patterns of newly synthesized proteins in imbibing after-ripenedyoung and aged Agrostemma embryos show differences in the dynamicsof cotyledons and axes. In the course of imbibition the terminationof some syntheses in the embryonal parts of aged seeds is delayed.As was shown by in vitro translation, mRNAs coding for the synthesisof some proteins are still present in the embryos when proteintranslation has already finished. Key words: Protein pattern, in vivo protein synthesis, in vitro protein synthesis, after-ripened embryos, aged embryos, Agrostemma githago     

Proteinbiosynthesen in dormanten und nachgereiften embryonen und samen von Agrostemma githago

Seed germination of Agrostemma githago is prevented by inhibitors of protein and RNA synthesis. Thus protein as well as RNA synthesis are essential prerequisites for germination. Early protein synthesis of Agrostemnia embryos can be completely inhibited by cycloheximide and cordycepin. During the aging of seeds there is a considerable decrease in germination capacity and protein synthesis. In dormant and afterripened embryos of Agrostemma githago¹⁴C-leucine and ¹⁴C-uracil are incorporated in protein and RNA respectively with nearly the same intensity, whereas RNA and protein synthesis of dormant seeds and embryos starts earlier than in those subjected to afterripening. ³H-uracil-labelled RNA from dormant and afterripened embryos are able to hybridize on oligo-dT-cellulose to the same extent. There is a similarity in the protein pattern of dormant and afterripened embryos revealed by electrophoresis in polyacrylamide gels of double-labelled proteins. According to these results dormancy of Agrostemma githago is not caused by a general but by a specific metabolic block.     

Release of dormancy during after-ripening of Agrostemma githago seeds

Freshly harvested seeds of Agrostemma githago L. do not germinate when they are imbibed at 20°C. The block is located in the embryo and is relased by dry storage at 20°C (after-ripening). Freshly harvested seeds complete only a small part of the processes that occur in after-ripened seeds during the lag phase prior to germination (radicle protrusion). After-ripening removed the block on lag phase processes much faster than the block on germination. This was shown both by direct determinations of the completion of lag phase processes and by measurements of the rate of axial protein synthesis, which approximately doubles when seeds are progressing through the lag phase. It is concluded that the percentage germination does not adequately reflect the extent to which the dormancy mechanism has been overcome.     

Protein Synthesis in Embryos of Dormant and Germinating Agrostemma githago L. seeds

The time course of protein synthesis in embryos of dormant and afterripened Agrostemma githago seeds was studied. In embryos of afterripened geminating seeds, protein synthesis increased in three successive stages: (a) concurrent with swelling; (b) during the lag phase between the completion of water uptake and the onset of growth; and (c) immediately after protrusion through the seed coat. Embryos of dormant seeds showed the first increase but not the second unless dormancy was broken by imbibition at 4°C. This indicates that dormancy affects processes prior to the onset of growth. The third increase was largely due to higher oxygen availability after the rupture of the seed coat and not to actual growth. It could also be elicited in dormant embryos by isolating them from the seeds.

Electrophoretic analysis of the newly synthesized proteins demonstrated that the patterns of dormant and afterripened embryos became significantly different in both axes and cotyledons only just prior to the onset of axis elongation. Thereafter, the differences became larger.

When afterripened or dormant seeds were transferred from a low, germination-permitting to a high, germination-inhibiting temperature, the seeds germinated at the high temperature if they had completed the lag phase to a sufficient extent at the low temperature. This shows that the processes during the lag phase were inhibited by the high temperature while the onset of growth was not affected.

     

Advantageous and Detrimental Effects of Osmotic Pre-Sowing Treatment on the Germination Performance of Agrostemma githago Seeds

Dormant and after-ripened seeds of Agrostemma githago (corn-cockle)were pretreated in polyethylene glycol 400 (PEG) solutions attemperatures which would have allowed germination if the seedshad been imbibed in water, viz. 4?C or 20?C for after-ripenedseeds, and 4?C for dormant seeds. Pretreated seeds germinatedfaster than untreated seeds. The maximum decrease of the T₅₀(time to 50% germination) was 66%. Furthermore, pretreated seedswere capable of germination at supra-optimal temperatures whichotherwise had inhibited germination completely (20?C for dormantseeds and 30?C for after-ripened seeds). The percentage germinationat a supra-optimal temperature was considerably higher whenthe seeds had been primed at a temperature at which they developedmore extension power. The advantageous effects of the osmotic pretreatment were lessthan might be expected when the osmoticum had inhibited onlycell elongation. This was largely, if not fully, due to a generaldetrimental effect of osmotic stress and not to a selectiveinhibition of the processes which occur during the pregerminativephase in preparation for growth. Thus, during priming seedscomplete all or almost all processes which occur in water-imbibedseeds prior to radicle emergence. Key words: Agroatemma githago, dormancy, germination, germination performance, osmotic stress, priming     

Proteomic analysis of seed dormancy in Arabidopsis

The mechanisms controlling seed dormancy in Arabidopsis (Arabidopsis thaliana) have been characterized by proteomics using the dormant (D) accession Cvi originating from the Cape Verde Islands. Comparative studies carried out with freshly harvested dormant and after-ripened non-dormant (ND) seeds revealed a specific differential accumulation of 32 proteins. The data suggested that proteins associated with metabolic functions potentially involved in germination can accumulate during after-ripening in the dry state leading to dormancy release. Exogenous application of abscisic acid (ABA) to ND seeds strongly impeded their germination, which physiologically mimicked the behavior of D imbibed seeds. This application resulted in an alteration of the accumulation pattern of 71 proteins. There was a strong down-accumulation of a major part (90%) of these proteins, which were involved mainly in energetic and protein metabolisms. This feature suggested that exogenous ABA triggers proteolytic mechanisms in imbibed seeds. An analysis of de novo protein synthesis by two-dimensional gel electrophoresis in the presence of [(35)S]-methionine disclosed that exogenous ABA does not impede protein biosynthesis during imbibition. Furthermore, imbibed D seeds proved competent for de novo protein synthesis, demonstrating that impediment of protein translation was not the cause of the observed block of seed germination. However, the two-dimensional protein profiles were markedly different from those obtained with the ND seeds imbibed in ABA. Altogether, the data showed that the mechanisms blocking germination of the ND seeds by ABA application are different from those preventing germination of the D seeds imbibed in basal medium.     

Untersuchungen über rnasen in kotyledonen von nachgereiften und dormantenagrostemma githago-samen

Activities of RNasea were studied in cotyledons of dormant and afterripenedAgrostemma githago seeds. Activity of RNase increases during imbibition and germination. This increase in activity cannot be observed in variants which are not able to germinate (dormant seeds and seeds blocked by higher temperature). The development of RNase activities during germination cannot be inhibited by concentrations of cycloheximide or actinomycine D completely preventing phosphatase synthesis. These results may be indicative for the assumption that the increase of RNase during germination is caused by enzyme activation and not by enzyme synthesis. Cytokinins and a combination of cycloheximide and gibberellic acid stimulate the activity of RNase in dormant cotyledons, whereas neither cycloheximide nor gibberellic acid, applicated by themselves, show any effect. Cytokinins and gibberellic acid do not influence the activity of RNase of afterripened cotyledons, abscisic acid inhibits the increase of enzyme activity. There are characteristic changes in the pattern of RNases during germination revealed by polyacrylamide gel electrophoresis. The increase in RNase activity of dormant cotyledons caused by cytokinins is accompanied by obvious changes in the RNase pattern on polyacrylamide gel. Treating dormant cotyledons with cytokinins dormancy is partially overcome. In consequence of the application of cytokinins the differences in the electrophoretic RNase pattern between dormant and afterripened cotyledons can be nearly balanced.     

Growth and Endogenous Gibberellin Content of Dormant Groundnut Embryonic-axes as Influenced by Leaching and GA(3)

Leacthing the excised embryonic-axes from dormant groundunt (Arachis hypogaea) seeds resulted in their growth. However, their growth was stunted compared to that of after-ripened ones. It is attributed to (1) the lower amount of gibberellin-like substances in the dry, dormant embryonic-axes than in the after-ripened ones, and (2) the inability of the former to sythesise tbe tibberetllin as indicated by the lower content compared to the after-ripened ones kept in water. Exogenousty supplied GA(3) (0.1 mg/1) increased both the endogenous gibberellin and growth of the dormant, leached embryonic-axes to the level of after-ripened ones.     

Inhibition of pregerminative processes in dormant and thermodormant Agrostemma githago seeds

Imbibed non-dormant seeds do not germinate immediately after completion of water uptake and reactivation of their metabolism. During the lag-period the seeds apparently undergo processes which are essential for germination. The extent to which these pregerminative processes occur in dormant seeds (freshly harvested seeds) and thermodormant seeds (afterripened seeds imbibed at a supra-optimal temperature) of Agrostemma githago was determined. The pregerminative processes were inhibited almost completely in dormant seeds, but only to 50% or less in thermodormant seeds. When seeds were progressing through the pregerminative processes, the axes showed a higher rate of protein synthesis than axes of blocked seeds. However, this increased rate of protein synthesis was a late event and neither necessary nor sufficient for germination.     

Possible involvement of volatile compounds in the after-ripening of cocklebur seeds

The mechanism of emergence from primary dormancy, the process of after-ripening, in cocklebur (Xanthium pennsylvanicum) seeds was examined in relation to the involvement of volatile compounds and to the relative humidity (RH) in which the seeds were stored. The after-ripening of these seeds proceeds only at water contents between 7 and 14% which are conditioned under RHs of 33% to 53% and are identified with water-binding region II. After-ripening of cocklebur seeds occurred even in water-binding region I. imposed by 12% RH. when exposed to HCN gas during the storage period. Exposure of dormant seeds to acetaldehyde (ethanal) retarded after-ripening. even in water-binding region II. thus decreasing germinability. This decrease of germinability by ethanal was found also in the after-ripened seeds, suggesting that ethanal accelerates seed deterioration rather than retarding the after-ripening. The contents of ethanal. ethanal and HCN were high only in the dormant seeds held at 12% RH. Regardless of RH. a possible conversion of ethanal to ethanol. perhaps via alcohol dehydrogenase. was far larger in dormant than in non-dormant seeds. In contrast, the reverse conversion of ethanol to ethanal was more profound in non-dormant seeds. Pre-exposure of both types of seeds to HCN reduced the contents of both ethanal and ethanol at 12% RH. The contents of various adenylales including ATP in seed tissues were higher in dormant seeds stored at 12% RH than in non-dormant seeds after-ripened at 44% RH. It is suggested that emergence of cocklebur seeds from primary dormancy by HCN treatment at 12% RH may result from the reduction in the contents of ethanal via an unknown mechanism incurring the consumption of ATP. This implies involvement of volatile compound metabolism at the water-binding region II in the after-ripening process of cocklebur seeds.     

Biosynthesis of Storage Proteins in Ripening Agrostemma githago L. Seeds

The synthesis of storage proteins in ripening Agrostemma githago seeds was studied by in vivo pulse and pulse-chase experiments with labeled amino acids and labeled glucosamine. It was found that storage proteins were not synthesized directly, but via cleavage of several large precursor proteins. Two disulfide-linked proteins of 38 and 25 kilodaltons were synthesized via a single large precursor protein. This precursor protein contained internal disulfide bridges, at least one of which is involved in holding the subunit structure together following cleavage of the precursor. A similar mode of biosynthesis was noted for two other disulfide-linked proteins of 36 and 22 kilodaltons. The half-life of the precursors was about 2 hours. This mode of processing is analogous to the synthesis of legumin in legumes and globulin in oats. A third pair of disulfide-bonded proteins (41 and 23 kilodaltons) was synthesized from a precursor protein in several steps. These included a legumin-like cleavage, whereafter the subunits remained disulfide-bonded. Then, from the largest subunit, a part was cleaved off, probably a storage protein of 17 kilodaltons. This 17-kilodalton protein was not disulfide-bonded to the 41 and 23-kilodalton complex. The first processing step was fast, the second slow: The half-lives of the precursors were about 3 and 10 hours, respectively. Finally, a group of 16- and 17-kilodalton proteins was synthesized by cleavage of large precursor proteins, likely in two steps. After cleavage, the proteins were not disulfide-bonded. The half-life of the precursors was short, less than 1 hour. In addition, for the 38-, 23-, and one of the 17-kilodalton proteins, a small decrease of relative molecular weight was observed as a last processing step. This was likely due to deglycosylation.     

Wheat seed proteins regulated by imbibition independent of dormancy status

Seed dormancy is an important trait in wheat (Trticum aestivum L.) and it can be released by germination-stimulating treatments such as after-ripening. Previously, we identified proteins specifically associated with after-ripening mediated developmental switches of wheat seeds from the state of dormancy to germination. Here, we report seed proteins that exhibited imbibition induced co-regulation in both dormant and after-ripened seeds of wheat, suggesting that the expression of these specific proteins/protein isoforms is not associated with the maintenance or release of seed dormancy in wheat.     

Abscisic acid,phaseic acid and gibberellin contents associated with dormancy and germination in barley

Analyses of abscisic acid (ABA), ent -kaurenoids and gibberellins (GAs) showed that there were major changes in the contents of these compounds associated with germination of after-ripened barley ( Hordeum vulgare cv. Schooner and cv. Proctor) grain but not in hydrated dormant grain. Embryos from dormant and after-ripened dry grain contained similar amounts of ABA, of ent -kaurenoids and of GAs, determined by gas chromatography-mass spectrometry-selected ion monitoring. In embryos of after-ripened grain, ABA content decreased rapidly after hydration and ABA appeared to be metabolized (inactivated) to phaseic acid (PA) rather than diffusing into the endosperm or the surrounding medium as previously thought. Similar changes in ABA occurred in hydrated dormant grain during germination in darkness. Accumulation of ent -kaurenoids and GAs, including GA_1, the first biologically active GA in the early 13-hydroxylation biosynthetic pathway, occurred to a much greater extent in after-ripened than in dormant grain and these changes occurred mainly after 18 h of hydration when ABA had already decreased and germination was occurring. The block in ent -kaurenoid and GA synthesis in dormant grain appeared to occur prior to ent -kaurene in the biosynthetic pathway. These results are consistent with the view that ABA is the primary effector of dormancy and that after-ripening involves the development of the ability to reduce the amount of ABA quickly following hydration. Accumulation of GAs does not appear to be causally related to loss of dormancy but it does appear to be related to germination.     

After-ripening and phytochrome action in seeds of dormant lines of wild oat (Avena fatua)

The effects of a short exposure to red, far-red or alternate red/far-red light on the germination of seeds after-ripened for different periods of time were studied in dormant lines of wild oat ( Avena fatua L.). Three stages were distinguishable in the after-ripening period in the response of germination to light. Seeds stayed dormant and showed no response to light during stage I. Phytochrome-mediated germination was observed in seeds during stage II. The phytochrome action disappeared during stage III, i.e. seeds fully germinated following treatments of all light qualities. When the seeds were imbibed in polyethylene glycol solutions, dark germination was reduced and phytochrome again had an effect, which suggested the involvement of phytochrome in water uptake of the seed.     

The role of abscisic acid in germination, storage protein synthesis and desiccation tolerance in alfalfa (Medicago sativa L.) seeds, as shown by inhibition of its synthesis by fluridone during development

Abscisic acid and osmoticum maintain maturation and proteinsynthesis of developing alfalfa embryos, individually and incombination. The in situ environment of developing alfalfa zygoticembryos is rich in ABA and low in osmotic potential. When ABAsynthesis was inhibited by treating the pods with fluridoneat an early stage of development, the seeds which subsequentlydeveloped contained low amounts of ABA, but had a similar osmoticpotential as untreated control seeds. The reduced ABA in seedsfrom fluridone-treated pods did not change the morphology exceptthe colour of seeds, nor did it induce viviparous germinationor affect storage protein synthesis. However, two nonstorageproteins which were synthesized in control seeds during earlyto mid-development were absent from fluridone-treated seeds.Control seeds containing these two proteins were desiccation-tolerant,whereas the fluridone-treated seeds which lacked them were desiccation-intolerant,at least until the deposition of storage proteins was nearlycomplete. Culture of isolated embryos on nutrient medium inducedgermination and curtailed storage protein synthesis in the embryos.Addition of either ABA or osmoticum to the nutrient medium preventedgermination and maintained storage protein synthesis. When fluridonewas added along with osmoticum, germination occurred, but storageprotein synthesis was maintained. Key words: Embryogenesis, Medicago sativa L., alfalfa, ABA, osmotic potential, fluridone, desiccation, storage protein synthesis     

Dormancy and Impotency of Cocklebur Seeds VIII. Lack of Germination Responsiveness in Primarily Dormant Seeds to Cyanide, Azide, Anoxia and Chilling

Germination responsiveness to KCN, NaN₃, chilling or anoxiaand respiration activity was compared between non-after-ripenedand after-ripened upper cocklebur (Xanthium pennsylvanicum Wallr.)seeds. The latter, coat-imposed dormant seeds, could germinatein response to the above chemicals and conditions, whereas theformer, primarily dormant seeds, could not respond. There waslittle difference in the respiratory properties of both typesof seeds. (Received June 22, 1981; Accepted October 30, 1981)     

Dormancy-associated embryonic mRNAs and proteins in imbibing Avena fatua caryopses

The mechanisms controlling seed dormancy maintenance and release are not understood. To characterize the molecular events accompanying dormancy release, two-dimensional gel electrophoresis was used to monitor changes in soluble proteins and in vitro translation products of embryonic mRNA populations during imbibition of dormant and nondormant (after-ripened) Avena fatua L. caryopses. No differences were observed between in vitro translation products of mRNA extracted from dry dormant and nondormant embryos. However, the expression patterns of several imbibition- and germination-associated mRNAs were temporally modulated during the first 24 h of imbibition. Two dormancy-associated mRNAs, represented by polypeptides D₁ and D₂, were differentially overexpressed in dormant embryos after 3 h of imbibition. mRNA levels for D₁ and D₂ were about 8- and 3-fold higher, respectively, in dormant embryos than in nondormant embryos after 3 h of imbibition. Overexpression of D₁ continued through 12 h of imbibition, while expression of both mRNAs fell to low and equivalent amounts in dormant and nondormant embryos after 24 h. Similar dormancy-associated changes in two soluble proteins were observed during imbibition. The results demonstrate that steady-state levels of specific mRNAs and proteins change during early imbibition of dormant and nondormant A. fatua embryos and indicate that these changes may be associated with differential gene expression responsible for the maintenance of dormancy.