Immunolocalization of FsPK1 correlates this abscisic acid-induced protein kinase with germination arrest in Fagus sylvatica L. seeds

An enzymatically active recombinant protein kinase, previously isolated and characterized in Fagus sylvatica L. dormant seeds (FsPK1), was used to obtain a specific polyclonal antibody against this protein. Immunoblotting and immunohistochemical analysis of FsPK1 protein in beech seeds showed a strong immunostaining in the nucleus of the cells located in the vascular tissue of the embryonic axis corresponding to the future apical meristem of the root. This protein kinase was found to accumulate in the seeds only when embryo growth was arrested by application of ABA, while the protein amount decreased during stratification, previously proved to alleviate dormancy, and no protein was detected at all when seed germination was induced by addition of GA(3). These results indicate that FsPK1 may be involved in the control of the embryo growth mediated by ABA and GAs during the transition from dormancy to germination in Fagus sylvatica seeds.     

Physical forces in dormancy and germination of xanthium seeds

The germination of seeds of Xanthium pensylvanicum Wallr. occurs in 2 phases, an initial passive phase of water uptake followed by an active phase of growth. These 2 phases have been separated experimentally, and shown to occur similarly in isolated cotyledons and embryonic axes. Measurements of the physical thrust generated by the entire seed and its separate components of cotyledon and axis reveal that non-dormant Xanthium seeds develop more than twice the thrust of dormant seeds, and that this difference develops principally in the second phase of enlargement of the axis. Measurement of the forces required for piercing the testa of these seeds establishes that whereas the thrust developed by non-dormant seed is adequate to cause testa rupture, that developed by dormant seeds is not. It is concluded that the dormancy of Xanthium involves an inadequacy in the embryo for rupture of the testa.     

Transcripts of a gene,encoding a small GTP-binding protein from Fagus sylvatica,are induced by ABA and accumulated in the embryonic axis of dormant seeds

A cDNA clone was selected from a cDNA library constructed using mRNA from ABA-treated Fagus sylvatica L. dormant seeds as a template. The clone is highly expressed in the presence of ABA and tends to disappear in stratified seeds. A search of sequence databases showed that the clone encodes a small GTP-binding protein. By means of in situ hybridization, the mRNA has been located in the apical meristem of the embryonic axis and in the central vascular cylinder. Its possible involvement in growth regulation in the embryonic axis of F. sylvatica is discussed.     

Ethylene as a Component of the Emanations From Germinating Peanut Seeds and Its Effect on Dormant Virginia-type Seeds

The embryonic axes of Spanish-type peanut seeds that do not exhibit dormancy to any extent were found to produce ethylene during germination. Virginia-type peanut seeds of the extremely dormant variety NC-13 produced low levels of ethylene when imbibed but not germinating. Treatments that released dormancy of NC-13 peanut seeds resulted in increased ethylene production by the embryonic axis. The estimated internal concentration of ethylene in Virginia-type peanut seeds was 0.4 ppm at 24 hr of germination. Fumigation with an external concentration of 3.0 to 3.5 ppm for 6 hr was sufficient to break dormancy of Virginia-type peanut seeds. These results suggest that ethylene is associated with the germination processes of non-dormant seeds and participates in the breaking of seed dormancy of dormant peanut varieties.     

Changes in protein synthesis during stratification and dormancy release in embryos of sugar maple (Acer saccharum)

Protein synthesis in dormant embryos of sugar maple ( Acer saccharum ) was investigated in seeds stratified at 4°C or incubated at 15°C. Seeds stratified at 4°C germinated after 27 days; seeds incubated at 15°C failed to germinate. Stratification increased the embryo's capacity for protein synthesis by day 11 as measured by in vivo incorporation of [³⁵S]-methionine into purified protein. At 4°C protein synthesis in the embryonic axis rose in a linear fashion prior to germination, whereas in cotyledons it increased until day 20 and then declined. Analysis of radiolabelled proteins by two-dimensional gel electrophoresis revealed that the levels of specific proteins were altered by temperature, primarily in the cotyledons. Several proteins were expressed in the cotyledons at 15°C but were absent in unstratified embryos and in embryos stratified at 4°C. That is, the expression of these proteins was repressed during stratification and release from dormancy. Levels of other proteins in the cotyledons declined at 4°C during stratification. We suggest that one or more of these proteins may be associated with the inhibition of growth of the embryonic axis imposed by the cotyledons.     

The Effect of Stratification and Thidiazuron Treatment on Germination and Protein Synthesis of Pyrus serotina Rehd cv. Niauli

Breakage of seed dormancy in Pyrus serotina Rhed cv. Niauliis achieved by cold treatment. The chilling time necessary forgermination of Pyrus serotina Rhed cv. Niauli seeds was determined,and synthesis of polypeptides during stratification and thidiazuron(TDZ) treatments was investigated. The chilling time requiredwas about 21 d. The longer the chilling time, the higher thegermination percentage. The effect of 200 µM TDZ on seedgermination was equivalent to 14-21 d of chilling, and TDZ couldbe used as a substitute for cold treatment. Use of in vivo ³⁵S-methionine label and two-dimensional gel electrophoretic analysisof polypeptides revealed that the patterns of untreated (dormant)and chilled embryonic axes differed significantly in their polypeptideprofiles. The difference became conspicuous after prolongedchilling exposure. During 28 d of stratification followed by5 d of incubation at 25 °C, changes were noted in 35 solublepolypeptides (ESPs) and 38 pellet polypeptides (EPPs) from theembryonic axis. The changes included increase, decrease or newlyinduced synthesis. Common patterns of changes in soluble andpellet polypeptides of embryonic axes, can be broken down intofour categories. The possible functions and roles of these fourcategories of polypeptides in breaking seed dormancy and germinationare discussed. Although the magnitude of polypeptide changesare not as extensive in seeds receiving TDZ treatment as afterstratification, similar polypeptide changes were co-inducedby the two treatments.Copyright 1994, 1999 Academic Press Stratification, protein synthesis, pear, Pyrus serotina, Thidiazuron, TDZ     

Non-Decarboxylating transformation of indol-3-ylacetic acid in apple seeds

An enzyme extract from apple(Pyrus malus Borb.) seeds which causes the disappearance of free indol-3-ylacetic acid (IAA) requires the presence of oxygen, but is not inhibited by cyanide. Using 1-¹⁴C-IAA it has been demonstrated that the IAA transformation is not accompanied by its decarboxylation. Decarboxylating IAA oxidase is absent during the whole period of apple seed cold stratification. Free IAA has not been detected in dormant apple seeds and in seeds stratified at low temperature. It appears during stratification at 25 °C. Ethyl ester of IAA and indol-3-ylacetyl aspartate have been identified in dormant and after-ripened seeds. Exogenous 1-¹⁴C IAA taken up by apple embryos is converted into conjugates with aspartate and short peptides containing an aspartate moiety.     

Effects of the embryonic axis and phytohormones on proteolysis of the storage protein in buckwheat seed

The role of the embryonic axis in regulation of proteolysis of the main storage protein was studied in buckwheat ( Fagopyrum esculentum ) seed. Polyacrylamide gel electrophoresis (PAGE) analysis revealed that removal of the embryonic axis had no effect on the first stage of hydrolysis, that is proteolytic modification, of 13S globulin. This modification took place in the growing seedlings also in the presence of cycloheximide, i.e. it was due to an enzyme present in dry seed. However, in isolated cotyledons the 13S globulin was not degraded completely. Incubation of isolated cotyledons with cytokinins, gibberellic acid and indoleacetic acid could not replace the excised embryonic axis. At the same time, proteolysis of the 13S globulin in the growing seedlings was strongly inhibited by casein hydrolyzate. It is suggested that a complete proteolysis of the modified storage protein is regulated by the concentration of hydrolysis products at the site of hydrolysis. The embryonic axis serves, most probably, as a site of efflux of the products of protein hydrolysis in the cotyledons during seedling growth and thus regulates the course of proteolysis.
Abscisic acid (10–100 μ M ) was without effect on modification of the 13S globulin, but suppressed the complete proteolysis of the protein by inhibiting, apparently, the synthesis of the cysteine proteinase in the growing seedlings.     

MECHANISM OF SEED DORMANCY IN AMBROSIA ARTEMISIIFOLIA

Dormancy in Ambrosia artemisiifolia seeds was broken by 8 weeks of stratification. Germination of nondormant seeds was greater in light than in continuous darkness. Embryos of freshly harvested seeds were nondormant. Leaching and scarification did not stimulate germination of the dormant seeds. Exogenous gibberellin (GA₃) slightly increased germination of intact dormant seeds, and the effect was greatly increased by scarification. Germination was greater in the light in both tests. Exogenous indoleacetic acid did not stimulate germination of dormant seeds. Endogenous gibberellin and auxin content increased during stratification, and there was also a significant increase in GA during post-stratification at a favorable germination temperature. Inhibitors in the dormant seeds decreased during stratification and post-stratification. The high concentration of chlorogenic acid present in dormant seeds increased slightly during stratification. An unknown phenol very similar to chlorogenic acid in fluorescence and U.V. absorption significantly increased after 2 weeks of stratification. A significant decrease in the concentration of a second unidentified phenol occurred after 2 weeks of stratification. It is proposed that dormancy in Ambrosia artemisiifolia may be controlled by an inhibitor-promoter complex. The dormant seed is characterized by high inhibitor and low promoter levels. In the nondormant seed the balance was shifted to favor the promoter. Evidence suggests that the inhibitor involved may be abscisic acid and the promoters may be gibberellin and auxin. The content of auxin may be partially controlled by the concentration of phenols.     

离体发育和萌发中花生种子不同部份内源ABA含量的变化

离体发育和萌发中花生种子不同部份的内源ＡＢＡ含量变化存在明显的差异．种皮和子叶内存在ＡＢＡ的Ｃ（４０）生物合成途径,胚轴内恻为Ｃ（１５）生物合成途径．种子内源ＡＢＡ含量变化和种子活力有密切关系．     

Protein changes between dormant and dormancy-broken seeds of Prunus campanulata Maxim

Seed dormancy is regulated by complex networks in order to optimize the timing of germination. However, the biochemical basis of the regulation of seed dormancy is still poorly understood. Many temperate timber species, which are of ecological and/or economic interest, are deeply dormant in seeds, such as Prunus campanulata. Freshly harvested seeds require warm plus cold stratification to break dormancy before they can begin to germinate. According to the results of germination, both warm and cold stratifications are the critical influences for breaking seed dormancy. Significant variations in seed proteins were observed by 2-DE before and after the breaking of seed dormancy. Among the 320, 455, and 491 reproducibly detected spots on the cotyledons, embryos, and testae, respectively, 71 dramatic changes in abundances were observed following warm and/or cold stratification. Among these protein spots, dehydrin, prunin 1 precursor, prunin 2 precursor, and prunin 2 were identified by MS and sequence comparison. The implications of protein changes in relation to the breaking of seed dormancy and germination are discussed. This is the first report of a proteomic analysis of dormancy breaking in woody plant seeds.     

The Effect of Stratification on the Endogenous Cytokinin Levels of Seed of Protect compacta and Leucadendron daphnoides

The soybean callus assay was used to study the effect of stratification on the cytokinin levels of the embryo dormant seed of Protea compacta R.Br. and the seed of Leucadendron dapbnoides Meisn., where dormancy is coat imposed. Chilling the seed for 30 days increased germination significantly, and resulted in a simultaneous increase in the butanol soluble cytokinins of both species. It would appear as if these compounds are either synthesized or released from a bound form in embryo dormant seed. In contrast, an interconversion from water soluble to butanol soluble cytokïnins appears to account for the increase where dormancy is coat imposed. The results also indicate that for germination to take place a threshold concentration of cytokinin may be required. It is suggested that the increase in butanol soluble cytokinins may lead to the breaking of dormancy, probably by increasing radicle elongation and/or cotyledon expansion.     

The influence of cytokinins on apple embryo photosensitivity and acid phosphatase activity during stratification

Kinetin (KIN) and benzyladenine (BA) stimulate to different extent the germination of apple embryos isolated from dormant seeds or seeds submitted to stratification. KIN is much more active in the replacement of light requirement in apple embryos germination. Both cytokinins decrease the photosensitivity of embryos isolated from the seeds stratified less than one month, but only BA accelerates the appearance of the second photosensitivity maximum, normally occuring on the 70th day of stratification. Both cytokinins stimulate the activity of acid phosphatase between the 30th and 50th day of apple seed stratification. The stimulation between the 50th day and the end of stratification is exerted only by KIN. These differences allow to discuss the specificity of action of particular cytokinins during the after-ripening and germination of apple embryos.     

Embryo Dormancy and Light Requirements in the Germination of Helipterum craspedioides (Compositae), an Arid-zone Annual

Seeds of Helipterum craspedioides require light for germination,and this may be given while the seed is not fully imbibed. Removalof the seed coat, or leaching, does not bring about germinationin the dark. Gibberellin enhances dark germination to some extent,whether or not the seed coat is removed. The light requirementtherefore resides in the embryo. Fresh seed is dormant in lightor dark. Excised embryos of these dormant seeds grow slowly,rarely giving mature plants, but a larger number of normal plantsis produced after gibberellin treatment. Dormancy is lost afterhigh temperature storage, but not stratification.     

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.

     

The role of lipases in the removal of dormancy in apple seeds

Summary It was found that the temperature optimum for apple (Malus domestica Borb.) seed acid lipase is the same as that for seed after-ripening process. The activity of the enzyme occurs between the 40th and 70th days of stratification, whereas the activity of alkaline lipase very low at that time appears about 20 days later. The changes of both enzyme activities were also studied during dark and light culture of embryos isolated from seeds after different times of stratification. Only the alkaline enzyme activity is under the control of light. It was concluded that essentially the same process, i.e. the hydrolysis of reserve fats is catalysed by two different enzymes: acid lipase acting during the cold-mediated breaking of embryo dormancy and alkaline lipase acting during the germination of dormant embryos, thus being under light control.