Regulation of alcohol dehydrogenase gene expression in barley aleurone by gibberellin and abscisic acid

The expression of the Adh1 gene (alcohol dehydrogenase, EC 1.1.1.1) was studied in the aleurone layer of barley ( Hordeum vulgare cv. Himalaya). Expression increased markedly during grain development at the levels of activity, enzyme protein and mRNA. mRNA content, but not enzyme activity, could be increased further by exogenous abscisic acid (ABA) when isolated, de-embryonated developing grains were pre-treated with gibberellic acid (GA₃) or fluridone. In isolated mature aleurone layers incubated with exogenous hormones, ADH mRNA was strongly up-regulated by ABA and down-regulated by GA₃ within 6 h. With ABA, this increase in mRNA was followed by an increase in ADH protein and activity, peaking at 18 h. With GA₃, the decrease in mRNA was accompanied by simultaneous decreases in protein and activity. In general, GA₃ counteracted the effect of ABA and vice versa. In the aleurone of germinating grain, ADH activity decayed in a distal direction from the embryo, consistent with down-regulation by gibberellin(s) diffusing from it. It was concluded that ADH gene expression in the aleurone of the intact grain is regulated by an ABA/gibberellin interaction.     

Modulation of germination of embryos isolated from dormant and nondormant barley grains by manipulation of endogenous abscisic acid

Dormant and non-dormant barley (Hordeum distichum L.) grains with identical genetic backgrounds were obtained by maturing grains under different climate conditions. When isolated embryos from dormant grains were incubated in a well containing a fixed volume of water (300 l), the germination rate and percentage were dependent on the embryo number per well. A higher embryo number per well was correlated with a lower germination rate and percentage. However, this was not the case for the embryos isolated from nondormant grains. During germination, the endogenous cis-abscisic acid (ABA) in isolated embryos from both dormant and nondormant grains was analyzed. The inhibitory effect on germination of a higher number per well of isolated dormant embryos was due to diffusion of endogenous ABA out of the embryos and accumulation of ABA in the incubation medium. Moreover, there was de-novo synthesis of ABA in embryos isolated from dormant grains during incubation but not in embryos isolated from nondormant grains. The inhibitory effect of ABA on germination of embryos isolated from dormant grains could be mimicked by addition of ABA or the medium in which dormant embryos had been placed. Embryos isolated from nondormant grains were insensitive to addition of ABA and medium from dormant embryos. Our results demonstrate that diffusion of endogenous ABA, de-novo ABA synthesis and ABA sensitivity play a role in the control of germination. It is proposed that dormancy-breaking treatments act via changes to these processes.Abbreviations ABA cis-abscisic acid - E/W embryo(s) per well Prof. K.R. Libbenga (Institute of Molecular Plant Sciences, Leiden University) is thanked for fruitful discussions. B.V.D. was partly supported by E.E.C. BIOTECH program PL 920175.     

Molecular cloning and characterization of an inorganic pyrophosphatase from barley

A cDNA clone with sequence homology to soluble inorganic pyrophosphatase (IPPase) was isolated from a library of developing barley grains. The protein encoded by this clone was produced in transgenic Escherichia coli, and showed IPPase activity. In nondormant barley grains, the gene appeared to be expressed in metabolically active tissue such as root, shoot, embryo and aleurone. During imbibition, a continuous increase of the steady state mRNA level of IPPase was observed in embryos of non-dormant grains. In the embryos of dormant grains its production declined, after an initial increase. With isolated dormant and nondormant embryos, addition of recombinant IPPase, produced by E. coli, enhanced the germination rate. On the other hand, addition of pyrophosphate (PPi), substrate for this enzyme, appeared to reduce the germination rate. A role for this IPPase in germination is discussed.     

Free and conjugated gibberellins in dormancy and germination of apple seeds

Halińska, A. and Lewak, St. 1987. Free and conjugated gibberellins in dormancy and germination of apple seeds.
The presence of gibberellin A₄ (GA₄) was confirmed in partly stratified seeds of apple ( Malus domestica Borb., cv. Antonówka) by mass spectrometry of the methyl ester. Levels of free and conjugated gibberellins A₄₊₇ and A₉ changed during drying of mature seeds, during cold and warm stratification, as well as during germination of dormant and non-dormant embryos. The temporary rise in GA₄₊₇ during cold stratification and during the culture of dormant embryos as well as the lack of it under conditions of warm stratification, allowed us to postulate a role for GA₄₊₇ in the removal of dormancy. In addition, GA₉ was absent in dormant embryos and increased during cold stratification and during the culture of non-dormant embryos. This suggests the involvement of GA₉, in induction of normal development of the seedling. The equivalence between changes in free and conjugated GAs suggests that formation and hydrolysis of conjugates are involved in the control of the physiologically active levels of free GA₄₊₇ and GA₉.     

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.     

Rapid Germination of a Barley Mutant Is Correlated with a Rapid Turnover of Abscisic Acid Outside the Embryo

In our study of the role of abscisic acid (ABA) in controlling the germination of barley grains, we tested a barley mutant line with a gigantum appearance (Hordeum distichum cv Quantum) for an ABA-insensitive phenotype by assaying germination in the presence of 10-4 M ABA. Dissected embryos of the mutant germinated at least 10 h earlier than did those of the wild type. The half-maximal concentrations of ABA inhibitory for germination were determined to be 5 x 10-4 M for the mutant and 10-6 M for the wild type. Expression of an ABA-induced Rab gene was studied to determine ABA responsiveness. The ABA concentration required for a half-maximal induction of Rab gene expression was 4 x 10-6 M in isolated embryos of both the mutant and wild type. This result suggests that ABA signal transduction pathways were not affected in the mutant. When isolated embryos were allowed to imbibe in water, ABA was released from the mutant and wild-type embryos at the same rate. However, the free ABA level in the incubation medium of the mutant showed a much faster decrease than that of the wild type, as demonstrated by two independent ABA assay methods (high-performance liquid chromatography and enzyme-linked immunosorbent assay). Our results suggest that turnover of ABA outside the embryo is a determining factor in the germination of barley seeds.     

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.     

Hormonal regulation of barley nuclease: investigation using a monoclonal antibody

Abstract. A monoclonal antibody prepared against barley ( Hordeum vulgare L., cv. Himalaya) nuclease (EC 3.1.30.2) was characterized with solid-state enzyme-linked immunosorbent assays and immuno-blotting. The antibody was specific for intracellular and secreted nuclease. Hormonal regulation of the synthesis and secretion of nuclease in isolated aleurone layers was investigated by immunoprecipitation of biosynthetically-labelled nuclease using polyclonal antibodies and by immunoblot analyses using the monoclonal antibody, respectively. Gibberellic acid (GA₃) induced the de novo synthesis and secretion of nuclease in a time-and concentration-dependent manner. Nuclease was detected in aleurone layers incubated in 1 mmol m⁻³ GA₃, after 24 h. The maximum rates of nuclease synthesis and secretion occurred 36–48 h after hormone treatment. A minimum concentration of 10⁻⁶ mol m⁻³ GA₃ was required for nuclease synthesis and secretion, whereas the maximum rate of nuclease secretion occurred at concentrations of 10⁻⁵ mol m⁻³ and higher. In the presence of abscisic acid, the synthesis and secretion of nuclease from GA₃-treated aleurone layers was almost completely inhibited. Based on these findings, the authors conclude that all nuclease within and secreted from aleurone layers treated with GA₃ is the result of its de novo synthesis.     

Interactions between hydrogen cyanide, gibberellin, abscisic acid and red light in germination of lettuce seeds

Germination of lettuce ( Lactuce sativa L. cy. Grand Rapids) seeds was promoted by red light and by pulse treatments with gibbercllie acid (GA₃) or hydrogen cyanide, whereas it was inhibited by short exposure of seeds to absusic acid (ABA). The eflects of unsalLirating red light and of 10 μ M GA₃ on lettuce germination were completely reversed the effect of ABA (100 μ M ). In contrast, hydrogen cyanide did not reverse the effect of 100 μ M ABA and only partly eliminated the effect of 10μ M ABA, independently of the sequence of treatments. Possible interactions between HCN GA₃, ABA and red light were discussed. It was concluded thai light GA₃ and HCN affect different mechanisms involved in lettuce germination: ABA counteracts the stimulatory action of all these faclors. being the most effective against cvanide Additional key words - Lactuca sativa, pholodormancy, phylohormoncs.     

The effect of some growth regulators on light-induced germination of Paulownia tomentosa seeds

Grubišié, D., Konjevié, R. and Neškovié, M. 1988. The effect of some growth regulators on light-induced germination of Paulownia tomentosa seeds. - Physiol. Plant. 72: 525–528.
The germination of Paulownia tomentosa Steud. (Empress tree) seeds can be induced either by red light (R) or by exogenously applied gibberellic acid (GA₃). The R induced germination is completely suppressed by far red irradiation, abscisic acid (ABA) or by growth retardants such as ancymidol, tetcyclacis and paclobutrazol, though not by AMO 1618 or chloro-choline chloride (CCC). The inhibition caused by far red light (FR) and growth retardants can be overcome by GA₃, while the ABA-induced inhibition can be reversed by fusicoccin (FC), which does not reverse the inhibition caused by FR or growth retardants. It is noteworthy that the germination of light insensitive wheat, corn, alfalfa and mung bean seeds is not inhibited by growth retardants.     

The Arabidopsis aleurone layer responds to nitric oxide, gibberellin, and abscisic acid and is sufficient and necessary for seed dormancy

Seed dormancy is a common phase of the plant life cycle, and several parts of the seed can contribute to dormancy. Whole seeds, seeds lacking the testa, embryos, and isolated aleurone layers of Arabidopsis (Arabidopsis thaliana) were used in experiments designed to identify components of the Arabidopsis seed that contribute to seed dormancy and to learn more about how dormancy and germination are regulated in this species. The aleurone layer was found to be the primary determinant of seed dormancy. Embryos from dormant seeds, however, had a lesser growth potential than those from nondormant seeds. Arabidopsis aleurone cells were examined by light and electron microscopy, and cell ultrastructure was similar to that of cereal aleurone cells. Arabidopsis aleurone cells responded to nitric oxide (NO), gibberellin (GA), and abscisic acid, with NO being upstream of GA in a signaling pathway that leads to vacuolation of protein storage vacuoles and abscisic acid inhibiting vacuolation. Molecular changes that occurred in embryos and aleurone layers prior to germination were measured, and these data show that both the aleurone layer and the embryo expressed the NO-associated gene AtNOS1, but only the embryo expressed genes for the GA biosynthetic enzyme GA3 oxidase.     

Developmental and organ-specific expression of an ABA- and stress-induced protein in barley

An mRNA species, HVA1, has been shown to be rapidly induced by abscisic acid (ABA) in barley aleurone layers (Hong, Uknes and Ho, Plant Mol Biol 11: 495–506, 1988). In the current work we have investigated the expression of HVA1 in other organs of barley plants. In developing seeds, HVA1 mRNA is not detected in starchy endosperm cells, yet it accumulates in aleurone layers and embryo starting 25 days after anthesis, and its level remains high in these organs in dry seeds. Although the levels of HVA1 mRNA are equivalent in the dry embryos of dormant and nondormant barley seeds, upon imbibition HVA1 mRNA declines much slower in the dormant than in the nondormant embryos. The HVA1 mRNA and protein levels are highly induced by ABA treatment in all organs of 3-day-old seedlings. However, the induction in the leaf of 7-day-old seedlings is less than one tenth the level observed in the leaf of 3-day-old seedlings. In the leaf, HVA1 mRNA and protein are induced mainly at the base. These observations indicate that the expression of HVA1 is under developmental regulation. Besides the HVA1 protein, a smaller protein (p20) of approximately 20 kDa cross-reacting with anti-HVA1 polyclonal antibodies, is induced by ABA in barley seedlings but not in seeds. HVA1 mRNA is induced by drought, NaCl, cold or heat treatment. Similar to ABA treatment, the drought induction of HVA1 occurs in all the tissues of 3-day-old seedling, but the induction decreases dramatically in the leaf of 7-day-old plants. The significance of organ-specific, developmentally regulated, and stress-induced expression of HVA1 is discussed.     

Interaction between Pfr and growth substances in the germination of light-requiring Kalanchoë seeds

Seeds of Kalanchoë blossfeldiana Poelln. cv. Feucrblüte, incubated on gibberellic acid, become very light-sensitive through a synergism between the far-red absorbing form of phytochrome and the growth substance, which results in high physiological activity of short far-red (FR) exposures. On 2 × 10^-3 M gibberellic acid (GA₃), one saturating FR pulse is somewhat more effective than one saturating red light (R) irradiation. Fluence-response curves for R and FR confirm this observation. At lower GA₃ concentrations, this difference disappears and the effects of one saturating R and FR pulse decrease in an identical way with the GA₃ concentration. When two saturating irradiations, separated by 24 h are given, the effect of FR falls off faster than that of R at low GA₃ concentrations. Consequently, the second irradiation must have a different impact in comparison with the first one. Of the other growth substances tested, only a mixture of gibberellins A4 and A₇ had an analogous, still more pronounced effect than GA₃. Abscisic acid (ABA) inhibits the phytochrome-mediated germination of Kalanchoë , both in the absence and presence of GA₃. An antagonism between ABA and GA₃ was demonstrated.     

Contribution of aleurone layer and scutellum to α-amylase synthesis and secretion in wheat and rice grains

Intact aleurone layers from wheat ( Triticum aestivum L. cv Camp Rémy) and rice ( Oryza sativa L. cv Cigalon) grains both contained and secreted more α-amylase (EC 3.2.1.1.) than did the corresponding scutellar tissues. This discrimination was already evident at the earliest stages of germination at which the tissues could be isolated, and became more pronounced upon subsequent germination and growth. Isoenzyme patterns obtained upon isoelectric focusing showed a considerable polymorphism of the α-amylases of each cereal. The enzyme polymorphism pattern was the same in the aleurone layer and in the scutellum, but some secondary constituents appeared to be more specific for the one or the other of the tissues. Moreover, the isozymes found in the tissues were the same as those found to be secreted. A third α-amylase antigen which differs from the well established α₁ and α₁₁ forms was identified in the germinating wheat grains. The presence of Ca²⁺ in the secretion medium favoured maximum secretion of α-amylases from the wheat scutellum and aleurone layers, whereas it inhibited the secretion of the enzymes from the rice aleurone layer.     

Temporal, spatial and hormonal regulation of the S-adenosylmethionine synthetase gene in petunia

Gibberellic acid (GA₃) promotes corolla elongation and pigmentation in petunia flowers. We have previously shown that G.A₃ induces pigmentation by activating specific genes of the anthocyanin biosynthetic pathway. The aim of the present work was to examine whether GA₃ induces also the expression of genes from other metabolic pathways in petunia corollas that may be associated with growth. Recently we reported the cloning of the petunia sam gene coding for S -adenosylmethionine synthetase (SAM-S). In the present work we show that sam expression is induced by GA₃ in both corollas and stems. The expression of the gene was correlated with corolla elongation. GA₃ and the cylokinin, N -6-benzyladenine (BA) promoted corolla growth and sam expression, whereas abscisic acid (ABA) inhibited corolla elongation and repressed sam mRNA accumulation. An analysis of sam expression in stems indicated a high level in young, elongating internodes and a very low level in the mature, non-elongating stem zone. The results of the present study show that the effect of GA₃ on gene expression in the corolla of petunia, is not restricted to the anthocyanin biosynthetic pathway, they also suggest a possible role for sam in GA₃-induced corolla and stem elongation.