The endogenous ABA contents of dormant and nondormant barley grains were determined following application of different compounds to break dormancy. The chemicals used for breaking of dormancy in intact dormant grains were weak and strong acids, alcohols, hydrogen peroxide, cyanide, nitrate, salicylic acid, gibberellic acid and fusicoccin. The dormancy-breaking compounds could be classified into two major groups: compounds that caused a decrease in endogenous ABA (class I) and compounds which did not affect endogenous ABA (class II). Class I compounds included gibberellic acid, ethanol, hydrogen peroxide, nitrate, salicylic acid; class II compounds were fusicoccin, acid (H2SO4), sodium azide, n-caproic acid. In addition, these dormancy-breaking compounds were able to stimulate the germination rate when applied to embryos isolated from dormant grains. The concentrations necessary for stimulation of germination of isolated embryos were much lower than the concentrations for breaking the dormancy of intact grains. After embryos were isolated from dormant grains and incubated in water, ABA was determined in both embryos and in the incubation media. The class I compounds stated above also reduced ABA content in the incubation medium of isolated embryos, while class II compounds had no effect on ABA content of the medium. External application of ABA could overcome the effect of dormancy-breaking compounds of class I but not of class II. The results suggest that in the presence of the agents belonging to class II, ABA responsiveness of isolated embryos from dormant grains is decreased, compared to nontreated embryos.

The family of 14-3-3 proteins is ubiquitous in eukaryotes and has been shown to exert an array of functions. We were interested in the possible role of 14-3-3 proteins in seed germination. Therefore, we studied the expression of 14-3-3 mRNA and protein in barley (Hordeum distichum L.) embryos during germination. With the use of specific cDNA probes and antibodies, we could detect individual expression of three 14-3-3 isoforms, 14-3-3A, 14-3-3B, and 14-3-3C. Each homolog was found to be expressed in barley embryos. Whereas protein levels of all three isoforms were constant during germination, mRNA expression was found to be induced upon imbibition of the grains. The induction of 14-3-3A gene expression during germination was different from that of 14-3-3B and 14-3-3C. In situ immunolocalization analysis showed similar spatial expression for 14-3-3A and 14-3-3B, while 14-3-3C expression was markedly different. Whereas 14-3-3A and 14-3-3B were expressed throughout the embryo, 14-3-3C expression was tissue specific, with the strongest expression observed in the scutellum and the L2 layer of the shoot apical meristem. These results show that 14-3-3 homologs are differently regulated in barley embryos, and provide a first step in acquiring more knowledge about the role of 14-3-3 proteins in the germination process.

Under the same mannitol pretreatment and culture conditions, regeneration efficiency in the barley cultivar (cv.) Igri was about 10 times higher than in the cv. Digger, a difference only partially reflected by a difference in viable microspores after anther pretreatment. Therefore, a comparative study between cvs. Igri and Digger was carried out under various pretreatment conditions. For both cultivars, under water, CPW buffer and mannitol pretreatment conditions, there was a positive correlation between microspore viability and regeneration efficiency in that mannitol > CPW buffer >> water. Mannitol pretreatment of cv. Igri produced a much higher endogenous abscisic acid (ABA) level than as to Digger. Addition of ABA stimulated both percentages of viability and regeneration efficiency except in the case of mannitol pretreatment. Under CPW buffer pretreatment conditions, addition of ABA significantly stimulated regeneration efficiency and was ABA concentration dependent. However, cv. Digger was less responsive to ABA than cv. Igri. In both cultivars, under less optimal pretreatment conditions (e.g., water and CPW buffer), the effect of ABA was to stimulate increased percentages of viability and/or to reduce the number of binucleate microspores. Moreover, in cv. Igri, direct culture of anthers for 4 days without pretreatment caused an increased number of binucleate microspores compared with microspores with pretreatment for 4 days. These binucleate microspores showed DNA degradation in the nuclei. However, with mannitol pretreatment binucleate microspores and DNA fragmentation in the nuclei of microspores was rarely observed. On the basis of our observations, we suggest that the difference in regeneration efficiency in cv. Igri and cv. Digger is related to the differences in endogenous ABA production levels under mannitol pretreatment and responsiveness to ABA. One of the effects of ABA is likely due to an inhibition of cell death.

Abstract.A panel of hybridoma products generated against pea (Pisum sativum L.) guard-cell protoplasts has been assayed for anti-abscisic acid (ABA) biological activity in barley (Hordeum vulgare L.) aleurone protoplasts. The effects of the antibodies on ABA-induced accumulation of mRNA transcribed from RAB-16, a gene responsive to ABA, were determined. Most of the antibodies, and culture medium, had no effect, but five monoclonal antibodies (MAbs) were found to inhibit ABA-induced RAB-16 gene expression and one MAb enhanced it. The effects of one inhibitory MAb, JIM19, were studied in some detail. These effects were specific to ABA-induced events, as incubation with JIM19 had no effect on the expression of a constitutively-expressed gene, GAPDH, encoding glyceraldehyde-3-phosphate dehydrogenase, and only a slight effect on the production of a-amylase induced by gibberellic acid. Increasing concentrations of ABA in the incubation medium partly overcame the inhibitory effect of JIM19. Immunolabelling and biological activity remained together during immuno-purification of JIM19 from hybridoma culture supernatant. Immunoblotting of JIM19 to membrane preparations from barley aleurone protoplasts revealed that JIM19 recognised a number of proteins.