Abscisic acid (ABA) induces a rapid and transient mitogen-activated protein (MAP) kinase activation in barley aleurone protoplasts. MAP kinase activity, measured as myelin basic protein phosphorylation by MAP kinase immunoprecipitates, increased after 1 min, peaked after 3 min, and decreased to basal levels after ~5 min of ABA treatment in vivo. Antibodies recognizing phosphorylated tyrosine residues precipitate with myelin basic protein kinase activity that has identical ABA activation characteristics and demonstrate that tyrosine phosphorylation of MAP kinase occurs during activation. The half-maximal concentration of ABA required for MAP kinase activation, 3 x 10-7 M, is very similar to that required for ABA-induced rab16 gene expression. The tyrosine phosphatase inhibitor phenylarsine oxide can completely block ABA-induced MAP kinase activation and rab16 gene expression. These results lead us to conclude that ABA activates MAP kinase via a tyrosine phosphatase and that these steps are a prerequisite for ABA induction of rab16 gene expression.
Abstract. 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 gl), 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 dormancybreaking treatments act via changes to these processes.
We investigated whether intracellular pH (pH;) is a causal mediator in abscisic acid (ABA)-induced gene expression. We measured the change in pHi by a "null-point" method during stimulation of barley (Hordeum vulgare cv Himalaya) aleurone protoplasts with ABA and found that ABA induces an increase in pHi from 7.11 to 7.30 within 45 min after stimulation. This increase is inhibited by plasma membrane H'-ATPase inhibitors, which induce a decrease in pH;, both in the presence and absence of ABA. This ABAinduced pHi increase precedes the expression of RAB-16 mRNA, as measured by northern analysis. ABA-induced pH; changes can be bypassed or clamped by addition of either the weak acids 5,5-dimethyl-2,4-oxazolidinedione and propionic acid, which decrease the pH;, or the weak bases methylamine and ammonia, which increase the pH;. Artificial pH; increases or decreases induced by weak bases or weak acids, respectively, do not induce RAB-16 mRNA expression. Clamping of the pH; at a high value with methylamine or ammonia treatment affected the ABA-induced increase of RAB-16 mRNA only slightly. However, inhibition of the ABA-induced pH; increase with weak acid or proton pump inhibitor treatments strongly inhibited the ABA-induced RAB-16 mRNA expression. We conclude that, although the ABA-induced the pHi increase is correlated with and even precedes the induction of RAB-16 mRNA expression and is an essential component of the transduction pathway leading from the hormone to gene expression, it is not sufficient to cause such expression.
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.
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