Study on signal transduction mechanisms involved in abscisic acid-responsive transcription

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In order to identify protein kinases responsible for the ABA-dependent phosphorylation of an ABRE binding factor, TRAB1, we performed a comprehensive analysis of the rice SnRK2 protein kinase family. Some kinases, such as fava bean AAPK and Arabidopsis OST1, of this family had been reported to be involved in the ABA regulation of stomata apertures. Thus we expected that TRAB1 kinase would be included in this kinase family. Rice genome encode 10 members of this family, which we named SAPK1 through 10. By expressing epitope-tagged SAPKs transiently in rice cultured cell protoplasts, we analyzed for their activity regulation and found that SAPK8, 9 and 10 were rapidly activated by ABA. We then showed that transient overexpression in cultured cell protoplasts of these ABA-activated SnRK2 protein kinases led to the activation of an ABRE regulated promoter, suggesting that these kinases are involved in the gene regulation pathway of ABA signaling. We further showed several lines of evidence … More that these ABA-activated SnRK2 protein kinases directly phosphorylate TRAB1 in response to ABA. Kinetic analysis of SAPK10 activation and TRAB1 phosphorylation indicated that the latter immediately followed the former. TRAB1 was found to be phosphorylated not only in response to ABA but also in response to hyperosmotic stress, which was interpreted as the consequence of phosphorylation of TRAB1 by hyperosmotically activated SAPKs. Physical interaction between TRAB1 and SAPK10 in vivo was demonstrated by a co-immunoprecipitation experiment. Finally, TRAB1 was phosphorylated in vitro by the ABA-activated SnRK2 protein kinases at Ser102, which is phosphorylated in vivo in response to ABA and critical for the activation function.ABA-activated SnRK2 PKs are also activated by hyperosmotic stress. We previously have demonstrated that this activation is mediated by the phosphorylation of the PK molecule. In this research, we showed that the ABA activation was also mediated by phosphorylation. However, we found that the mechanisms are different between ABA and hyperosmotic activation. This finding was based on the observations that the synergistic activation of the PKs was achieved by the ABA and hyperosmotic stimulation at the signal strengths where the response to each stimulation is saturated, and that a small internal deletion at the C-terminal regulatory domain of SAPK10 responded to hyperosmotic stress but not to ABA.Another important progress was that we identified by the yeast two-hybrid screening a SAPK interacting protein that would function as a scaffold for signaling factors, which may serve for effective signaling and signal sorting. Less