Increased dehydrin promoter activity caused by HvSPY is independent of the ABA response pathway

A barley SPINDLY protein, HvSPY, is a negative regulator of gibberellin (GA) action. It is also found to be a positive regulator of the promoter of a barley dehydrin (Dhn) gene which is abscisic acid (ABA) upregulated. To investigate whether HvSPY acts through the ABA signaling pathway to upregulate the Dhn promoter, functional characterization was carried out by co-bombardment experiments. These experiments used Dhn promoter-GUS reporter constructs and an effector construct to overexpress HvSPY protein in barley aleurone. ABA dose-response experiments with and without HvSPY overexpression showed that the induction by HvSPY occurred in addition to the ABA effect. Gibberellic acid (GA3) did not reduce the induction by ABA, but it had a small, although significant, effect on the ability of HvSPY to upregulate. The induction of promoter activity of Dhn by HvSPY required the intact protein, and a small deletion in the tetratricopeptide repeat (TPR) region reduced this ability significantly. When a promoter region containing an element for ABA responsiveness was mutagenized or deleted, the mutant promoters lost ABA responsiveness but remained responsive to HvSPY. In addition, HvSPY did not increase promoter activities of other ABA-upregulated genes. Taken together, these results indicate that HvSPY and ABA both regulate promoter activity of Dhn, and that HvSPY acts independently of the ABA signaling pathway.