Gibberellin-Responsive Elements in the Promoter of a Barley High-pl a-Amylase Gene

Deletion analysis has previously shown that the major gibberellic acid (GA)and abscisic acid (ABA)-responsive elements in the promoter of a high-pl a-amylase gene of barley are located downstream of -174 (Jacobsen and Close, 1991). We have used transient expression assays in barley aleurone protoplasts to identify sequences between -174 and +53 that confer GA and ABA responsiveness on expression of a P-glucuronidase reporter gene. Using a-amylase promoter fragments and synthetic oligonucleotides fused to minimal promoters, we have shown that the hormone-responsive region is located between -174 and -108. A single copy of this region fused to a minimal a-amylase promoter (-41) conferred both GAand ABA-responsive expression on the reporter gene comparable to the positive control, Am(-174)IGN. Multiple copies of this region were able to activate even greater levels of expression. Site-directed mutagenesis was used to determine the functional importance of the conserved motifs (-l69pyrimidine box, -143TAACAAA box, and -r24TATCCAC box) and nonconserved intervening sequences within the region between -174 and -108. Our results showed that both the TAACAAA and TATCCAC boxes play an important role in GA-regulated expression. We propose that the TAACAAA box is a gibberellin response element, that the TATCCAC box acts cooperatively with the TAACAAA box to give a high level of GA-regulated expression, and that together these motifs form important components of a gibberellin response complex in high-pl a-amylase genes. The TAACAAA box also appears to be the site of action of ABA. In addition, we have identified a sequence that acts as a repressor of GA action and that resembles a cAMP response element.