Gibberellin-responsive elements in the promoter of a barley high-pl alpha-amylase gene
1992
Gubler, F. | Jacobensen, J.V.
Deletion analysis has previously shown that the major gibberellic acid (GA)- and abscisic acid (ABA)-responsive elements in the promoter of a high-pI alpha-amylase gene of barley are located downstream of -174 (Jacobsen and Close, 1991). We have used transient expression assays in barley aleurone protoplasts to identity sequences between -174 and +53 that confer GA and ABA responsiveness on expression of a beta-glucuronidase reporter gene. Using alpha-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 alpha-amylase promoter (-41) conferred both GA- and 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 ((-169)pyrimidine box, (-143)TAACAAA box, and (-124)TATCCAC 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-pI alpha-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.
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