Gibberellin-responsive elements in the promoter of a barley high-pI alpha-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-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 identify 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 (-169pyrimidine box, -143TAACAAA box, and -124TATCCAC 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Gubler
F
F
Commonwealth Scientific and Industrial Research Organization, Division of Plant Industry, Canberra, ACT, Australia.
Jacobsen
J V
JV
D001483:Base Sequence; D004252:DNA Mutational Analysis; D004274:DNA, Recombinant; D017343:Genes, Plant / Q000187:drug effects / Q000235:genetics*; D005875:Gibberellins / Q000494:pharmacology*; D001467:Hordeum / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics*; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D000516:alpha-Amylases / Q000187:drug effects / Q000235:genetics*
D004274:DNA, Recombinant; D005875:Gibberellins; D000516:alpha-Amylases