STRUCTURE AND PROMOTER ANALYSIS OF AN ABA-REGULATED AND STRESS-REGULATED BARLEY GENE, HVA1

A single-copy barley gene, HVA1, encoding a class 3 late embryogenesis -abundant protein, can be induced by either treatment with abscisic ac id (ABA) or by stress conditions such as drought, cold, heat and salin ity. We have isolated an HVA1 genomic clone containing about 400 bp of 5'-upstream sequence, a single 109 bp intron, and the full coding seq uence. Linker scan mutagenesis and transient expression studies were u sed to test the function of four HVA1 promoter elements conserved in A BA-responsive genes. Mutations in two of these elements, the C box and the putative ABRE 1 (ABA-responsive element) containing an ACGT core, resulted in no significant change in transcription level or ABA induc tion. In contrast, mutations of the other two elements, putative ABRE 2 and 3 cause the level of transcription to drop to 10-20 % of that ob tained with the wild-type promoter indicating that the high level of e xpression of HVA1 is dependent on both pABRE 2 and 3. Interestingly, d espite their low level of expression, the mutated promoters still gave more than 20-fold induction in response to ABA treatment. We suggest that the ABA induction of barley HVA1 gene is governed by a complex co nsisting of pABRE 2 and 3 working together to regulate the absolute le vel of expression, and either of these elements or a possible third el ement may regulate ABA inducibility. Phylogenetic analysis by parsimon y indicates that the barley HVA1 and wheat pMA2005 sequences share a r ecent common ancester. These two genes are closely related to the carr ot Dc3 and cotton D-7 genes with which they share a similar structural gene organization.