Structure and promoter analysis of an ABA- and stress-regulated barley gene, HVA1

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Abstract
A single-copy barley gene, HVA1, encoding a class 3 late embryogenesis-abundant protein, can be induced by either treatment with abscisic acid (ABA) or by stress conditions such as drought, cold, heat and salinity. We have isolated an HVA1 genomic clone containing about 400 bp of 5′-upstream sequence, a single 109 bp intron, and the full coding sequence. Linker scan mutagenesis and transient expression studies were used to test the function of four HVA1 promoter elements conserved in ABA-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 induction. In contrast, mutations of the other two elements, putative ABRE 2 & 3 cause the level of transcription to drop to 10–20% of that obtained with the wild-type promoter indicating that the high level of expression of HVA1 is dependent on both pABRE 2 & 3. Interestingly, despite 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 consisting of pABRE 2 & 3 working together to regulate the absolute level of expression, and either of these elements or a possible third element may regulate ABA inducibility. Phylogenetic analysis by parsimony indicates that the barley HVA1 and wheat pMA2005 sequences share a recent common ancester. These two genes are closely related to the carrot Dc3 and cotton D-7 genes with which they share a similar structural gene organization.