FUNCTIONAL DISSECTION OF AN ABSCISIC-ACID (ABA)-INDUCIBLE GENE REVEALS 2 INDEPENDENT ABA-RESPONSIVE COMPLEXES EACH CONTAINING A G-BOX AND ANOVEL CIS-ACTING ELEMENT

To elucidate the mechanism by which abscisic acid (ABA) regulates gene expression, the promoter of the barley ABA-responsive HVA22 gene has been analyzed by both loss- and gain-of-function studies. Previous rep orts indicate that G-box sequences, which are present in genes respond ing to a variety of environmental and physiological cues, are involved in ABA response. However, our data suggest that G-box sequences are n ecessary but not sufficient for ABA response, Instead, an ABA response complex consisting of a G-box, namely, ABRE3 (GCCACGTACA), and a nove l coupling element, CE1 (TGCCACCGG), is sufficient for high-level ABA induction, and replacement of either of these sequences abolishes ABA responsiveness. We suggest that the interaction between G-box sequence s, such as ABRE3 in the HVA22 gene, and CE-type sequences determines t he specificity in ABA-regulated gene expression. Our results also demo nstrate that the ABA response complex is the minimal promoter unit gov erning high-level ABA induction; four copies of this 49-bp-long comple x linked to a minimal promoter can confer more than 100-fold ABA-induc ed gene expression. In addition to ABA response complex 1, composed of ABRE3 and CE1, the HVA22 promoter contains another ABA response compl ex. The ABA responsiveness of this ABA response complex 2 relies on th e interaction of a G-box (ABRE2; CGCACGTGTC) with another yet unidenti fied coupling element, These two complexes contribute incrementally to the expression level of HVA22 in response to ABA.