Functional Dissection of an Abscisic Acid (ABA)-lnducible Gene Reveals Two lndependent ABA-Responsive Complexes Each Containing a G-Box and a Nove1 cis-Acting Element

To elucidate the mechanism by which abscisic acid (ABA) regulates gene expression, the promoter of the barley ABAresponsive HVA22 gene has been analyzed by both lossand gain-of-function studies. Previous reports indicate that G-box sequences, which are present in genes responding to a variety of environmental and physiological cues, are involved in ABA response. However, our data suggest that G-box sequences are necessaty but not sufficient for ABA fesponse. Instead, an ABA response complex consisting of a G-box, namely, ABRE3 ( G C C e A C A ) , and a nove1 coupling glement, CE1 (TGCCACCGG), is sufficient for high-leve1 ABA induction, and replacement of either of these sequences abolishes ABA responsiveness. We suggest that the interaction between G-box sequences, such as ABRE3 in the HVA22 gene, and CE-type sequences determines the specificity in ABA-regulated gene expression. Our results also demonstrate that the ABA response complex is the minimal promoter unit governing high-leve1 ABA induction; four copies of this 49-bplong complex linked to a minimal promoter can confer more than 100-fold ABA-induced gene expression. In addition to ABA response complex 1, composed of ABRE3 and CE1, the HVA22 promoter contains another ABA response complex. The ABA responsiveness of this ABA response complex 2 relies on the interaction of a G-box (ABREP; C G C g G T C ) with another yet unidentified coupling element. These two complexes contribute incrementally to the expression leve1 of HVA22 in response to ABA.