Functional definition of ABA-response complexes: the promoter units necessary and sufficient for ABA induction of gene expression in barley ( Hordeum vulgare L.).

Abscisic acid (ABA)-response promoter complexes (ABRCs), consisting of an ACGT core-containing element (ACGT box) and a coupling element (CE), have been shown to be necessary and sufficient for ABA induction of gene expression in cereal plants. In this work, the component elements of two ABRCs are defined in terms of base sequence, orientation, and distance from each other. The ACGT element requires the sequence 5'-ACGTGGC-3' and the elements CE1 and CE3 require the sequences CCACC and GCGTGTC, respectively. The ACGT element and CE3 are next to each other in the barley ABA-inducible gene HVA1, and lengthening the distance between them gradually decreases their activity in conferring ABA response. On the other hand, the ACGT element and CE1 are separated by about 20 bp in the promoter of another ABA-inducible gene, HVA22, and need to be separated by multiples of 10 bp in order to confer high ABA induction, suggesting that these two elements have to be located in the same side of the DNA double helix. Although the coupling between an ACGT box and a CE is sufficient for ABA induction, two copies of the ACGT element are equally active. However, two copies of CE3 appear to be less active. Specific interactions between ABRC and nuclear proteins have been detected. In vitro binding activities of nuclear proteins to an ABRC and to its mutant forms appear to be proportional to the biological activities of these sequences in vivo. Our data suggest that the specific response to ABA is determined by the presence of two ACGT boxes or an ACGT box plus a CE as well as by the flanking sequences of the ACGT boxes and the CEs.