Involvement of multiple cis-elements in the regulation of GA responsive promoters:: Definition of a new cis-element in the Amy32b gene promoter of barley (Hordeum vulgare)

The Amy32b gene is a member of the low-pi cr-amylase gene family of barley, whose expression is tightly regulated by hormones in the aleurone layer. Four cis-elements are known to be important for the GA induction of this gene: GARE, amylase box, pyrimidine box, and O2S, These sequences are located between - 101 and - 149 relative to the transcription start site. In the present work, we have created a series of Amy32b promoter-GUS reporter constructs introducing mutations in the -79 to - 93 region. Using a transient expression system, we have functionally defined an additional region (-81 to -89) essential for the GA activation of the Amy32b promoter. This region is highly conserved among barley, wheat, and wild oat low-pi cr-amylase promoters. Interestingly, in contrast with the variability in the relative distances among other cis-elements, this region maintains a nearly constant distance to GARE, which suggests that the function of these elements might be coupled. The involvement of this and other sequences in the transactivation of Amy32b by a transcription factor, GAMyb, has also been studied. Our results indicate that the only indispensable element for the GAMyb transactivation of the alpha -amylase promoter is GARE, The present work brings new evidence to the proposed model that considers the GAMyb-GARE interaction as a critical point for the GA induction of alpha -amylase genes, hut also strengthens the notion that multiple sequences are required for full regulation of alpha -amylase promoters.