GIBBERELLIN TREATMENT STIMULATES NUCLEAR FACTOR-BINDING TO THE GIBBERELLIN RESPONSE COMPLEX IN A BARLEY ALPHA-AMYLASE PROMOTER

The promoters of a majority of cereal alpha-amylase genes contain thre e highly conserved sequences (gibberellin response element, box I, and pyrimidine box). Recent studies have demonstrated the functional impo rtance of four regions that either coincide with or are immediately pr oximal to these three conserved elements as well as an upstream Opaque -2 binding sequence. In this study, we describe the characterization o f nuclear protein factors from barley aleurone layers whose binding ac tivity toward gibberellin response complex sequences from the barley l ow-pl alpha-amylase gene (Amy32b) promoter is stimulated by gibberelli n A(3) (GA(3) treatment. Barley proteins isolated from crude nuclear e xtracts prepared from aleurone layers incubated wither without GAB wer e fractionated by anion exchange fast protein liquid chromatography an d studied using band shift assays, sequence-specific competitions, and DNase I footprinting. A GA(3)-dependent binding activity eluting at 2 10 mM KCl was shown to bind specifically to the gibberellin response e lement and the closely associated box 1. DNase I footprinting with the proteins in this fraction indicated interactions with sequences in th e gibberellin response element and box I. A second DNA binding activit y eluting at 310 mM KCl was present constitutively in extracts prepare d from tissues incubated both in the absence and in the presence of ho rmone. Proteins in this fraction were able to bind to many DNA sequenc es and, in general, were largely nonspecific. DNase I footprinting wit h the proteins in this fraction indicated a large area of protection w ith a single unoccupied region located at the 3' end of box I. The pos sible function of such an activity in hormone regulation of the alpha- amylase genes is discussed.