DNase1 footprints suggest the involvement of at least three types of transcription factors in the regulation of alpha-Amy2/A by gibberellin

To elucidate the mechanisms by which alpha-amylase genes are expressed in w ild oat aleurone, two genes, alpha-Amy2/A and alpha-Amy2/D, were isolated. Both were shown to be positively regulated by gibberellin (GA) during germi nation and both contain the conserved cis-acting elements Box 2, GA-respons e element (TAACAGA) and TATCSATSS (where S is C or G). In addition, they po ssess a conserved initiator element (CATCA) that is present in both alpha-A my2 and alpha-Amy1 genes, and also in a number of other plans TATA-containi ng and TATA-less promoters. DNase1 footprint analysis showed the alpha-Amy2 /A promoter to be a complex array of binding sites for a number of differen t classes of DNA-binding proteins. Our data suggest that the area around th e initiator element (Inr) is bound by a large complex of general transcript ion factors, that the TATA box is bound by the TFIID complex, that Box 2 is bound by one or more WRKY proteins and that the GA-response element is bou nd by one or more MYBs. TWO other elements containing the core sequence CCA TGG/C are bound by nuclear protein and this sequence is the core of the Sph element. The regulation of alpha-Amy2 genes by GA therefore involves an in terplay of at least three different types of transcription factor.