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

To elucidate the mechanisms by which α-amylase genes are expressed in wild oat aleurone, two genes, α-Amy2/A and α-Amy2/D, were isolated. Both were shown to be positively regulated by gibberellin (GA) during germination and both contain the conserved cis-acting elements Box 2, GA-response element (TAACAGA) and TATCSATSS (where S is C or G). In addition, they possess a conserved initiator element (CATCA) that is present in both α-Amy2 and α-Amy1 genes, and also in a number of other plans TATA-containing and TATA-less promoters. DNase1 footprint analysis showed the α-Amy2/A promoter to be a complex array of binding sites for a number of different classes of DNA-binding proteins. Our data suggest that the area around the initiator element (Inr) is bound by a large complex of general transcription 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 bound by one or more MYBs. TWO other elements containing the core sequence CCATGG/C are bound by nuclear protein and this sequence is the core of the Sph element. The regulation of α-Amy2 genes by GA therefore involves an interplay of at least three different types of transcription factor.