The bifactorial endosperm box of gamma-zein gene: characterisation and function of the Pb3 and GZM cis-acting elements

Summary The proximal region of the γ-zein promoter (γZ) has a functional bifactorial prolamin box element containing twocis-acting elements, a prolamin-box motif (Pb3) and a GCN4-like motif (GZM). By particle bombardment of maize endosperms with 5′ deletions and internal deletions of γZfused to the GUS gene, we have shown that a 135 bp region containing the bifactorial element is involved in the transcriptional activation of the γZpromoter. However, the 135 bp region was unable to activate the γZpromoter in the absence of a 84 bp downstream sequence. Usingin vivofootprinting and gel mobility shift assays with 15 DAP endosperm nuclear extracts, we have demonstrated the presence oftrans-acting factors that interact with Pb3 and GZM target sites. Base-substitution mutations within Pb3 and GZM decreased transcription activity of the γZpromoter suggesting a co-ordinated function between the twocis-acting elements. Two additionalcis-motifs upstream of the bifactorial prolamin element have been identified: a motif with high homology to the AACA elements of rice glutelin genes and an AZM motif containing an ACGT core which binds nuclear proteins other than the Opaque 2 (O2).