Divergent Transactivation of Maize Storage Protein Zein Genes by the Transcription Factors Opaque2 and OHPs

Maize transcription factors (TFs) opaque2 (O2) and the O2 heterodimerizing proteins (OHP1 and OHP2) originated from an ancient segmental duplication. The 22-kDa (z1C) and 19-kDa (z1A, z1B, and z1D) alpha-zeins are the most abundant storage proteins in maize endosperm. O2 is known to regulate alpha-zein gene expression, but its target motifs in the 19-kDa alpha-zein gene promoters have not been identified. The mechanisms underlying the regulation of alpha-zein genes by these TFs are also not well understood. In this study, we found that the O2 binding motifs in the alpha-zein gene promoters are quite flexible, with ACGT being present in the z1C and z1A promoters and a variant, ACAT, being present in the z1B and z1D promoters. OHPs recognized and transactivated all of the alpha-zein promoters, although to much lower levels than did O2. In the presence of O2, the suppression of OHPs did not cause a significant reduction in the transcription of alpha-zein genes, but in the absence of O2, OHPs were critical for the expression of residual levels of alpha-zeins. These findings demonstrated that O2 is the primary TF and that OHPs function as minor TFs in this process. This relationship is the converse of that involved in 27-kDa gamma-zein gene regulation, indicating that the specificities of O2 and the OHPs for regulating zein genes diverged after gene duplication. The prolamine-box binding factor by itself has limited transactivation activity, but it promotes the binding of O2 to O2 motifs, resulting in the synergistic transactivation of alpha-zein genes.