Evidence for direct activation of an anthocyanin promoter by the maize C1 protein and comparison of DNA binding by related Myb domain proteins

The enzyme-encoding genes of two classes of maize flavonoid pigments, anthocyanins and phlobaphenes, are differentially regulated by distinct transcription factors. Anthocyanin biosynthetic gene activation requires the Myb domain C1 protein and the basic helix-loop-helix B or R proteins. in the phlobaphene pathway, a subset of C1-regulated genes, including a1, are activated by the Myb domain P protein independently of B/R. We show sequence-specific binding to the al promoter by C1 in the absence of B. Activation is decreased by mutations in the C1 DNA binding domain or in al sequences bound by C1, providing direct evidence for activation of the anthocyanin biosynthetic genes by C1. The two C1 binding sites in the a1 promoter are also bound by P. One site is bound with higher affinity by P relative to C1, whereas the other site is bound with similar lower affinity by both proteins. Interestingly, either site is sufficient for C1 plus B/R or P activation in vivo, demonstrating that differences in DNA binding affinities between P and C1 are insufficient to explain the differential requirement for B. Results of DNA binding site-selection experiments suggest that C1 has a broader DNA binding specificity than does P, which may help C1 to activate a more diverse set of promoters.