Role of the maize transcription factor R in the regulation of anthocyanin biosynthesis

The maize flavonoid biosynthetic pathway is one of the best characterized plant model systems to study the combinatorial regulation of gene expression. Flavonoids are important secondary metabolites and important for the plant, such as in the case for anthocyanins as attractants to pollinators as well as for humans due to a number of biological activities. Anthocyanins in maize are regulated by the cooperation of the R2R3-MYB domain protein C1 and the basic helix-loop-helix (bHLH) protein R. In contrast, phlobaphene pigments, derived from a separate flavonoid biosynthetic branch, are regulated by the R2R3-MYB domain protein P1, which can activate transcription in the absence of a (known) bHLH. Our laboratory has established that the bHLH transcription factor R plays a key role in determining the biological specificity of C1. I describe here how R might contribute to regulatory specificity, on one hand by forming a platform for protein-protein interactions, and on another, by binding to different sites in the regulatory regions of its target genes depending on the interacting partners. Using a variety of techniques I have investigated three protein-protein interacting regions of R. I demonstrate that the highly conserved bHLH domain of R is involved in transcriptional regulation and histone functions. I show that R interacts with the EMSY-like protein RIF1 specifically via the bHLH domain and that this interaction is required for the regulation of endogenous flavonoid genes. RIF1 is part of the C1/R regulatory complex and I discuss how RIF1 links transcriptional regulation of flavonoid biosynthetic genes with chromatin function. In addition, I show that the region adjacent to the bHLH domain has structural similarity to a leucine zipper and that the extended bHLH-LZ-like region is able to homodimerize. The bHLH-LZ-like mediated dimerization is required for activation of a synthetic pG-box::Luc promoter::reporter construct in transient expression studies and for binding to a synthetic G-box probe, as well as to the Bz1 and C2 promoter in vitro. Furthermore, I demonstrate that the ACT domain at the C-terminus of R homodimerizes. This domain is necessary for anthocyanin pigment formation, for transcriptional activation of at least four anthocyanin biosynthetic genes and important for DNA-binding to the A1 and Bz1 promoters. I show that interplay between the functional domains described here is necessary for transcriptional activation and DNA-binding. I am also characterizing R-interacting partners which possibly tether R to as yet unknown target genes and therefore might show the involvement of R in other cellular processes.;Taken together, these studies emphasize the importance of the bHLH transcription factor R in combinatorial regulation of gene expression of anthocyanin biosynthetic genes and open new possibilities for R to function in other cellular processes. Moreover, these studies highlight the complexity of biochemical pathway regulation and show novel mechanisms of how one TF can participate in several regulatory complexes.