PIF3 Regulates Anthocyanin Biosynthesis in an HY5-dependent Manner with Both Factors Directly Binding Anthocyanin Biosynthetic Gene Promoters in Arabidopsis

Phytochromes are red/far-red light receptors that regulate various light responses by initiating the transcriptional cascades responsible for changing the expression patterns of 10-30% of the entire plant transcriptome. Several transcription factors that are thought to participate in this process have been identified, but the functional relationships among them have not yet been fully elucidated. Here we investigated the functional relationship between two such transcription factors, PIF3 and HY5, and their effects on anthocyanin biosynthesis. Our results revealed that PIF3 and HY5 do not regulate each other at either the transcriptional or the protein levels in continuous light conditions, suggesting that they are not directly linked within phytochrome-mediated signaling. We found that both PIF3 and HY5 positively regulate anthocyanin biosynthesis by activating the transcription of the same anthocyanin biosynthetic genes, but the positive effects of PIF3 required functional HY5. Chromatin immunoprecipitation analyses indicated that both PIF3 and HY5 regulate anthocyanin biosynthetic gene expression by directly binding to different regions of the gene promoters in vivo. Additional experiments revealed that PIF3 bound the promoters regardless of light and HY5. Collectively, these data show that PIF3 and HY5 regulate anthocyanin biosynthesis by simultaneously binding anthocyanin biosynthetic gene promoters at separate sequence elements.