PeMYB4L interacts with PeMYC4 to regulate anthocyanin biosynthesis in Phalaenopsis orchid

Phalaenopsis spp., one genus of Orchidaceae, have become very popular worldwide for their fascinating flowers with various colors and pigmentation patterns. Several R2R3-MYB transcription factors have been reported to function in anthocyanin accumulation in Phalaenopsis spp. However, its molecular mechanism underlying the detailed regulatory pathway remains poorly understood. In this study, we identified a novel subgroup 2 R2R3-MYB transcription factor PeMYB4L, the expression profile of which was concomitant with red color formation in Phalaenopsis spp. flowers. Virus-induced gene silencing (VIGS) and transient overexpression assay verified that PeMYB4L promotes anthocyanin accumulation in flower tissues. In addition, PeMYB4L could directly regulates the expression of Phalaenopsis spp. chalcone synthase gene (PeCHS) through MYBST1 (GGATA) binding site. It's interesting that the basic-helix-loop-helix (bHLH) protein PeMYC4 shows opposite expression pattern from PeMYB4L in anthocyanin accumulation. Furthermore, PeMYC4 was verified to form MYB-bHLH complex with PeMYB4L, and attenuated the expression of PeCHS and weakened anthocyanin production, indicating a novel regulatory model of MYB-bHLH complex. Our findings uncover the detailed regulatory pathway of MYB-bHLH, and might provide a new insight into the complicated anthocyanin pigmentation in Phalaenopsis spp.