PagMYB128 regulates anthocyanin biosynthesis by directly activating PagMYB116 in Populus alba x Populus glandulosa

MYB transcription factors regulate the biosynthesis of anthocyanin, a crucial flavonoid in plants. In this research, we identified PagMYB128 as a new activator of anthocyanin production in the hybrid poplar (Populus alba x Populus glandulosa). The PagMYB128 protein was located in the nucleus and has higher expression level in stems. Dominant repression of PagMYB128 led to a reduction of anthocyanin content in the petiole and stems. Overexpression of PagMYB128 resulted in the opposite phenotype. We found that PagMYB128 promoted anthocyanin biosynthesis by activating PagCHS1, PagF3'5'H1, PagDFR2, and PagANR1, which encoded the essential enzymes of anthocyanin biosynthesis. Further, PagMYB116 was identified as a potential direct target of PagMYB128 by ChIP-seq. Through ChIP-qPCR, Y1H, and Split-LUC assays, it was demonstrated that PagMYB128 could directly interact with the MYB-site located in the promoter region of PagMYB116, thereby exerting control over the transcription of PagMYB116. Moreover, through the poplar transient transformation method, we found that PagMYB116 could activate anthocyanin biosynthetic enzyme genes PagF3'5'H1 and PagANR1, through direct binding to their promoters. These findings indicate that the PagMYB128-MYB116 module for transcriptional regulation plays a crucial part in the synthesis of anthocyanin, enriching our comprehension of the networks responsible for regulating anthocyanin transcription.