PnMYB4a , a R2R3 transcription factor, modulates coumarin biosynthesis during melanization in the culms of Phyllostachys nigra

Culm colour is one of the primary traits determining the ornamental value of bamboos. The black pigment shown to accumulate in Phyllostachys nigra was previously characterized as melanin, but the underlying regulatory mechanism for its deposition remains elusive. Herein, metabolomics data show that coumarins serve as precursor substrates for melanization in P. nigra, and RNA-seq and weighted gene correlation network analysis (WGCNA) revealed that the transcription factor PnMYB4a is integral to coumarin biosynthesis. Overexpression of PnMYB4a promotes coumarin production in both Oryza sativa and Arabidopsis thaliana. Pn4CL4-2, a biosynthetic gene involved in coumarin biosynthesis, was identified as a direct target gene of PnMYB4a. Additionally, PnWRKY8 and PnWRKY72-2 were found to interact with PnMYB4a both in vivo and in vitro. Both PnWRKY8 and PnWRKY72-2 positively promoted coumarin accumulation in transgenic rice, but they repressed the expression of Pn4CL4-2 by directly binding to W-box elements within its promoter. Interestingly, PnMYB4a was found to alleviate the inhibitory effect on Pn4CL4-2 by forming complexes with both PnWRKY8 and PnWRKY72-2. Collectively, these findings revealed a PnMYB4a-PnWRKY8/PnWRKY72-2 regulatory module involved in the coumarin biosynthetic pathway in P. nigra and provide new insights into the mechanisms of melanization and coloration in bamboo species.