Transcription factor NtMYB59 targets NtMYB12 to negatively regulate the biosynthesis of polyphenols in Nicotiana tabacum

MYB12 is a key regulator that has been shown to promote the accumulation of various phenylpropanoid compounds in plants. However, the regulation of MYB12 gene is largely unknown. In this study, we found that overexpression of the NtMYB59 gene significantly inhibited the accumulation of chlorogenic acid (CGA), flavonols, and anthocyanins in tobacco, while knock-down and knock-out of NtMYB59 significantly increased the contents of these polyphenol compounds. Transcriptome analysis between WT and NtMYB59-OE plants revealed several differentially expressed genes (DEGs) encoding crucial enzymes in the phenylpropanoid pathway and the transcription factor NtMYB12. ChIP-seq assay further indicated that NtMYB12 might be a direct target of NtMYB59. Subsequent yeast one-hybrid, electrophoretic mobility shift assay, and Dual-Luciferase assays confirmed that NtMYB59 directly binds to the promoter of NtMYB12 to inhibit its expression. Moreover, loss-function of NtMYB59 significantly promoted the accumulation of flavonols and anthocyanins in ntmyb59, but their contents in ntmyb59/ntmyb12 double mutants were significantly lower than that of WT and ntmyb59 plants, indicating that the regulation of NtMYB59 on flavonoids biosynthesis depends on the activity of NtMYB12. Our study revealed that NtMYB59 regulates the expression of NtMYB12, and provided new potential strategies for modulating phenylpropanoids biosynthesis in tobacco.