The TT2-type MYB transcription factor JrMYB12 positively regulates proanthocyanidin biosynthesis in red walnut

Proanthocyanidins (PAs) are important phenolic secondary metabolites. Red walnut ‘RW-1’ is rich in PAs, but little information is available on the regulation mechanism of PA biosynthesis. Here, we reported a MYB transcription factor from ‘RW-1’, designated JrMYB12, which acted as a positive regulator in PAs accumulation. Sequence alignment showed JrMYB12 protein belongs to TT2-type R2R3 MYB transcription factor because of its TT2 domain. The expression of JrMYB12 gene was positively correlated with the accumulation of PAs and the expressions of PA-related genes (JrLAR and JrANR) in red walnut. JrMYB12 protein localized to the nuclear and showed transcriptional self-activation activity. The expression of key genes related to PA biosynthesis (AtCHS, AtDFR, AtANS, and AtANR) was up-regulated in transgenic JrMYB12 overexpression Arabidopsis lines, and the content of PAs in leaves and seed coat increased significantly. However, the overexpression of JrMYB12 gene did not cause the change of the key anthocyanin related gene AtUFGT expression in Arabidopsis. Yeast two-hybrid showed that JrMYB12 and JrbHLH42 proteins had obvious interaction, and dual-luciferase assays showed that the two proteins could form a complex to significantly enhance the promoter activity of PAs biosynthesis structural genes JrLAR and JrANR alone or together. Collectively, our research revealed the regulatory mechanism of PA biosynthesis in red walnut, and will be useful for manipulating proanthocyanidins accumulation in walnut and other plants.