The Mechanism of TT2-Type MYB Transcription Factor JrMYB1L in Anthocyanin Biosynthesis in ‘Jinghong 1’ Walnuts

Red walnuts have been widely studied because of their strong antioxidant activity and ornamental value. However, research on the mechanism of anthocyanin biosynthesis in walnuts remains in the initial stage. The regulatory mechanism of TT2-type <i>R2R3-MYB</i> transcription factors in anthocyanin biosynthesis in walnuts is also unclear. Therefore, this study used 'D2-1' and 'Jinghong 1' walnuts as plant materials. The testa of 'Jinghong 1' was red, and its anthocyanin content was significantly higher than that of 'D2-1', mainly composed of cyanidin-3-O-arabinoside, cyanidin-3-O-galactoside, and cyanidin-3-O-glucoside. Differentially expressed genes between 'D2-1' and 'Jinghong 1' testa were enriched in phenylpropanoid biosynthesis and flavonoid biosynthesis. Next, this study identified a TT2-type <i>R2R3-MYB</i> transcription factor <i>JrMYB1L</i>, which was involved in regulating the anthocyanin biosynthesis in the testa of 'Jinghong 1'. The overexpression of <i>JrMYB1L</i> could promote anthocyanin accumulation in walnut leaves and activate the expression of <i>JrCHS</i>, <i>JrCHI</i>, <i>JrF3H</i>, <i>JrDFR</i>, <i>JrANS</i>, <i>JrUFGT</i>, <i>JrLAR,</i> and <i>JrANR</i>. In addition, yeast two-hybrid results proved that JrMYB1L, JrbHLH42, and JrWD40 proteins could interact with each other. The results of yeast one-hybrid and dual-luciferase assays indicated that <i>JrMYB1L</i> could activate the expression of <i>JrCHS</i> and <i>JrUFGT</i> by binding to their promoters. Based on the above results, this study proposed a possible regulatory mechanism. <i>JrMYB1L</i> activated the expression of <i>JrCHS</i> and <i>JrUFGT</i> in the form of JrMYB1L-JrbHLH42-JrWD40 complex, thereby promoting anthocyanin accumulation in the testa of 'Jinghong 1'. In summary, this study lays a theoretical foundation for revealing the regulatory mechanism of anthocyanin biosynthesis in red walnut and contributes to the breeding of new varieties of red walnuts with more edible and ornamental value.