JrATHB-12 Mediates JrMYB113 and JrMYB27 to Control the Different Types of Red Walnut1

Red walnut has broad market prospects because it is richer in anthocyanin than ordinary walnut. However, the anthocyanin biosynthetic mechanism of red walnut is still unknown. We studied two types of red walnut, called red walnut 1 (R1), with a red pericarp and seed coat, and red walnut 2 (R2), with a red seed coat only. R1 mostly contained cyanidin-3-O-galactoside, while R2 contained a certain amount of each of cyanidin-3-O-galactoside, cyanidin-3-O-arabinoside, and cyanidin-3-O-glucoside. The LDOX-2 (LOC109007163) and LDOX-3 (LOC109010746), encoding genes of leucoanthocyanidin dioxygenase/anthocyanidin synthase (LDOX/ANS), were preliminarily appraised as crucial genes for anthocyanin biosynthesis in R1 and R2, respectively. The MYB differential genes analysis showed that MYB27 and MYB113 are specifically expressed in the red parts of R1 and R2, respectively, and are regarded as candidate regulatory genes. Ectopic expression in Arabidopsis and transient injection in walnut showed that both MYB27 and MYB113, located in the nucleus, promoted anthocyanin accumulation, MYB27 promoted the LDOX-2 expression, and MYB113 promoted the expressions of LDOX-3 and UAGT-3. Yeast one-hybrid and electrophoretic mobility shift assays showed that MYB27 could only bind the LDOX-2 promoter, while MYB113 could bind the promoters of LDOX-3 and UAGT-3. In addition, we also identified an HD-Zip transcription factor ATHB-12, which is specifically expressed in the pericarp. After silencing the expression of ATHB-12, the R2 pericarp turned red, and the MYB113 expression increased. Further experiments showed that ATHB-12 could specifically interact with MYB113 and bind to its promoter. This suggests that MYB27 controls R1 coloration by regulating LDOX-2, while MYB113 controls R2 coloration by regulating LDOX-3 and UAGT-3, but the ATHB-12 can specifically bind and inhibit the MYB113 of R2 pericarp so that it becomes unpigmented. This study reveals the anthocyanin biosynthesis mechanisms in different types of red walnut and provides a scientific basis for the selection and breeding of red walnut varieties.