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 (LOC109007163) and (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 and are specifically expressed in the red parts of R1 and R2, respectively, and are regarded as candidate regulatory genes. Ectopic expression in and transient injection in walnut showed that both MYB27 and MYB113, located in the nucleus, promoted anthocyanin accumulation, MYB27 promoted the expression, and MYB113 promoted the expressions of and . Yeast one-hybrid and electrophoretic mobility shift assays showed that MYB27 could only bind the promoter, while MYB113 could bind the promoters of and . In addition, we also identified an HD-Zip transcription factor ATHB-12, which is specifically expressed in the pericarp. After silencing the expression of , the R2 pericarp turned red, and the 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 , while MYB113 controls R2 coloration by regulating and , but the ATHB-12 can specifically bind and inhibit the 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.