Two amino acid changes in the R3 repeat cause functional divergence of two clustered MYB10 genes in peach

R2R3-MYB genes play a pivotal role in regulating anthocyanin accumulation. Here, we report two tandemly duplicated R2R3-MYB genes in peach, PpMYB10.1 and PpMYB10.2, with the latter showing lower ability to induce anthocyanin accumulation than the former. Site-directed mutation assay revealed two amino acid changes in the R3 repeat, Arg/Lys⁶⁶ and Gly/Arg⁹³, responsible for functional divergence between these two PpMYB10 genes. Anthocyanin-promoting activity of PpMYB10.2 was significantly increased by a single amino acid replacement of Arg⁹³ with Gly⁹³. However, either the Gly⁹³ → Arg⁹³ or Arg⁶⁶ → Lys⁶⁶ substitutions alone showed little impact on anthocyanin-promoting activity of PpMYB10.1, but simultaneous substitutions caused a significant decrease. Reciprocal substitution of Arg/Gly⁹³ could significantly alter binding affinity to PpbHLH3, while the Arg⁶⁶ → Lys⁶⁶ substitution is predicted to affect the folding of the MYB DNA-binding domain, instead of PpbHLH3-binding affinity. Overall, the change of anthocyanin-promoting activity was accompanied with that of bHLH-binding affinity, suggesting that DNA-binding affinity of R2R3-MYBs depends on their bHLH partners. Our study is helpful for understanding of functional evolution of R2R3-MYBs and their interaction with DNA targets.