A novel R2R3-MYB transcription factor PpMYB5 assisting Ppbbx24-del positively regulates anthocyanin biosynthesis in ‘Red Zaosu’ pear

Anthocyanins are vital pigments that play a crucial role in the coloration of various fruits. Our previous study identified a mutant Ppbbx24-del protein in the 'Red Zaosu' pear that positively regulates anthocyanin biosynthesis. However, this mutant protein exhibited nucleo-cytoplasmic localization due to the lack of the NLS domain. We hypothesized that a transcription factor in 'Red Zaosu' pear interacts with Ppbbx24-del, facilitating its nuclear translocation for regulatory function. In this study, a PpMYB5 was screened by Y2H assay using the Ppbbx24-del as bait, which was an R2R3-MYB transcription factor and significantly up-expressed in 'Red Zaosu' compared to 'Zaosu'. Pull-down, Y2H and BiFC assays confirmed that PpMYB5 could interact with both mutant Ppbbx24-del and common PpBBX24. Notably, co-expression experiments revealed that PpMYB5 facilitated the nuclear translocation of Ppbbx24-del. Transient expression assays in 'Zaosu' pear fruits demonstrated that PpMYB5 alone failed to induce anthocyanin accumulation, but its co-expression with Ppbbx24-del significantly enhanced the anthocyanin content of fruit peel compared to Ppbbx24-del alone. This synergistic effect was accompanied by significant upregulation of key anthocyanin biosynthetic genes, including <i>PpCHS</i> and <i>PpCHI</i>. Additionally, dual-luciferase assays demonstrated that PpMYB5 not only enhanced the activation effect on the promoters of <i>PpCHS</i> and <i>PpCHI</i> by Ppbbx24-del but also had the same effect on the promoter of <i>PpMYB5</i>. Our findings indicate that PpMYB5 and Ppbbx24-del form a crucial regulatory module that finely regulates anthocyanin synthesis in pear.