R2R3-MYB transcription factor PhMYB2 positively regulates anthocyanin biosynthesis in Pericallis hybrida

Cineraria (Pericallis hybrida) is an ideal species for anthocyanin accumulation studies in ornamental plants due to its bright flower colors and diverse anthocyanin branching metabolic pathways. However, the regulatory pattern of anthocyanin metabolism was still unclear in previous research on the flower color of cineraria. In order to explore the anthocyanin synthesis mechanism of pure color cultivar of cineraria, we isolated a novel R2R3-MYB gene PhMYB2 based on the previous transcriptome of blue cineraria. PhMYB2 was significantly expressed in carmine and blue cineraria, and its expression profile corresponded with the pattern of anthocyanin accumulation in the ray florets. PhMYB2 could activate the transcription of anthocyanin biosynthetic genes PhF3 & PRIME;H1, PhF3 & PRIME;5 & PRIME;H, PhDFR3, and PhANS when transiently silenced PhMYB2 in cineraria leaves by virus-induced gene silencing (VIGS). Meanwhile, PhMYB2 could interact with PhbHLH17, which belonged to TT8-clade, while PhbHLH17 could interact with a possible R2R3-MYB repressor PhMYB4.Y1H and dual-luciferase assays revealed that PhMYB2 binds and activates the promoter of PhDFR3, and when PhMYB2 interacts with PhbHLH17, the complex can significantly enhance the activity of the PhDFR3 promoter. Furthermore, when PhMYB4 co transformed with PhMYB2 and PhbHLH17, the activity of the PhDFR3 promoter was significantly reduced. Based on this, we speculated that PhMYB4 might fine-tune the anthocyanin synthesis by interacting with PhbHLH17 and competing with PhMYB2. The above results clarified the mechanism of PhMYB2-PhbHLH17 promoting anthocyanin synthesis and improved the anthocyanin regulatory network in cineraria.