Genome-Wide Analysis of the R2R3-MYB Family in Perilla frutescens and the Regulatory Model of the PfMYB33-PfbHLH11 Complex in Anthocyanin Biosynthesis

Perilla frutescens (L.) leaves exhibit diverse coloration due to differential anthocyanin accumulation, yet the underlying transcriptional regulatory mechanisms remain largely unclear. R2R3-MYB transcription factors and their complexes are known to play central roles in plant anthocyanin biosynthesis. Here, we integrated metabolomics, transcriptomics, gene family analysis, and molecular interaction assays to elucidate the metabolic basis and key regulatory modules of leaf coloration. Targeted and untargeted metabolomics indicated that the differential accumulation of cyanidin-derivative anthocyanins primarily drives leaf color variation. Transcriptomics revealed differential expression patterns of anthocyanin structural genes across varying leaf colors. Genome-wide analysis identified 164 PfMYB genes, which were classified into 32 subfamilies. Through combined analyses, PfMYB33 (subgroup 6) was identified as a core gene highly correlated with anthocyanin accumulation. Both PfMYB33 and its interacting partner, PfbHLH11, localize to the nucleus. Yeast one-hybrid and dual-luciferase assays showed that PfMYB33 directly binds to the promoters of multiple anthocyanin biosynthetic genes and acts as an activator of DFR2 but a repressor of 5GT2. Transient co-expression of PfMYB33 and PfbHLH11 in P. frutescens leaves resulted in the strongest purple pigmentation and the highest anthocyanin accumulation. This study constructs a metabolic profile of anthocyanin accumulation in P. frutescens leaves and proposes a novel regulatory model wherein the PfMYB33-PfbHLH11 complex coordinates downstream structural genes. These findings provide valuable insights into the molecular mechanisms governing leaf coloration in P. frutescens .