PfMYB66 targets PfANS1 and its functional partner PfFOMT1 enhances PfANS1 -driven Perilla leaf coloring

Abstract

Perilla frutescens, a typical anthocyanin-enriched medicinal and edible herb, displays obvious leaf color divergence. However, the molecular regulatory mechanisms governing this trait in isogenic backgrounds remain largely unclear. Here, we integrated transcriptomic, metabolomic, and functional analyses to elucidate the molecular basis of purple (DP), light-purple (LP), and green (GR) leaf phenotypes. We identified PfANS1, a key anthocyanin biosynthetic gene encoding anthocyanidin synthase, whose expression correlated positively with anthocyanin accumulation and purple pigmentation. Through transcription factor screening, we characterized PfMYB66, an R2R3-MYB transcription factor that directly binds to the promoters of both PfANS1 and PfFOMT1 (Perilla frutescens flavonoid O-methyltransferase 1) and activates their expression, as demonstrated by yeast one-hybrid, chromatin immunoprecipitation, electrophoretic mobility shift assay (EMSA), and dual-luciferase assays. Transient overexpression of PfMYB66 significantly elevated the transcript abundance of PfANS1 and PfFOMT1, resulting in higher contents of cyanidin-3-O-glucoside (C3G), myricitrin (Myr) and reduced salvianolic acid C (Sal C), accompanied by intensified purple pigmentation in perilla leaves. Conversely, virus-induced gene silencing of PfMYB66 reduced anthocyanin content and purple intensity. Notably, although overexpression of PfFOMT1 alone does not increase anthocyanin accumulation and purple intensity, overexpression of PfANS1 alone does; however, co-expression of PfANS1 and PfFOMT1 resulted in significantly stronger pigmentation than overexpression of either gene alone, demonstrating the possibility of a synergistic effect. Physiological assays revealed that purple leaves exhibited enhanced antioxidant enzyme activities, accompanied by higher levels of MDA, H2O2 and O2− than green leaves. Our findings establish the PfMYB66-PfANS1/PfFOMT1 regulatory module as a central determinant of leaf color formation in P. frutescens, providing new insights into tissue-specific anthocyanin regulation and offering potential targets for molecular breeding of ornamental and functional food crops.