The transcription factor CaMYB108a negatively regulates anthocyanin and other flavonoid biosynthesis in chili pepper (Capsicum annuum L.) fruit

Fruit color is a key horticultural trait that determines the economic value of chili pepper ( Capsicum annuum L.), which is predominantly governed by the accumulation of flavonoid compounds, especially anthocyanins. The R2R3-MYB transcription factor subfamily plays a crucial role in regulating anthocyanin and flavonoid biosynthesis, yet its specific regulatory mechanisms in chili peppers remain largely elusive. In this study, using the known anthocyanin-related light-responsive factor CaHY5 as bait, we identified it interacting with R2R3-MYB protein through yeast two-hybrid screening, designated CaMYB108a. Phylogenetic and genomic analyses supported its classification within the MYB family. Functional validation demonstrated that CaMYB108a acts as a negative regulator of anthocyanin accumulation and total flavonoid compounds in chili pepper fruits. Overexpression of CaMYB108a significantly suppressed the expression of key anthocyanin biosynthetic structural genes, reduced anthocyanin accumulation, and decreased total flavonoid and quercetin contents. Conversely, silencing CaMYB108a markedly enhanced the expression of anthocyanin-related genes, promoted anthocyanin accumulation, and increased total flavonoid content and quercetin levels, whereas luteolin showed a slight decrease, indicating differential regulation across distinct branches of the flavonoid pathway. Further investigation revealed that CaMYB108a directly binds to the promoter regions of key anthocyanin biosynthetic genes CaCHI , CaF3'5'H , and CaDFR , repressing their transcription. This study uncovers the negative regulatory role of CaMYB108a in anthocyanin and flavonoid accumulation in chili pepper fruits and provides novel insights and a genetic target for improving fruit coloration. • CaMYB108a interacted with the light-responsive factor CaHY5 and identified its evolutionary position within the R2R3-MYB family clarified. • Functional validation demonstrated that CaMYB108a negatively regulated anthocyanin and other flavonoid biosynthesis in chili pepper fruits. • The molecular mechanism of an R2R3-MYB transcription factor negatively controlling anthocyanin and other flavonoid synthesis was confirmed in chili pepper fruits. • It provides a potential molecular breeding target for improving chili pepper fruit coloration.