Transcriptomic and metabolomic analyses reveal the mechanism of anthocyanin metabolism in H18 pepper leaves and the function of CaDFR1

Anthocyanins play a crucial role in plant growth, development, reproduction, and stress response. Additionally, anthocyanins enhance the quality of fruits and vegetables due to their antioxidant properties. While numerous previous studies have been conducted on anthocyanins, limited information exists regarding their composition and the role of the anthocyanin pathway gene dihydroflavonol 4-reductase (DFR) in chili pepper leaves. In this study, we used a purple leaf pepper cultivar H18 in which the anthocyanin content in leaves decreases with plant growth and development. Targeted anthocyanin metabolite assays revealed that the contents of delphinidin, malvidin, and petunidin derivatives followed the same trend as the overall anthocyanin content, with delphinidin derivatives being the predominant component of H18 pepper leaves. Transcriptome sequencing was performed on H18 leaves at four different stages. The results showed that differentially expressed genes (DEGs) at various stages were primarily associated with biological processes and flavonoid metabolic pathways. Through phylogenetic tree and expression analysis, we identified three candidate genes involved in DFR function. Substrate catalysis assays of CaDFRs demonstrated that only CaDFR1 was active, catalyzing dihydroquercetin (DHQ), dihydromyricetin (DHM), and dihydrokaempferol (DHK). VIGS-mediated silencing of CaDFR1 resulted in a significant decrease in anthocyanin levels in H18 pepper leaves and stems, along with a reduction in the expression levels of other candidate functional genes in the anthocyanin metabolic pathway. This study identifies the key anthocyanin components in the leaves of H18 peppers and validates the function of CaDFR1, providing a theoretical foundation for modifying anthocyanin content in pepper plants through molecular breeding.