Transcriptomics integrated with metabolomics to characterize key pigment compounds and genes related to anthocyanin biosynthesis in Zanthoxylum bungeanum peel

Zanthoxylum bungeanum is an important condiment with high economic value and its peel color is one of the main quality indexes. However, the key pigment compounds and related genes are still unclear affecting the quality control of the plants. In this study, the contents of four types of pigments were measured in Z. bungeanum and flavonoids were identified as the most important pigments. Based on the targeted flavonoid metabolomics of Z. bungeanum peels, 14 key pigment compounds were screened out from 152 flavonoids, among which cyanidin-3-O-rutinoside and cyanidin-3-O-glucoside were the most critical compounds for peel color. They were further verified to be present in nine varieties of Z. bungeanum by HPLC fingerprints. The 14 compounds were all associated with flavonoid and anthocyanin biosynthesis pathways and the 39 differentially expressed genes related to these pathways were annotated and screened based on transcriptomics. The genes ZbDFR, ZbANS, and ZbUFGT were identified as three key genes for anthocyanin synthesis in Z. bungeanum peels. Further qRT-PCR results confirmed the reliability of transcriptomics and the accuracy of gene screening. Subsequent protein induced expression demonstrated that ZbANS and ZbUFGT were expressed after 12 h induced by IPTG while ZbDFR was expressed after 15 h. Further transient and stable transformation analysis confirmed that both anthocyanin content and the expression of ZbDFR were significantly increased in overexpression Z. bungeanum leaves and Nicotiana benthamiana. The functional effect of stable transformation of ZbDFR was more significant than that of transient transformation with a 7.67-fold/1.49-fold difference in total anthocyanin content and a 42.37-fold/12.32-fold difference in the expression of ZbDFR. This study provides new insights into the chemical composition and the molecular mechanisms of Z. bungeanum peel color and lays an effective foundation for the color quality control, multi-purpose utilization of Z. bungeanum and the creation of new germplasm.