CsWRKY22 positively regulates quercetin biosynthesis by activating the CsFLSd promoter in tea plants (Camellia sinensis).

key messageCsWRKY22 acts as a positive regulator of CsFLSd through binding to proCsFLSd, promoting quercetin accumulation in tea plants, which contributes to the astringent taste of tea. Flavonol derivatives, including quercetin, are nutritionally valuable dietary components with health benefits. In tea plants, their high accumulation not only contributes to the beverage's nutritional quality but also influences its characteristic astringent taste. To gain a deeper insight into the mechanisms underlying flavonols biosynthesis in tea plants, this study first identified a candidate flavonol synthase-encoding gene, CsFLSd, via bioinformatic mining of the tea plant transcriptome. Functional validation showed that targeted manipulation of CsFLSd expression directly modulated quercetin levels: transient suppression of CsFLSd expression in tea plant leaves resulted in a significant reduction in quercetin content. Heterologous stable overexpression of CsFLSd in Arabidopsis thaliana further confirmed its role in promoting quercetin accumulation. To dissect the upstream regulation of CsFLSd, a Yeast One-Hybrid (Y1H) screen was performed using a tea plant cDNA library, leading to the identification of CsWRKY22 as a potential interacting partner of the CsFLSd promoter. Subsequent validation experiments revealed that CsWRKY22 could specifically bind to the CsFLSd promoter. Functional analyses demonstrated that transient overexpression of CsWRKY22 in tea plant leaves elevated the quercetin content, whereas transient suppression of its expression reduced quercetin accumulation, indicating that CsWRKY22 acts as a positive regulator of quercetin biosynthesis. Collectively, these findings characterize an important regulatory step in quercetin biosynthesis in tea plants, defined by the CsWRKY22-proCsFLSd module that controls quercetin production. Additionally, this study provides valuable insights into flavonols biosynthesis in tea plants, advancing the understanding of flavonoid metabolic regulation in tea plants.