A novel CsRNF-CsHSF24-CsF3'5'H2 module regulates epigallocatechin gallate biosynthesis in Camellia sinensis.

Tea is rich in tea polyphenols, vitamins, amino acids, and other components that have many benefits to health. Catechins are divided into dihydroxy catechins and trihydroxy catechins based on the hydroxylation pattern of their B-ring. Although genes involved in catechin biosynthesis have been extensively studied and several transcription factors have been reported to regulate this process, the regulatory mechanisms controlling catechin biosynthesis remain incompletely understood. In particular, the role of post-translational modifications of transcription factors in catechin biosynthesis is still largely unknown. In this study, we identified tea plant heat shock transcription factor (CsHSF24) as an important positive regulator of epigallocatechin gallate (EGCG) biosynthesis. Specifically, we found that CsHSF24 binds to the promoter of CsF3'5'H2, which is a key controller of EGCG biosynthesis in tea plants, as its transcriptional activator. In addition, CsRNF functions as a RING-finger E3 ubiquitin ligase to ubiquitinate CsHSF24, leading to its degradation through the 26S proteasome pathway. Degradation of CsHSF24 reduces CsF3'5'H2 expression, thereby inhibiting EGCG biosynthesis. Additionally, we identified a naturally occurring 14-bp deletion in the CsF3'5'H2 promoter of the low-EGCG cultivar "Siqiu." Although located ~450 bp upstream of the canonical HSE, this deletion functions as a critical structural hinge; its absence disrupts the spatial architecture required for robust transcriptional activation, thereby reducing both CsF3'5'H2 expression and EGCG accumulation. This discovery reveals a natural structural variant that links genetic polymorphism to metabolic diversity via the CsRNF-CsHSF24-CsF3'5'H2 regulatory module.