Abstract: Tea plant (Camellia sinensis) accumulates abundant secondary metabolites under cold stress, some of which are thought to play important roles in enhancing cold tolerance. To explore novel secondary metabolites involved in cold tolerance, we conducted an untargeted metabolomics analysis of tea plants under cold stress treatment. This revealed a novel acylated flavonoid, kaempferol-3-O-(6″-p-coumaroyl)-glucoside (KCG), in which accumulation positively correlated with stress severity. The compound was purified and structurally characterized using nuclear magnetic resonance (NMR) spectroscopy. Exogenous application of this flavonoid significantly improved cold tolerance in tea plants, indicating its role as a defensive metabolite. Transcriptome sequencing identified candidate acyltransferases, with tea hydroxycinnamoyl transferase (CsHCT) emerging as a key biosynthetic gene. In vitro assays confirmed that recombinant CsHCT catalyzes the formation of KCG from kaempferol-3-O-glucoside and p-coumaroyl-CoA. Overexpression of CsHCT in tea seedlings and Arabidopsis thaliana resulted in markedly elevated levels of this flavonoid and cold resistance of these plants, validating its in vivo role. Our findings elucidate the biosynthesis of acylated flavonoids in tea plants and highlight CsHCT as a genetic target for enhancing cold resistance. This study provides foundational insights for advancing cold-resistant tea breeding programs.

Key words: biosynthesis, Camellia sinensis, cold stress, CsHCT, kaempferol-3-O-(6″-p-coumaroyl)‐glucoside, metabolomics