The CsMYB44–CsICE1 module mediates flavonoid and catechin biosynthesis to regulate cold tolerance in tea plants ( <i>Camellia sinensis</i> )

The tea plant is an economically important perennial crop whose yield and quality are severely constrained by cold stress. With the increasing frequency of extreme weather events, elucidating the molecular basis of cold tolerance is therefore essential for safeguarding tea production and its associated economic value. Catechins contribute to cold tolerance in tea plants, but the underlying regulatory mechanisms remain poorly understood. Here, we integrated transcriptomic, metabolomic, and physiological approaches to identify the CsMYB44-CsICE1 module, which regulates cold-induced flavonoid and catechin biosynthesis to enhance cold tolerance. Weighted gene co-expression network analysis (WGCNA) revealed that CsICE1 was strongly positively correlated with catechin accumulation and cold tolerance, whereas CsMYB44 was negatively correlated with these traits. Overexpression of CsICE1 in Arabidopsis improved cold tolerance by elevating flavonoid levels and upregulating antioxidant and cold-responsive genes, including AtSOD, AtPOD, and AtCBF1. Conversely, overexpression or silencing CsICE1 in tea plants significantly affected cold sensitivity. Overexpression of CsMYB44 reduced cold tolerance in Arabidopsis, while its silencing enhanced tolerance in tea plants. Yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase assays demonstrated that CsMYB44 directly binds to the promoters of CsICE1, CsCHS (chalcone synthase), CsFLS (flavonol synthase), and CsANR (anthocyanidin reductase) to repress their expression. In contrast, CsICE1 activates the transcription of CsCHS, CsFLS, and CsANR. RT-qPCR analysis further indicated that short-term cold stress suppresses CsMYB44 expression, thereby releasing CsICE1, which subsequently upregulates CsCHS, CsFLS, and CsANR, promoting flavonoid and catechin accumulation and ultimately mitigating cold-induced damage. Collectively, these findings uncover a novel cold-tolerance mechanism in tea plants.