CsbHLH18 Directly Activates CsTSI to Promote Theanine Biosynthesis in Tea Plants (Camellia sinensis L.)

Theanine, a unique nonproteinogenic natural amino acid predominantly accumulated in tea plants (Camellia sinensis), is a key determinant of tea flavor and quality, with broad applications. Theanine synthase (CsTSI) is a key enzyme in theanine biosynthesis. However, the transcriptional regulation of CsTSI, particularly by bHLH transcription factors, remains largely unexplored. Through coexpression network screening and tissue-specific qPCR, we identified CsbHLH18 as a putative regulator of CsTSI. Yeast one-hybrid (Y1H) and dual-luciferase reporter assays confirmed the direct interaction between CsbHLH18 and the CsTSI promoter. An electrophoretic mobility shift assay (EMSA) further identified the specific binding motif of CsbHLH18 as "CAAATG". Transient overexpression and virus-induced gene silencing (VIGS) in tea plants demonstrated that CsbHLH18 positively regulates theanine biosynthesis by activating CsTSI transcription. These findings elucidate a previously uncharacterized molecular mechanism underlying theanine biosynthesis and provide theoretical insights into molecular breeding and cultivation of high-quality tea varieties.