Sucrose signaling regulates theanine accumulation via the CsYABBY2-CsMYB5-CsPDX2.1 module in tea plants

Theanine is a specialized non-protein amino acid that determines the quality of tea (Camellia sinensis), yet the regulatory mechanisms controlling its hydrolysis remain unknown. Here we show that sucrose acts as a signal to modulate theanine accumulation by regulating a transcriptional repressor module that suppresses the theanine hydrolase gene CsPDX2.1. Using a yeast one-hybrid screen, we identified two transcription factors, CsYABBY2 and CsMYB5, that directly bind to distinct cis-elements (TAATGATT and TAACCA) in the CsPDX2.1 promoter. We demonstrate that CsYABBY2 and CsMYB5 interact to form a complex, and that co-expression of both proteins synergistically enhances repression of CsPDX2.1 transcription. Functional analyses via virus-induced gene silencing and transient overexpression confirmed that this module negatively regulates CsPDX2.1 expression and positively controls theanine accumulation. Importantly, exogenous sucrose suppresses CsYABBY2 and CsMYB5 expression, thereby derepressing CsPDX2.1 expression and reducing theanine levels, suggesting that sucrose functions as a regulatory factor affecting this pathway. Our findings reveal a sucrose-responsive (CsYABBY2-CsMYB5-CsPDX2.1) module that mediates theanine accumulation, providing a molecular basis for understanding varietal differences in theanine content.