The transcription factor CsS40 negatively regulates TCS1 expression and caffeine biosynthesis in connection to leaf senescence in Camellia sinensis .

Caffeine is considered as one of the most important bioactive components in the popular plant beverages tea, cacao, and coffee, but as a wide-spread plant secondary metabolite its biosynthetic regulation at transcription level remains largely unclear. Here, we report a novel transcription factor () as a caffeine biosynthesis regulator, which was discovered during screening a yeast expression library constructed from tea leaf cDNAs for activation of tea caffeine synthase () promoter. Besides multiple hits of the non-self-activation CsS40 clones that bound to and activated promoter in yeast-one-hybrid assays, a split-luciferase complementation assay demonstrated that acts as a transcription factor to activate the gene and EMSA assay also demonstrated that CsS40 bound to the gene promoter. Consistently, immunofluorescence data indicated that CsS40-GFP fusion was localized in the nuclei of tobacco epidermal cells. The expression pattern of in 'Fuding Dabai' developing leaves was opposite to that of ; and knockdown and overexpression of in tea leaf calli significantly increased and decreased expression levels, respectively. The expression levels of were also negatively correlated to caffeine accumulation in developing leaves and transgenic calli of 'Fuding Dabai'. Furthermore, overexpression of reduced the accumulation of xanthine and hypoxanthine in tobacco plants, meanwhile, increased their susceptibility to aging. expression in tea leaves was also induced by senescence-promoting hormones and environmental factors. Taken together, we showed that a novel senescence-related factor CsS40 negatively regulates and represses caffeine accumulation in tea cultivar 'Fuding Dabai'. The study provides new insights into caffeine biosynthesis regulation by a plant-specific senescence regulator in tea plants in connection to leaf senescence and hormone signaling.