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 Camellia sinensis Senescnece 40 (CsS40) 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 (TCS1) promoter. Besides multiple hits of the non-self-activation CsS40 clones that bound to and activated TCS1 promoter in yeast-one-hybrid assays, a split-luciferase complementation assay demonstrated that CsS40 acts as a transcription factor to activate the CsTCS1 gene and EMSA assay also demonstrated that CsS40 bound to the TCS1 gene promoter. Consistently, immunofluorescence data indicated that CsS40-GFP fusion was localized in the nuclei of tobacco epidermal cells. The expression pattern of CsS40 in 'Fuding Dabai' developing leaves was opposite to that of TCS1; and knockdown and overexpression of CsS40 in tea leaf calli significantly increased and decreased TCS1 expression levels, respectively. The expression levels of CsS40 were also negatively correlated to caffeine accumulation in developing leaves and transgenic calli of 'Fuding Dabai'. Furthermore, overexpression of CsS40 reduced the accumulation of xanthine and hypoxanthine in tobacco plants, meanwhile, increased their susceptibility to aging. CsS40 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 TCS1 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.

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