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

Abstract

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.

miR828a-CsMYB114 Module Negatively Regulates the Biosynthesis of Theobromine in Camellia sinensis

“…19,21 In addition, a novel senescenceassociated transcription factor, CsS40, was found to regulate caffeine biosynthesis by downregulating the biosynthetic gene TCS1. 20 However, there is limited understanding regarding the regulatory mechanisms of theobromine biosynthesis. It has been reported that CsMYB184 (TEA029017.1) seemed to be involved in the biosynthesis of theobromine, but this needs to be further verified.…”

“…Theobromine has received more and more attention as an important alkaloid in tea plant. Up to this point, only CsNAC7 and CsMYB184 have been functionally identified as regulators of caffeine biosynthesis, with each one specifically enhancing the expression of the critical biosynthetic genes NMT1 and TCS1 , respectively. , In addition, a novel senescence-associated transcription factor, CsS40, was found to regulate caffeine biosynthesis by downregulating the biosynthetic gene TCS1 . However, there is limited understanding regarding the regulatory mechanisms of theobromine biosynthesis.…”

“…In vitro experiments, employing techniques such as dual luciferase, yeast one-hybridization, trans-activation analysis, and electrophoretic mobility shift assay (EMSA), confirmed the interaction of MYB184 to the MYB1AT-MYBPLANT motif, which is integral to regulating caffeine biosynthesis in tea plants . A novel senescence-associated factor, CsS40, has been reported to negatively regulate TCS1 and inhibit the accumulation of caffeine in the tea cultivar 'Fuding Dabai' . In addition, CsNAC7 positively regulates the caffeine synthase gene yhNMT1 , promoting the accumulation of caffeine within tea plants .…”

“…19 A novel senescence-associated factor, CsS40, has been reported to negatively regulate TCS1 and inhibit the accumulation of caffeine in the tea cultivar 'Fuding Dabai'. 20 In addition, CsNAC7 positively regulates the caffeine synthase gene yhNMT1, promoting the accumulation of caffeine within tea plants. 21 The purification and characterization of theobromine has been comprehensively understood, and have successfully isolated and cloned both the N-methyltransferase (NMT) gene and its promoter.…”

Zinc Treatment of Tea Plants Improves the Synthesis of Trihydroxylated Catechins via Regulation of the Zinc-Sensitive Protein CsHIPP3

“…According to the method of Yao et al, each leaf tissue culture seedling was cut into four parts and infected inside a resuspension (containing 100 μmol/L AS) containing no-load, 35S: CsHIPP3 , 35S: CsF3′5′H1 , and co-overexpression bacterial solution for 20 min. The leaves were grown on MS solid medium supplemented with 1.0 mg/L NAA and 0.5 mg/L 6-BA for 3 days in the dark after being dried using sterile absorbent paper.…”