Tea (Camellia sinensis [L.] O. Kuntze) is an important global economic crop and is considered to enhance health. However, the functions of many genes in tea plants are unknown. Virus-induced gene silencing (VIGS) mediated by tobacco rattle virus (TRV) is an effective tool for the analysis of gene functions, although this method has rarely been reported in tea plants. In this study, we established an effective VIGS-mediated gene knockout technology to understand the functional identification of large-scale genomic sequences in tea plants. The results showed that the VIGS system was verified by detecting the virus and using a real-time quantitative reverse transcription PCR (qRT-PCR) analysis. The reporter gene CsPOR1 (protochlorophyllide oxidoreductase) was silenced using the vacuum infiltration method, and typical photobleaching and albino symptoms were observed in newly sprouted leaves at the whole plant level of tea after infection for 12 d and 25 d. After optimization, the VIGS system was successfully used to silence the tea plant CsTCS1 (caffeine synthase) gene. The results showed that the relative caffeine content was reduced 6.26-fold compared with the control, and the level of expression of CsPOR1 decreased by approximately 3.12-fold in plants in which CsPOR1 was silenced. These results demonstrate that VIGS can be quickly and efficiently used to analyze the function of genes in tea plants. The successful establishment of VIGS could eliminate the need for tissue culture by providing an effective method to study gene function in tea plants and accelerate the process of functional genome research in tea.

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.

Phosphorus (P) is the macronutrients essential for the development and growth of plants, but how external inorganic phosphate (Pi) level and signaling affect tea plant growth and characteristic secondary metabolite biosynthesis are not understood. Theanine is major secondary metabolites, and its contents largely determine tea favor and nutrition qualities. Here, we found theanine contents in tea leaves and roots declined as Pi concentration increased in tea plants after Pi feeding. The transcriptome analysis of global gene expression in tea leaves under Pi feeding suggested a wide range of genes involved in Pi/N transport and responses were altered. Among them, CsSPX3 and CsPHL7 transcript levels in response to Pi feeding to tea plants, their expression patterns were generally opposite to these of major theanine biosynthesis genes, indicating possible regulatory correlations. Biochemical analyses showed that CsSPX3 interacted with CsPHL7, and CsPHL7 negatively regulated theanine biosynthesis genes CsGS1 and CsTS1. Meanwhile, VIGS and transient overexpression systems in tea plants verified the functions of CsSPX3 and CsPHL7 in mediating Pi-feeding-repressed theanine biosynthesis. This study offers fresh insights into the regulatory mechanism underlying Pi repression of theanine biosynthesis, and the CsSPX3-CsPHL7-CsGS1/CsTS1 module plays a role in high Pi-inhibition of theanine production in tea leaves. It has an instructional significance for guiding the high-quality tea production in tea garden fertilization.

Tea plant trichomes not only contribute to the unique flavor and high quality of tea products but also provide physical and biochemical defenses for tea plants. Transcription factors play crucial roles in regulating plant trichome formation. However, limited information about the regulatory mechanism of transcription factors underlying tea plant trichome formation is available. Here, the investigation of trichome phenotypes among 108 cultivars of Yunwu Tribute Tea, integrated with a transcriptomics analysis of both hairy and hairless cultivars, revealed the potential involvement of CsGeBPs in tea trichome formation. In total, six CsGeBPs were identified from the tea plant genome, and their phylogenetic relationships, as well as the structural features of the genes and proteins, were analyzed to further understand their biological functions. The expression analysis of CsGeBPs in different tissues and in response to environmental stresses indicated their potential roles in regulating tea plant development and defense. Moreover, the expression level of CsGeBP4 was closely associated with a high-density trichome phenotype. The silencing of CsGeBP4 via the newly developed virus-induced gene silencing strategy in tea plants inhibited trichome formation, indicating that CsGeBP4 was required for this process. Our results shed light on the molecular regulatory mechanisms of tea trichome formation and provide new candidate target genes for further research. This should lead to an improvement in tea flavor and quality and help in breeding stress-tolerant tea plant cultivars.

Background: Micronutrient malnutrition affects millions of people due to a lack of Zn and Se. Methods: The process conditions for the manufacture of glycine−chelated sodium selenite (Se−Gly) and zinc sulfate heptahydrate (Zn−Gly) were studied. The effects of ligand concentration, pH, reaction ratio, reaction temperature, and reaction time on fertilizer stability were assessed. The effects of Zn−Gly and Se−Gly on tea plants were determined. Results: Orthogonal experiments showed that the optimal preparation conditions for Zn−Gly (75.80 % Zn chelation rate) were pH 6.0, ligand concentration 4 %, reaction ratio 1:2, reaction time 120 min, reaction temperature 70 ℃. The optimal preparation conditions for Se−Gly (56.75 % Se chelation rate) were pH 6.0, ligand concentration 10%, reaction ratio 2:1, reaction time 40 min, temperature 50 ℃. Each chelate was completely soluble in water and verified by infrared spectroscopy and ultraviolet spectroscopy. Conclusions: Zn−Gly and Se−Gly increased the Zn and Se content in tea plants, and foliar application was more effective than soil application. Combined application of Zn−Gly and Se−Gly was more effective than Zn−Gly or Se−Gly alone. Our findings suggest that Zn−Gly and Se−Gly provide a convenient method of addressing human Zn and Se deficiency.