Tea is the most popular nonalcoholic beverage worldwide for its pleasant characteristics and healthful properties. Catechins, theanine and caffeine are the major natural products in tea buds and leaves that determine tea qualities such as infusion colors, tastes and fragrances, as well as their health benefits. Shading is a traditional and effective practice to modify natural product accumulation and to enhance the tea quality in tea plantation. However, the mechanism underlying the shading effects is not fully understood. This study aims to explore the regulation of flavonoid biosynthesis in Camellia sinensis under shading by using both metabolomic and transcriptional analyses. While shading enhanced chlorophyll accumulation, major catechins, including C, EC, GC and EGC, decreased significantly in tea buds throughout the whole shading period. The reduction of catechins and flavonols were consistent with the simultaneous down-regulation of biosynthetic genes and TFs associated with flavonoid biosynthesis. Of 16 genes involved in the flavonoid biosynthetic pathway, F3’H and FLS significantly decreased throughout shading while the others (PAL, CHSs, DFR, ANS, ANR and LAR, etc.) temporally decreased in early or late shading stages. Gene co-expression cluster analysis suggested that a number of photoreceptors and potential genes involved in UV-B signal transductions (UVR8_L, HY5, COP1 and RUP1/2) showed decreasing expression patterns consistent with structural genes (F3’H, FLS, ANS, ANR, LAR, DFR and CHSs) and potential TFs (MYB4, MYB12, MYB14 and MYB111) involved in flavonoid biosynthesis, when compared with genes in the UV-A/blue and red/far-red light signal transductions. The KEGG enrichment and matrix correlation analyses also attributed the regulation of catechin biosynthesis to the UVR8-mediated signal transduction pathway. Further UV-B treatment in the controlled environment confirmed UV-B induction on flavonols and EGCG accumulation in tea leaves. We proposed that catechin biosynthesis in C. sinensis leaves is predominantly regulated by UV through the UVR8-mediated signal transduction pathway to MYB12/MYB4 downstream effectors, to modulate flavonoid accumulation. Our study provides new insights into our understanding of regulatory mechanisms for shading-enhanced tea quality.

Theanine is an important quality parameter referring to tea quality. Applying nitrogen fertilizers is one strategy to improve the level of theanine; however, the effect of plant growth-promoting rhizobacteria on theanine synthesis in tea roots has been less studied. In this study, the bacteria isolated from Qimen County with the maximum potassium (K) solubilization were identified as Bacillus by biochemical and molecular analyses. We show that tartaric and pyruvic acids produced by Bacillus were important components related to K solubilization in vitro. Pot experiments and enzymatic assays in vitro showed that inoculation with Bacillus-secreted organic acids increased the level of available potassium in the soil. The increased K level activated recombinant CsTSI activity (theanine biosynthesis enzyme) and increased ethylamine content (the synthesis precursor of theanine), resulting in promoted theanine synthesis in tea roots. Therefore, our study indicates that Bacillus can be a potential bioinoculant for biofortification of tea.

Tea plant is known to be a hyper-accumulator of fluoride (F). Over-intake of F has been shown to have adverse effects on human health, e.g., dental fluorosis. Thus, understanding the mechanisms fluoride accumulation and developing potential approaches to decrease F uptake in tea plants might be beneficial for human health. In the present study, we found that pretreatment with the anion channel inhibitor NPPB reduced F accumulation in tea plants. Simultaneously, we observed that NPPB triggered Ca²⁺ efflux from mature zone of tea root and significantly increased relative CaM in tea roots. Besides, pretreatment with the Ca²⁺ chelator (EGTA) and CaM antagonists (CPZ and TFP) suppressed NPPB-elevated cytosolic Ca²⁺ fluorescence intensity and CaM concentration in tea roots, respectively. Interestingly, NPPB-inhibited F accumulation was found to be significantly alleviated in tea plants pretreated with either Ca²⁺ chelator (EGTA) or CaM antagonists (CPZ and TFP). In addition, NPPB significantly depolarized membrane potential transiently and we argue that the net Ca²⁺ and H⁺ efflux across the plasma membrane contributed to the restoration of membrane potential. Overall, our results suggest that regulation of Ca²⁺-CaM and plasma membrane potential depolarization are involved in NPPB-inhibited F accumulation in tea plants.