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 metabolism is a necessary biological process during the planting and production of tea that determines tea quality. There is currently little knowledge about the transcriptional regulation of theanine metabolism in tea plants. In this study, we demonstrated that γ-glutamyl-transpeptidase CsGGT4, as a homologous protein of the theanine hydrolase CsGGT2, exhibited a higher theanine synthesis catalytic efficiency. Homology modeling and molecular docking showed that differential protein structures between CsGGT2 and CsGGT4 implied their different biological functions in tea plants. Theanine content correlated significantly with the expression of CsGGT2, CsGGT4 and the transcription factor CsMYB73 in tea shoots from different seasons. Additionally, CsMYB73 was confirmed to act as a nucleus-localized transcription factor (TF), directly interacts with the CsGGT2 and CsGGT4 promoters, serving as an activator of CsGGT2 and a suppressor of CsGGT4. Consequently, this leads to a negative association with theanine accumulation in tea shoots. Furthermore, the continuous increase in CsMYB73 produced a significantly increase in CsGGT2 expression and inhibited CsGGT4 expression. The present study reveals that the degradation of theanine has been observed to increase, concomitantly with the inhibition of theanine synthesis, resulting in a significant decline in the accumulation of theanine in tea shoots during the process of seasonal greening in ‘Huangkui’ leaves. This study contributes to the broader comprehension of the intricate transcriptional regulatory hierarchy that governs the metabolism of theanine in tea shoots, offering novel approaches for managing tea plantations and enhancing tea quality.