Integrated metabolomic and transcriptomic analysis reveals green light-promoted caffeine biosynthesis in Camellia sinensis (tea plant)

Abstract In our previous study, green light (GL) had a great influence on plant seedling developmental processes via phytochromes, which are key photoreceptors of red light (RL). However, the role of GL in regulating plant secondary metabolism is still elusive. In this work, we examined the specific effects of GL treatment in tea plant (Camellia sinensis) via conjoint analysis of metabolome and transcriptome. Compared with RL treatment, GL-specific metabolites were mainly enriched in caffeine (CF)-related secondary metabolites. Transcriptome analysis further demonstrated that GL treatment upregulated the expression levels of caffeine synthase genes (CsTCS1) and CsTCS2, thereby promoting the accumulation of CF content. Subsequently, weighted gene co-expression network analysis identified five GL-responsive transcription factor candidates, namely CsbZIP43, CsbHLH75, CsWRKY40, CsWRKY12, and CsPRE6. Among them, CsbZIP43 positively regulated caffeine biosynthesis by activating CsTCS1 expression. Further experimental validation proved that CsbZIP43 directly binds to the promoter region of CsTCS1 via G-box motif. In addition, we constructed a heterologous expression system in soybean co-expressing both CsTCS1 and CsTCS2 and proved that CsTCS1/TCS2 are sufficient to produce the CF. Overall, the metabolomic and transcriptomic analysis indicated an important role of GL in regulating the CF metabolic pathway in tea plants. Therefore, we proposed that CsbZIP43 positively regulates the CF accumulation under GL conditions through direct activation of CsTCS1, laying the foundation for developing caffeine-enriched functional foods.