Effects of nitrogen supply on flavonol glycoside biosynthesis and accumulation in tea leaves (Camellia sinensis)

Widely distributed in tea plants, the flavonoid flavonol and its glycosylated derivatives have important roles in determining tea quality. However, the biosynthesis and accumulation of these compounds has not been fully studied, especially in response to nitrogen (N) supply. In the present study, 'Longling 43' potted tea seedlings were subjected to N deficiency (Og/pot), normal N (4g/pot) or excess N (16g/pot). Quantitative analyses using Ultra Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry (UPLC-QqQ-MS/MS) revealed that most flavonol glycosides (e.g., Quercetin-3-glucoside, Kaempferol-3-rgalactoside and Kaempferol-3-glucosyl-rhamnsoyl-glucoside) accumulated to the highest levels when treated with normal N. Results from metabolomics using Gas Chromatography-Mass Spectrometer (GC-MS) suggested that the levels of carbohydrate substrates of flavonol glycosides (e.g., sucrose, sucrose-6-phosphate, D-fructose 1,6-bisphosphate and glucose-1-phosphate) were positively correlated with flavonol glycoside content in response to N availability. Furthermore, Quantitative Real-time PCR analysis of 28 genes confirmed that genes related to flavonoid (e.g., flavonol synthase 1, flavonol 3-O-golnrtosyltransferase) and carbohydrate (e.g., sucrose phosphate synthase, sucrose synthase and glucokinase) metabolism have important roles in regulating the biosynthesis and accumulation of flavonol glycosides. Collectively, our results suggest that normal N levels promote the biosynthesis of flavonol glycosides through gene regulation and the accumulation of substrate carbohydrates, while abnormal N availability has inhibitory effects, especially excess N.