Metabolomics analysis reveals the metabolic and functional roles of flavonoids in light-sensitive tea leaves

Abstract

Background: As the predominant secondary metabolic pathway in tea plants, flavonoid biosynthesis increases with increasing temperature and illumination. However, the concentration of most flavonoids decreases greatly in light-sensitive tea leaves when they are exposed to light, which further improves tea quality. To reveal the metabolism and potential functions of flavonoids in tea leaves, a natural light-sensitive tea mutant (Huangjinya) cultivated under different light conditions was subjected to metabolomics analysis.

Results: The results showed that chlorotic tea leaves accumulated large amounts of flavonoids with ortho-dihydroxylated B-rings (e.g., catechin gallate, quercetin and its glycosides etc.), whereas total flavonoids (e.g., myricetrin glycoside, epigallocatechin gallate etc.) were considerably reduced, suggesting that the flavonoid components generated from different metabolic branches played different roles in tea leaves. Furthermore, the intracellular localization of flavonoids and the expression pattern of genes involved in secondary metabolic pathways indicate a potential photoprotective function of dihydroxylated flavonoids in light-sensitive tea leaves.

Conclusions: Our results suggest that reactive oxygen species (ROS) scavenging and the antioxidation effects of flavonoids help chlorotic tea plants survive under high light stress, providing new evidence to clarify the functional roles of flavonoids, which accumulate to high levels in tea plants. Moreover, flavonoids with ortho-dihydroxylated B-rings played a greater role in photo-protection to improve the acclimatization of tea plants.

Keywords: Camellia sinensis; Flavonoids; Light sensitive; Metabolism; Photo-protection.

Fig. 1

Phenotypic characterization of chlorotic and green leaves under full sunlight (left) and shaded (right, 60% light intensity) conditions

Fig. 2

PLS-DA score plots (a) and permutation test (b) of metabolites analyzed by UPLC-Q-TOF/MS in young tea shoots

Fig. 3

Quantitative RT-PCR validation. PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4Cl, 4-coumarate--CoA ligase; CHI, chalcone isomerase; CHS, chalcone synthase; F3′5′H, flavonoid 3′, 5′-hydroxylase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-monooxygenase; FLS, flavonol synthase; DFR, dihydroflavonol-4-reductase; ANR, anthocyanidin reductase; ANS, anthocyanidin synthase; LAR, leucoanthocyanidin reductase; CAT: catalase; SOD, superoxide dismutase

Fig. 4

Histochemical localization of catechin and flavonol in Huangjinya leaves. a, b, c, d show the normal green leaves of Huangjinya shaded with 60% (NG); e, f, g, h show the chlorotic leaves of HJY exposed to 100% sunlight (EM); b and f show staining with vanillin-HCl reagent; the red signal represents phenolic compounds (mainly catechins; d and h were stained with NaturstoVreagenz A (confocal laser scanning microscopy), and the green fluorescence signal represents flavonol

Fig. 5

Schematic representation of phenylpropanoid/flavonoid pathway as affected by the chlorosis mutation. Red and green font indicate up- and down-regulated genes, respectively, in the chlorotic leaves compared to shaded plants. Significantly changed metabolites are shown with a barplot (E and G indicate chlorotic and green leaves, respectively) while compounds that were not identified in this study are shown in gray font. PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4Cl, 4-coumarate--CoA ligase; CHI, chalcone isomerase; CHS, chalcone synthase; F3′5′H, flavonoid 3′, 5′-hydroxylase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-monooxygenase; FLS, flavonol synthase; DFR, dihydroflavonol-4-reductase; ANR, anthocyanidin reductase; ANS, anthocyanidin synthase; LAR, leucoanthocyanidin reductase

Fig. 6

Production and scavenging of free radicals. Red and green font indicate up- and down-regulated genes / metabolites in the chlorotic leaves compared to shaded plants