Metabolomics analysis reveals the metabolic and functional roles of flavonoids in light-sensitive tea leaves
© The Author(s). 2017
Received: 14 November 2016
Accepted: 9 March 2017
Published: 20 March 2017
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.
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.
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.
KeywordsCamellia sinensis Flavonoids Light sensitive Metabolism Photo-protection
Flavonoids, the main secondary metabolites in plants, are the most important quality-related compounds, and they comprise 20–40% of the dry matter in young shoots of tea plant . These flavonoid compounds also contribute to the color, taste and aroma of brewed tea. The contents of catechins (flavan-3-ol), flavonols, flavonol glycosides, anthocyanin and leucoanthocyanidin account for the majority of flavonoids in tea plant. The basic molecular structure / carbon skeleton of flavonoids is C6-C3-C6, and they are classified as mono-, di- and tri-hydroxylated based on their hydroxylated B-rings. The synthesis and accumulation of flavonoids occur in response to environmental cues . Numerous studies have shown that the biosynthesis of flavonoids (expression of structural genes, activity of some important enzymes and concentrations of flavonoids) increases under high light intensity [2, 3]. However, sub-groups of flavonoids in tea plants can be differentially affected [4, 5]. The fundamental role of flavonoids to cope with light stress may rely on their many potential functions .
The flavonoids metabolic pathway has been widely accepted to be involved in the regulation mechanisms of plants to various stressful conditions . Flavonoids are the main regulators of plant growth and defense, and they are induced and biosynthesized as the result of a long-term natural selection and acclimatization process [8–12]. The main physiological functions of flavonoids in tea plant are scavenging reactive oxygen species and increasing tolerance to adapt to environmental change, e.g., as antioxidants in photoprotection. The antioxidant activity of flavonoids is attributed to their reaction with free radicals as a hydrogen donor. However, flavonoids with different molecular structures have great differences in their antioxidant activity, which is highly correlated with the substituent positions and the amount of hydroxyl groups on the B-rings. The more hydroxylation of the flavonol, the more hydrogen atoms can be provided for binding free radicals, and the more antioxidant activity; e.g., the antioxidant activity of myricetin is higher than that of kaempferol. Moreover, the tautomeric interconversions of ortho-dihydroxylated B-ring flavonoids make them more efficient at dissipating excess energy  and scavenging reactive oxygen species [6, 14, 15].
Camellia sinensis (L.) O. Kuntze cv. Huangjinya, a natural light-sensitive tea mutant, shows high levels of free amino acids and a low content of polyphenols, which improves the quality of brewed green tea and imparts a higher economic value to the chlorotic varieties than to the non-chlorotic varieties [16–19]. The young leaves of Huangjinya display a yellow color and show normal growth despite being chlorotic under high light conditions . Moreover, Huangjinya shows a totally different metabolic response to light compared to the normal tea species. For example, as the main type of secondary metabolism in tea plant, the biosynthesis of flavonoids increases with increasing temperature and illumination . However, the concentration of most flavonoids decreases greatly in Huangjinya leaves when they are exposed to light. The characteristics of specific genes and chemicals such as total polyphenols, total amino acids, and pigments biosynthesis in Huangjinya has been reported in the previous studies [17, 20]. Although less attention has been paid to the metabolic regulation of flavonoids in this mutant, such an analysis may help to clarify the mechanisms by which flavonoids highly accumulate in tea plants.
Light is one of the most important environmental factors, providing energy and external stimuli for growth and development in plants . Changes in light intensity alter a complex set of molecular events within the chloroplast [5, 22]. However, light energy beyond the acceptable range of the reaction center causes light damage, leading to photoinhibition and decreasing the photochemical and carbon assimilation rates [23, 24]. Thus, photoprotection, either by scavenging harmful reactive intermediates or dissipating excess energy to protect cells from death under light stress, is essential for photosynthesis . Chloroplast-located flavonoids [10, 26], phenylpropanoids  and anthocyanins  are effective sunlight attenuators which play vital roles in protecting chloroplasts when facing with a superabundant radiant energy. The key antioxidant function of flavonoids to excess ultraviolet radiation b (UV-B)/sunlight irradiance have been well documented [29–31]. These studies are further confirmed by the fact that flavonoids with an ortho-dihydroxy structure in the B-ring are preferentially accumulated, compared to flavonoids having a mono-hydroxy substitution [32, 33]. The carotenoid or xanthophyll cycle also participates in protecting plants from high light intensity by thermal dissipation . Moreover, organisms show chlorosis when the carotenoid biosynthesis pathway is disrupted  or is inhibited by norflurazon .
Herein, the hypothesis of the present work was that the metabolism of flavonoids, especially the ‘different branch’, may be regulated by genetic and environmental stimuli in light-sensitive tea leaves and that it is highly correlated with the potential function of flavonoids in tea leaves. An experiment was therefore designed in which samples of tea leaves from a light-sensitive mutant cultivated under two different light conditions were compared using metabolomics analysis.
Plant material and shading treatment
Rooted-cuttings of the natural mutant Camellia sinensis (L.) cv. Huangjinya, which was officially released as a clone in Zhejiang province in 2008 were obtained free of charge from the owner of the mutant Yuyao Deshi Tea Plantation (located in Yuyao county, Zhejiang province). Each of four seedlings were planted in pots (~10 L capacity) filled with commercial growth medium consisting of perlite, vermiculite, and peat at the Tea Research Institute, Chinese Academy of Agricultural Sciences (TRI, CAAS, N 30°10′, E 120°5′) in May of 2013. The pots were placed in open with full sunlight and watered regularly. In March 2014, 60 pots of tea plants with uniform young shoots were selected for the experiment: Half of the pots were treated with high-density polyethylene tape two-pin net (60% sun-shading), and the remaining 30 pots were exposed to full sunlight for ten days. Samples (young shoots with bud and two expanding leaves) were randomly selected, frozen with liquid nitrogen and then stored in a −70 °C ultra-freezer. The sampling was repeated six times each from shaded and unshaded plants.
Ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS) based metabolomics analysis
All 12 obtained samples (six biological repetitions for the shaded and unshaded groups) were used for metabolomics analysis. The metabolites were extracted from young shoots using a mixture of 75% methanol and 1% formic acid as described by Zhang et al. . A 2 μL sample was injected into a UPLC-Q-TOF/MS (Waters, UPLC/Xevo G2-S Q-TOF) and separated with an HSS T3 column as described by Zhang et al. . TransOmics software (Version 1.0; Waters) was applied for data preprocessing. Metabolite peaks were assigned to the accurate mass measurements using online metabolite databases as described in Zhang et al. , and the retention times were compared with those in the published literature . A matrix was exported for further statistical analyses. The unit variance was scaled for further statistical analysis using SIMCA-P (version 13.0, Umetrics, Umea, Sweden). Unsupervised principal component analysis (PCA) and supervised projection to latent structure discriminant analysis (PLS-DA) were carried out to dissect the overall variance of metabolites and the composition differences of the samples, respectively. The combination of p(corr) and variable importance in the projection (VIP) values from the PLS-DA were used as a coefficient for metabolite selection (VIP > 1.0 and |p(corr)| > 0.5). Student’s t-test (P < 0.05) and one-way analysis of variance (ANOVA) using the SPSS (version 15.0, SPSS Inc., Chicago, IL) were performed for statistical analysis.
Quantitative real-time PCR analysis
Total RNA was isolated using a plant RNA extraction kit (Tiangen, China). PrimeScriptTM RT reagent kit (TaKaRa) was applied to synthesize cDNA. Quantitative real-time PCR (qRT-PCR) was performed on the Applied Biosystems 7300 machine (Carlsbad, CA, USA). The primer pairs used for qRT-PCR are shown in Additional file 1: Table S1, and GAPDH was used as the reference gene. For each target gene, triplicate reactions were performed. Relative transcript levels were calculated against that of the internal control GAPDH using the formula 2-ΔΔCt. All data are shown as the mean ± SD (n = 3).
Quantitative determination of chlorophylls, carotenoids, and catechins
To determine the contents of chlorophyll and carotenoids, leaf discs with an area of 86.59 mm2 were removed using a perforated metallic cylinder. Tea infusions were analyzed on a reverse phase high-performance liquid chromatographic (HPLC) system (Waters 2695) coupled to a diode array detector (Waters 2998) as described in . Catechins were also quantified by HPLC, and the separations were performed using a C18 reverse-phase column (250 × 4.6 mm i.d., Phenomenex, Torrance, CA, USA) as described by Wu et al. .
Tissue localization of phenolic compounds
Samples prepared by standard freehand sectioning and stained with 1% (w/v) vanillin-HCl reagent were used to study the localization of phenolic compounds. Photos of the sample sections were taken by a microscope (XQT-2, COIC) before and after staining .
The localization of flavonoids was determined by staining the sections with NaturstoVreagenz A and observing using confocal laser scanning microscopy (CLSM, Zeiss LSM 710 NLO) by the method described in .
Phenotype and content of chlorophyll and carotenoids in Huangjinya leaves
Concentrations of the main catechins and carotenoids in young shoots of chlorotic (full sunlight) and green (shaded) tea plants
Shaded green (G) plants
Chlorotic (C) plants
Catechins (mg/g fresh weight)
0.01 ± 0.00a
0.06 ± 0.01b
0.09 ± 0.03
0.16 ± 0.03
0.26 ± 0.04a
0.08 ± 0.01b
1.73 ± 0.12a
2.36 ± 0.21b
0.08 ± 0.02
0.10 ± 0.01
0.59 ± 0.04a
0.72 ± 0.04b
0.29 ± 0.05
0.24 ± 0.06
9.32 ± 0.51a
7.82 ± 1.1b
5.24 ± 0.23
5.34 ± 0.21
Carotenoids and chlorophyll (μg/cm2 leaf area)
16.44 ± 0.17a
1.21 ± 0.03b
50.90 ± 3.39a
11.15 ± 0.12b
3.45 ± 0.11a
1.78 ± 0.06b
8.08 ± 0.14a
13.02 ± 0.81b
3.22 ± 0.17a
10.12 ± 0.19b
128.84 ± 1.31a
5.83 ± 0.16b
34.46 ± 2.26a
9.01 ± 0.12b
Overview of metabolomic profiling
Significantly changed (VIP > 1 and |p(corr)| > 0.65 from partial least squares discriminant analysis) intercellular metabolites induced by chlorosis
Log2 (C/G) c
Quercetagetin 3′-methylether 7-glucoside
Quercetin 3-rutinoside 7-galactoside
Differences in flavonoid metabolism between chlorotic and normal green leaves
Analysis of metabolomics using UPLC-Q-TOF/MS revealed that the concentrations of flavan-3-ol, flavonols, and flavonol glycosides were greatly affected by shading (Table 2). Fifteen metabolites annotated as part of flavonoid metabolism were decreased in the chlorotic leaves compared with the green leaves, and these metabolites included (−)-epicatechin 8-C-glucoside, 6-galloylglucose, and epicatechin-ent-epicatechin. However, other flavonoids were not significantly reduced (e.g., epicatechin gallate, catechin) or were even increased (e.g., anthocyanidins, rutin, and catechin gallate) in the chlorotic leaves. It is interesting to note that the chlorotic leaves contained a higher level of dihydroxyflavonoids, mainly as quercetin and its glycosides (quercetin 3-galactoside, quercetin 4′,7-diglucoside, quercetin 3-rutinoside 7-galactoside etc.) than the green leaves, whereas the chlorotic leaves possessed a lower level of myricetrin glycoside (myricetin 3-arabinoside, myricetin 3,3′-digalactoside etc.) than did the green leaves (Table 2).
In the validation of quantitative analysis using HPLC, the chlorotic leaves showed lower contents of trihydroxy catechins (particularly the gallocatechin, epigallocatechin gallate) compared to shaded green leaves. However, the contents of dihydroxy catechins (catechin gallate, epicatechin) were significantly higher in the chlorotic leaves than in the shaded green shoots (Table 1).
Furthermore, metabolites involved in the metabolism of benzenoids, phenylpropanoids and polyketides, the branch upstream of catechin and flavonoid metabolism, were significantly downregulated in the chlorotic leaves (Table 2). Such as coumaric acid, caffeic acid, quinic acid, shikimic acid and chicoric acid.
The intracellular localization of flavonoids and Gene expression related to the antioxidant system in chlorotic tea leaves
The expression level of the gene encoding catalase (CAT) was 1.4-fold higher in shaded green leaves than in chlorotic leaves (Fig. 3). By contrast, the gene encoding superoxide dismutase (SOD) were repressed in chlorotic leaves.
The accumulation of small molecules with antioxidative activity plays important roles in mitigating ROS accumulation . These low-molecular-weight antioxidants include sugars, a-tocopherols, glutathione, amino acids (e.g., proline), ascorbic acid, carotenoids and quinic acid derivatives (e.g., chlorogenic acid). In our study, the tea mutant did not develop functional chloroplasts (data not shown) and lacked chlorophyll under light conditions (Table 1), suggesting that the mutant without shading was exposed to light stress. Many phenylpropanoid  and anthocyanin  compounds are effective sunlight attenuators and protect photosynthetic organs (chloroplasts) faced with a superabundance of radiant energy. Flavonoids play a vital role in protecting plants to excess UV-B or sunlight irradiance [29–31]. Flavonoids such as quercetin and dihydroxy catechins also serve multiple functions in higher plants under distinct environmental conditions . Flavonoids with a dihydroxy structure in the B-ring accumulate preferentially in response to high doses of ultraviolet radiation (UV) or sunlight radiation [10, 32, 33]. Therefore, the physiological mechanisms in leaves enduring light stress may be associated with dihydroxy flavonoids. In our study, chlorotic leaves contained higher levels of dihydroxyflavonoids, mainly as catechins (catechin gallate, epicatechin) and quercetin and its glycosides, whereas the levels of myricetrin and kaempferol glycoside were lower than in green leaves (Fig. 5, Tables 1 and 2). The similar results have been observed under sun light and partially shading treatments for normal tea plants , which suggest that the functions of flavonoids to ROS are not only available in chlorotic tea leaves, but also in all normal tea species. We also found that the dihydroxy flavonoids were mainly distributed intracellularly in the leaf epidermal cells and in the light-receiving area, whereas chlorophyll and catechin were distributed throughout the cells of the chlorotic tea mutant.
Generally, plants possess diverse photoprotection mechanisms, including the dissipation of absorbed light energy as thermal energy by non-photochemical quenching (NPQ). Photoinhibitory quenching (qI), quenching due to state transitions (qT), and high-energy-state-quenching (qE) are the three main components of NPQ, among which qI is related to the slow conversion of the xanthophyll cycle pigment zeaxanthin to violaxanthin, while qE depends on the existence of special xanthophyll molecules (i.e., lutein and zeaxanthin) bound to the PSII antenna proteins [41, 42]. We observed a significant increase in the zeaxanthin and carotene contents in the chlorotic leaves, although the total content of carotenoids decreased by 54% (Table 1). The xanthophyll cycle protects plants from high light intensity by converting violaxanthin into zeaxanthin, which participates in the thermal dissipation of excess absorbed light energy . We also noted higher transcript levels of violaxanthin de-epoxidase and zeaxanthin epoxidase (EC: 18.104.22.168, 22.214.171.124, a key gene in xanthophyll cycle that protects plants from high-intensity light) in chlorotic leaves compared with green (shaded) leaves with transcriptomic analysis (data not shown here). Suggesting that the xanthophyll cycle plays an important role in protecting ‘Huangjinya’ from high light intensity. This result is highly consistent with the findings of Li et al. . The tea mutants ‘White leaf No.1’ and ‘Huangjinya’ both showed reduced carotenoid and zeaxanthin content in chlorotic leaves compared with ‘Fuding dabaicha’ [17, 43]. Thus, ‘Huangjinya’ shows a significant difference in carotenoid levels compared with other tea mutants (mainly the albino mutants). We hypothesized that the variation in carotenoid composition and biosynthesis is a specific light protective mechanism in light-sensitive tea mutants. However, serious damage to cell membrane structures in chlorotic leaves suggests that antioxidants and the xanthophyll cycle were insufficient to protect tea plants from photodamage.
In this study, the variation in gene expression patterns and metabolites between chlorotic leaves and normal green leaves (leaves under shading treatment) in the ‘Huangjinya’ tea mutant was uncovered using metabolomics analysis. Our results suggest that high-intensity light stress caused photooxidation in chlorotic leaves and induced multi-operational photoprotection mechanisms for scavenging ROS, including the activation of dihydroxy flavonoids and xanthophyll cycle pathways, which also reversed the photodamage in mutant leaves, helping them endure light stress. Moreover, the differential accumulation of metabolites and differential gene expression suggested that dihydroxy flavonoids have great differences in terms of their functional roles in tea leaves. Especially, the accumulation and location of quercetin and its glycosides (compare with myricetrin and kaempferol glycoside) in chlorotic leaves suggest their great contribution to photo-protection and their unique functional roles in ROS scavenging in tea leaves.
flavonoid 3′, 5′-hydroxylase
- H2O2 :
High-performance liquid chromatography
Projection to latent structure discriminant analysis
Quantitative real-time PCR
Quenching due to state transitions
Reactive oxygen species
Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry
Variable importance in the projection
Thanks are given to Prof. Zongmao Chen and Dr. Xinzhong Zhang for assistance in using the UPLC-Q-TOF/MS and Prof. Kairong Wang for providing the mutant clone.
This work was financially supported by the Chinese Academy of Agricultural Sciences through Agricultural Sciences Innovation Project (CAAS-ASTIP-2016-TRICAAS) and the Ministry of Agriculture of China through the Earmarked Fund for the China Agriculture Research System (CARS 23).
Availability of data and materials
All data generated or analysed during this study are included in this published article and its supplementary information files.
QFZ and MYL gathered samples, participated in the study design, performed sample analysis, interpreted the results and drafted the manuscript. JYR conceived of the study, provided funding, gave guidance on experimental design and revised the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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