Article pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Comprehensive Dissection of Metabolic Changes in Albino and Green Tea Cultivars Chun-Fang Li,†,‡ Jian-Qiang Ma,† Dan-Juan Huang,† Chun-Lei Ma,† Ji-Qiang Jin,† Ming-Zhe Yao,† and Liang Chen*,† †
Key Laboratory of Tea Biology and Resources Utilization, Ministry of Agriculture, Tea Research Institute of the Chinese Academy of Agricultural Sciences, Hangzhou, Zhejiang 310008, People’s Republic of China ‡ School of Agriculture and Food Science, Zhejiang Agriculture and Forestry University, Lin’an, Hangzhou, Zhejiang 311300, People’s Republic of China S Supporting Information *
ABSTRACT: Albino tea cultivars are special mutants of tea plants with white or yellow leaf color. In this study, three albino tea cultivars, including ‘Anji Baicha’, ‘Huangjinya’, and ‘Baijiguan’, and two green tea cultivars, ‘Longjing 43’ and ‘Fuding Dabaicha’, were applied to metabolite profiling by gas chromatography−mass spectrometry and ultraperformance liquid chromatography− mass spectrometry. Multivariate analyses revealed significantly different metabolite phenotypes in leaves among albino cultivars and green cultivars. The differential metabolite-related pathways included galactose metabolism, tryptophan metabolism, phenylpropanoid biosynthesis, and flavonoid biosynthesis. For the young leaves of albino cultivars, the sugar (sorbitol and erythrose) and amino acid (mainly proline, isoleucine, ornithine, aspartic acid, threonine, and valine) concentrations increased, whereas gallocatechin and epigallocatechin gallate concentrations decreased. These results reveal the divergence in metabolic profiling between tea plant cultivars with different leaf colors. With the development of leaves, the concentrations of flavonoids increased largely in the older leaves of albino cultivars. KEYWORDS: albino tea cultivar, UPLC−Q−TOF/MS, GC × GC−TOF/MS, metabolomics
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INTRODUCTION Tea has been used as a natural medicine in China for centuries and is now one of the most popular beverages in the world. Modern findings have shown that teas contain great amounts of polyphenols, caffeine, theanine, and vitamins, which possess many healthy functions. The chemical composition of tea is influenced by many factors, such as genetic background, growth environment, and horticultural practices. Albino tea cultivars are special mutants of the tea plant with white or yellow leaf color. In comparison to green tea cultivars, the shoots of albino cultivars contained higher levels of amino acids and lower levels of polyphenols and caffeine.1 Thus, green tea processed from the new shoots of albino cultivars shows a strong umami taste and a fresh aroma that has become popular.2 There are two types of albino tea cultivars: one is temperature-sensitive, and another is light-sensitive. ‘Anji Baicha’ (‘Aj’), which is a temperature-sensitive albino tea cultivar, exhibits white shoots when the environmental temperature is below 20 °C in early spring. When the temperature is above 20 °C, the albino phenotype disappears.3 Light-sensitive albino tea cultivars, such as ‘Huangjinya’ (‘Hj’) and ‘Baijiguan’ (‘Bj’), exhibit yellow shoot under strong sunlight conditions in summer. When the sunlight intensity is reduced, the shoots turn green.4 The albino pheromone was mainly caused by the inhibition of the etioplast−chloroplast transition and the decreased content of chlorophyll a, chlorophyll b, and carotenoids.3,5,6 The new shoots of albino cultivars contain high levels of amino acids and moderate levels of polyphenols, which convey the high sensory quality of green tea. However, the © XXXX American Chemical Society
potential metabolomic mechanisms in albino tea cultivars have not yet been studied. A metabolomic analysis was performed in this study to give a broad overview of the metabolic differences between albino tea cultivars and green tea cultivars. The comprehensive metabolite profiles, including primary and secondary metabolites, and the albino tea cultivars, including ‘Aj’, ‘Hj’, and ‘Bj’, and green tea cultivars, including ‘Lonjing 43’ (‘Lj’) and ‘Fuding Dabaicha’ (‘Fd’), were used to investigate the metabolic changes between albino and green tea cultivars. Furthermore, because metabolomics are also closely related to the development of leaves, the detailed metabolic shifts in different stages of leaves were also analyzed.
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MATERIALS AND METHODS
Plant Materials. Four-year-old plants of Camellia sinensis (L.) O. Kuntze cv. ‘Aj’, ‘Hj’, and ‘Bj’ and normal green tea cultivars ‘Lj’ and ‘Fd’ were grown at the China National Germplasm Hangzhou Tea Repository (CNGHTR) of the Tea Research Institute of the Chinese Academy of Agricultural Sciences (TRICAAS). The third and fifth leaves in the ‘Aj’ shoots were collected on May 20, 2015, which were in white color; the third and fifth leaves in ‘Hj’ and ‘Bj’ were collected on June 10, 2015, which were in yellow color; and the third and fifth leaves of normal green cultivars ‘Lj’ and ‘Fd’ were harvested on May 25, 2015, which were in green color (Figure 1). A leaf sample Received: Revised: Accepted: Published: A
December 1, 2017 January 30, 2018 February 3, 2018 February 3, 2018 DOI: 10.1021/acs.jafc.7b05623 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
U.S.A.), kept at 40 °C. Mobile phase A was water with 0.1% formic acid, and mobile phase B was acetonitrile with 0.1% formic acid. The flow was 0.4 mL/min, and the gradient profile was from 0 to 3 min, 5% B; from 3 to 13 min, linear gradient to 95% B; and from 13 to 15 min, wash at 95% B. Mass spectra were acquired using electrospray ionization over the range of m/z 119−966. The drying gas temperature was 350 °C, and the cone gas flow was 11 L/h. Metabolomic analysis based on GC × GC−TOF/MS was performed following the principle previously described by Lisec et al.,8 with some modifications. Plant samples (60 mg) were added to 360 μL of cold methanol containing 20 μL of internal standard (0.03 mg mL−1 L-2-Cl-phenylalanine) and ground at 60 Hz for 2 min in a tissuelyser. The metabolites were extracted for 30 min in an ultrasonic bath, followed by the addition of 200 μL of chloroform and 400 μL of ddH2O, and then extracted for 30 min in an ultrasonic bath. The metabolites were centrifuged at 15000g for 10 min at 4 °C, and the upper phases of the aqueous methanol extract were collected. The supernatant (700 μL) was transferred to a new 2 mL Eppendorf tube and blow-dried in a vacuum concentrator with moderate nitrogen gas without heating. The dried samples were derivatized according to the previously described method.9 A 1 μL aliquot of derivatized solution was injected into the Agilent 7890A-5975C gas chromatography−mass spectrometry (GC−MS) system (Agilent, Santa Clara, CA, U.S.A.) in splitless mode. The metabolites were separated on a nonpolar DB-5 capillary column (30 m × 250 μm inner diameter, J&W Scientific, Folsom, CA, U.S.A.). The carrier gas was high-purity helium, and the flow rate was 1.0 mL/min. The GC programming began at 60 °C, which rose by 8 °C/min to 125 °C, then 4 °C/min to 210 °C, followed by 5 °C/min to 270 °C, 10 °C/min to 305 °C, and a final 3 min maintenance at 305 °C. The electron impact (EI) ion source was maintained at 260 °C with a filament bias of −70 V. A full scan was proceeded at the rate of 20 spectra/s in the MS setting mode (m/z 50−600). Aliquots of all samples were mixed as quality control (QC) samples and analyzed with the same method as the analytical samples. The QCs were injected at regular intervals (every 12 samples) throughout the analytical run to assess repeatability. Data Processing and Multivariate Data Analysis. Raw data acquired from the UPLC−Q−TOF/MS analysis were transformed into a general format using Agilent MassHunter Qualitative. Then, the general format data were imported into XCMS (http://metlin.scripps. edu/download/) to produce a peak table that included information on the MS intensity, mass-to-charge ratio (m/z), and retention time of each metabolite in the sample (Table S1 of the Supporting Information). The MS data from GC × GC−TOF/MS were analyzed using ChromaTOF software (version 4.34, LECO, St. Joseph, MI, U.S.A.). First, after alignment with the Statistic Compare component, the CSV file was obtained with three-dimensional data sets, including peak intensities, retention time−m/z, and sample information (Table S2 of the Supporting Information). In total, there were 1098 detectable peaks in the GC−MS data. The internal standard was used for data quality and reproducibility control. Pseudo-positive peaks, such as peaks caused by noise, column bleed, and the BSTFA derivatization procedure, were removed from the data set, and peaks from the same metabolite were combined. In total, there were 328 detectable metabolites of samples in the GC−MS data. Finally, the data set was normalized using the sum intensity of the peaks in each sample. Multivariate data analyses were processed by inputting the data sets of UPLC−Q−TOF/MS and GC × GC−TOF/MS into the SIMCA-P + 14.0 software package (Umetrics, Umea, Sweden). Data were Pareto-scaled and visualized by plotting the principal component scores, and each coordinate represented an individual biological sample. Orthogonal partial least squares discriminant analysis (OPLSDA) was performed to classify and discriminate the various cultivars at different leaf stages. Cross-validation of the developed models was performed on the basis of the default software options and explained by variation (R2X and R2Y) and predictive ability [Q(cum)2]. Overfitting of the OPLS-DA models was checked using a permutation test. These analyses employed a default 7-fold internal cross validation
Figure 1. Leaves and chlorophyll concentrations of albino tea cultivars and green cultivars: (A) leaf phenotypes of third and fifth leaves of albino tea cultivars and green cultivars. ‘Anji Baicha’ (‘Aj’), ‘Baijiguan’ (‘Bj’), ‘Huangjinya’ (‘Hj’), ‘Fuding Dabaicha’ (‘Fd’), and ‘Lonjing 43’ (‘Lj’) and (B) chlorophyll concentrations. Student’s t test was employed to compare chlorophyll concentrations in leaves of albino tea cultivars (‘Aj’, ‘Bj’, and ‘Hj’) and green cultivars (‘Fd’ and ‘Lj’). All measurements were analyzed in three independent biological replicates. “∗” indicates a significant difference (p < 0.05), and “∗∗” indicates a significant difference (p < 0.01). consisted of pooled leaves (3−4 per plant) collected from four randomly selected tea plants. There were six biological replicates for each chemical assay. All excised samples were immediately frozen in liquid nitrogen and stored at −80 °C before analysis. All of the albino tea cultivars used in this study were with the albino phenotype in the third and fifth leaves. For ‘Aj’, not all of the leaves were completely with the albino phenotype. For example, the leaves were yellow−green color in the one leaf and a bud stage and green in the mature leaves. The metabolic profiling was also changed with the development of the leaf. In this study, we designed to exclude the differential metabolites that were caused with the development by comparing the metabolic profilings of the third and fifth leaves. Chlorophyll a and Chlorophyll b Content Measurement. The chlorophyll a and chlorophyll b contents were measured according to the method of Arnon.7 First, chlorophyll a and chlorophyll b were extracted with 80% acetone from 100 mg of fresh leaf samples in three independent biological replicates. Second, the extracts were measured using a spectrophotometer at 645 and 663 nm. Student’s t test was used for comparisons of chlorophyll a and chlorophyll b in the leaves of ‘Aj’, ‘Bj’, and ‘Hj’ and those in ‘Lj’ and ‘Fd’. Metabolomic Analyses Based on Ultraperformance Liquid Chromatography Coupled to a Hybrid Quadrupole Orthogonal Time-of-Flight Mass Spectrometer (UPLC−Q−TOF/MS) and Two-Dimensional Gas Chromatography Coupled to Time-ofFlight Mass Spectrometry (GC × GC−TOF/MS). The leaves were subjected to metabolomic analyses based on UPLC−Q−TOF/MS and GC × GC−TOF/MS. For metabolomic analysis based on UPLC−Q− TOF/MS, the metabolites in 50 mg tea plant samples were added to 1 mL of 70% methanol and 20 μL of internal standard (0.03 mg mL−1 L2-Cl-phenylalanine) and then ground at 60 Hz for 2 min. Then, the metabolites were extracted for 30 min in an ultrasonic bath and centrifuged at 15000g for 10 min, and the upper phase of the aqueous methanol extract was collected. The extract was filtered through a 0.22 μm polytetrafluoroethylene (PTFE) filter, and 3 μL of the extract was injected into Agilent 1290 Infinity ultrahigh-performance liquid chromatography (UHPLC). Metabolites were separated on a Waters Acquity HSS T3 column (2.1 μm, 100 × 2.1 mm, Milford, MA, B
DOI: 10.1021/acs.jafc.7b05623 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry from which the R2 and Q2 values reflect the goodness of fit and predictability. Then, a supervised orthogonal projection to the latent structure discriminant analysis (OPLS-DA) was used to extract the maximum amount of information from the data set and to isolate the metabolites responsible for differences among the different cultivars at different leaf stages. The metabolites for which the variable influence on projection (VIP) value was greater than 1 contributed greatly to the separation of sample groups in the OPLS-DA models.10 The concentrations of metabolites were analyzed with Student’s t test (parametric tests); the metabolites with a p value less than 0.05 were differential metabolites. Differential metabolite GC−MS data were annotated with the National Institute of Standards and Technology (NIST) 05 standard mass spectral database and Fiehn database and then manually checked for similarity greater than 70%. If the annotation results of the two databases were not consistent, a high degree of similarity for the metabolite name was chosen. Differential metabolites in the high performance liquid chromatography−mass spectrometry (HPLC−MS) data were annotated with METLIN (https://metlin.scripps.edu/) and the Human Metabolome Database (http://www.hmdb.ca/). Some differential metabolite annotation results were validated by reference standards (Figure S1 of the Supporting Information). Finally, the differential metabolites were mapped to metabolic pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to interpret the biological significance associated with color changes in leaves. To reduce the false discovery rate, enrichment p values were computed by hypergeometric distribution and p values less than 0.01 were selected.
of the Supporting Information) and GC−MS (Table S2 of the Supporting Information) were analyzed separately through principal component analysis (PCA) (Figure 2). The PCA
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RESULTS Phenotypic Characterization of Three Albino Cultivars and Two Green Cultivars. In early spring, at low temperatures and low light intensities, the new fresh shoots of all three albino cultivars were yellowish or yellow−green. When the shoots grow to the stage with three or five fully opened leaves in late spring, the leaf color of ‘Aj’ turned white and the leaf color of ‘Bj’ and ‘Hj’ remained yellow, while the leaves of the normal green cultivars ‘Fd’ and ‘Lj’ were dark green (Figure 1A). The leaf color of ‘Aj’ gradually turned normal green over the following 2 weeks.2,6 The leaves of the other albino cultivars ‘Bj’ and ‘Hj’ remained yellowish. The albino cultivars are advantageous, because they can be processed into a unique green tea with a special color and brisk taste, using the yellowish or white fresh leaves. The concentrations of chlorophyll a, chlorophyll b, and chlorophyll a + b in the third and fifth leaves of ‘Aj’, ‘Bj’, and ‘Hj’ were significantly lower than that in the corresponding leaves of ‘Fd’ and ‘Lj’ (p < 0.05; Student’s t test) (Figure 1B). The reduction of chlorophyll a and chlorophyll b contents might be the main cause of the color difference between albino cultivars and green cultivars. Previous research revealed that the albino phenotype of ‘Aj’ was closely related to tea plant chlorophyll a and chlorophyll b biosynthesis, which is inhibited at low temperatures and recovers as the temperature increases.11 Reduced chlorophyll a and chlorophyll b synthesis induces a complex perturbation of metabolite accumulation in the tea plant; that is, more amino acids and fewer polyphenols are found in albino leaves.1,12 Metabolic Profiling Differences between Albino Cultivars and Normal Green Cultivars. Metabolite profiling of 60 tea plant leaf samples from three albino cultivars (‘Aj’, ‘Bj’, and ‘Hj’) and two normal green cultivars (‘Fd’ and ‘Lj’) was performed using HPLC−MS and GC−MS, respectively. The total metabolites in the third and fifth leaves from albino cultivars and green cultivars obtained by HPLC−MS (Table S1
Figure 2. PCA score plots derived from (A) HPLC−MS data and (B) GC−MS data. Data were derived from six independent biological replicates of third and fifth leaves of albino cultivars and green cultivars.
results showed that the metabolites obtained by HPLC−MS varied in the cultivars, with the formation of distinct clusters, and in particular, the metabolites in the albino cultivars and green cultivars clustered into two distinct parts (Figure 2A). However, the PCA results obtained by GC−MS were not clearly clustered according to different cultivars but rather showed overlapping of some samples (Figure 2B), which might be because metabolites detected by GC−MS differed less among cultivars. To investigate metabolic differences between albino cultivars (‘Aj’, ‘Bj’, and ‘Hj’) and normal green cultivars (‘Fd’ and ‘Lj’), supervised orthogonal projection to latent structure discriminant analysis (OPLS-DA) was used to classify and discriminate the leaves of each albino cultivar and green cultivar at the same leaf stage based on the HPLC−MS data (Table S1 of the Supporting Information) and GC−MS (Table S2 of the Supporting Information) data, demonstrating the greatest differences between the albino cultivars and normal green cultivars (Figures S1−S24 of the Supporting Information). Comparing Metabolomic Analyses of ‘Aj’ and ‘Lj’ Leaves at Different Stages. On the basis of the OPLS-DA models, key differential metabolites that contributed to the classification of ‘Aj’ and ‘Lj’ were identified (Tables S3 and S4 of the Supporting Information). These differential metabolites were mainly carbohydrates, amino acids, organic acids, flavonols, and their glycosides. The content of carbohydrates related to carbon metabolism, such as maltose, was lower in the third leaves of ‘Aj’ than in that of ‘Lj’, while erythrose was C
DOI: 10.1021/acs.jafc.7b05623 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 3. Differential metabolites from GC−MS were enriched to distinct KEGG pathways. Differential metabolites in third or fifth leaves between albino tea cultivars (‘Aj’, ‘Bj’, and ‘Hj’) and green cultivars (‘Lj’ and ‘Fd’) were mapped to distinct metabolic pathways. Enrichment p values were computed from a hypergeometric distribution. A p value cutoff of 0.01 was selected to reduce the false discovery rate.
higher in ‘Aj’ than in ‘Lj’ (Figure 5 and Table S3 of the Supporting Information). For the fifth leaves, erythrose was higher in ‘Aj’ than in ‘Lj’, while lyxose and lactulose were lower in ‘Aj’ than in ‘Lj’. For the free amino acids, the contents of glutamine and leucine were higher in the third leaves of ‘Aj’ than in the third leaves of ‘Lj’. For the fifth leaves, the content of homoserine was higher in ‘Aj’ than in ‘Lj’. For the secondary metabolites, the contents of flavan-3-ols and flavonols and their glycosides showed significant differences in ‘Aj’ compared to ‘Lj’ (Figure 6 and Tables S3 and S4 of the Supporting Information). Flavan-3-ols, including galloylcatechin (GC), catechin (C), epigallocatechin gallate (EGCG), and epicatechin (EC), were lower in the third leaves of ‘Aj’ than in ‘Lj’ (Figure 6 and Tables S3 and S4 of the Supporting Information). The contents of quercetin derivatives, such as quercetin 7-xyloside and quercetin 3-(6′′′-p-coumarylglucosyl)rhamnoside-7-glucoside, were higher in the third leaves of ‘Aj’ than in ‘Lj’, while the content of quercetin 3-rhamnosylrhamnosyl-glucoside was lower in the third leaves of ‘Aj’ than in ‘Lj’. For the contents of cyanidin derivatives, they were higher in the third leaves of ‘Aj’ than those in ‘Lj’. For the fifth leaves, the content of derivatives of peonidin, kaempferol, cyanidin, delphinidin, EGCG, and epicatechin gallate (ECG) were lower in ‘Aj’ than in ‘Lj’ (Figure 6 and Tables S3 and S4 of the Supporting Information). Metabolite differences in the third leaves from ‘Aj’ and ‘Lj’ were mainly enriched in metabolic pathways, including flavonoid biosynthesis, biosynthesis of alkaloids derived from histidine and purine, aminoacyl-tRNA biosynthesis, and valine, leucine, and isoleucine biosynthesis. Metabolite differences between the fifth leaves of ‘Aj’ and ‘Lj’ were mainly enriched in metabolic pathways, including sulfur metabolism, biosynthesis of phenylpropanoids, biosynthesis of alkaloids derived from histidine and purine, and the citrate cycle [tricarboxylic acid (TCA) cycle] (Figures 3 and 4). Comparing Metabolomic Analyses of ‘Aj’ and ‘Fd’ Leaves at Different Stages. For free amino acids, the contents of glutamine, proline, valine, and isoleucine were
higher in the third leaves of ‘Aj’ than in ‘Fd’ (Figure 5 and Table S4 of the Supporting Information). For the fifth leaves, the content of galactose was lower in ‘Aj’ than in ‘Fd’ (Figure 5 and Table S3 of the Supporting Information). In the secondary metabolism of the third leaves, the contents of flavan-3-ols and flavonols and their glycosides showed significant differences (VIP > 1, and p < 0.05) in ‘Aj’ compared to ‘Fd’ (Figure 6 and Table S4 of the Supporting Information). Flavan-3-ols, such as ECG, was lower in ‘Aj’ than in ‘Fd’, while the content of kaempferol was higher in ‘Aj’ than in ‘Fd’. For the fifth leaves, the content of ECG was lower in ‘Aj’ than in ‘Fd’. Metabolite differences between the third leaves of ‘Aj’ and ‘Fd’ were mainly enriched in metabolic pathways, including tryptophan metabolism, aminoacyl-tRNA biosynthesis, terpenoid backbone biosynthesis, and zeatin biosynthesis. Metabolite differences in the fifth leaves of ‘Aj’ and ‘Fd’ were mainly enriched in metabolic pathways, including alanine, aspartate, and glutamate metabolism and terpenoid backbone biosynthesis. Comparing Metabolomic Analyses of ‘Bj’ and ‘Lj’ Leaves at Different Stages. For the primary metabolism of the third leaves of ‘Bj’ and ‘Lj’, the contents of lactulose, sorbitol, and erythrose related to carbon metabolism were higher in ‘Bj’ than in ‘Lj’ (Figure 5 and Table S3 of the Supporting Information). For the fifth leaves, the content of sorbitol was higher in ‘Bj’ than in ‘Lj’, while lyxose, maltose, and glucose were lower in ‘Bj’ than in ‘Lj’. For the free amino acids in the third leaves, the contents of ornithine and homoserine were higher in ‘Bj’ than in ‘Lj’, while the contents of glutamic acid and lysine were lower in ‘Bj’ than in ‘Lj’. For the fifth leaves, the content of lysine was lower in ‘Bj’ than in ‘Lj’. In the secondary metabolism of third leaves, the contents of flavan-3-ols and flavonols and their glycosides showed significant differences in ‘Bj’ compared to ‘Lj’ (Figure 6 and Table S4 of the Supporting Information). Flavan-3-ols, such as EGCG, had lower contents in ‘Bj’ than in ‘Lj’ (Figure 6 and Table S4 of the Supporting Information). For the fifth leaves, D
DOI: 10.1021/acs.jafc.7b05623 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 4. Differential metabolites from HPLC−MS were enriched in distinct KEGG pathways. Differential metabolites in third or fifth leaves between albino tea cultivars (‘Aj’, ‘Bj’, and ‘Hj’) and green cultivars (‘Lj’ and ‘Fd’) were mapped to distinct metabolic pathways. Enrichment p values were computed from a hypergeometric distribution. A p value cutoff of 0.01 was selected to reduce the false discovery rate.
and Table S3 of the Supporting Information). For the fifth leaves, the content of lactulose was higher in ‘Bj’ than in ‘Fd’, while glucose was lower in ‘Bj’ than in ‘Fd’. For the free amino acids in the third leaves, the content of lysine was lower in ‘Bj’ than in ‘Fd’, while the contents of asparagine and ornithine were higher in ‘Bj’ than in ‘Fd’. For the fifth leaves, the content of lysine was lower in ‘Bj’ than in ‘Fd’. In the secondary metabolism of the fifth leaves, the contents of flavan-3-ols and flavonols and their glycosides showed significant differences in ‘Bj’ compared to ‘Fd’, For example, the contents of EGC, C, EGCG, GC, and kaempferol were higher in ‘Bj’ than in ‘Fd’ (Figure 6 and Tables S3 and S4 of the Supporting Information). Metabolite differences in the third leaves of ‘Bj’ from the third leaves of ‘Fd’ were mainly enriched in several metabolic pathways, including biosynthesis of phenylpropanoids and caffeine metabolism. Metabolite differences in the fifth leaves of ‘Bj’ and ‘Fd’ were mainly enriched in metabolic pathways,
(−)-epigallocatechin (EGC), C, EC, EGCG, and GC had higher contents in ‘Bj’ than in ‘Lj’, while the contents of xanthine and proanthocyanidin were lower in ‘Bj’ than in ‘Lj’ (Figure 6 and Table S4 of the Supporting Information). Metabolite differences in the third leaves of ‘Bj’ and ‘Lj’ were mainly enriched in several metabolic pathways, including caffeine metabolism, cysteine and methionine metabolism, and sulfur metabolism. Metabolite differences in the fifth leaves of ‘Bj’ from the fifth leaves of ‘Lj’ were mainly enriched in metabolic pathways, including biosynthesis of phenylpropanoids, biosynthesis of alkaloids derived from histidine and purine, citrate cycle (TCA cycle), flavone and flavonol biosynthesis, flavonoid biosynthesis, oxidative phosphorylation, and galactose metabolism. Comparing Metabolomic Analyses of ‘Bj’ and ‘Fd’ Leaves at Different Stages. For the primary metabolism of the third leaves of ‘Bj’ and ‘Fd’, the content of lactulose related to carbon metabolism was lower in ‘Bj’ than in ‘Fd’ (Figure 6 E
DOI: 10.1021/acs.jafc.7b05623 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 5. Heat map of primary metabolite concentrations displayed on a metabolic pathway representation for different leaf stages of various cultivars. Log10 ratios of concentrations in each cultivar at each stage are given by shades of red or green according to the scale bar. Data represent mean values of six biological replicates for each cultivar and stage. Abbreviations not already defined are as follows: gly, glycine; val, valine; leu, leucine; ile, isoleucine; thr, threonine; lys, lysine; asp, aspartic acid; orn, ornithine; and glu, glutamic acid.
Figure 6. Heat map of secondary metabolite concentrations displayed on a metabolic pathway representation at different stages for various cultivars. Log10 ratios of concentrations in each cultivar at each stage are given by shades of red or green according to the scale bar. Data represent mean values of six biological replicates for each cultivar and stage.
including biosynthesis of plant hormones, flavonoid biosyn-
Comparing Metabolomic Analyses of ‘Hj’ and ‘Lj’ Leaves at Different Stages. For the primary metabolism of the third leaves of ‘Hj’ and ‘Lj’, the contents of sorbitol, galactose, lactulose, and allose related to carbon metabolism were higher in ‘Hj’ than in ‘Lj’, while maltose was lower in ‘Hj’
thesis, pentose phosphate pathway, biosynthesis of alkaloids derived from histidine and purine, biosynthesis of phenylpropanoids, and the citrate cycle (TCA cycle). F
DOI: 10.1021/acs.jafc.7b05623 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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than in ‘Lj’ (Figure 5 and Tables S3 and S4 of the Supporting Information). For the fifth leaves, the contents of galactose and lactulose were lower in ‘Hj’ than in ‘Lj’. For the free amino acids in the third leaves, the contents of asparagine, ornithine, and leucine were higher in ‘Hj’ than in ‘Lj’, while the content of lysine was lower in ‘Hj’ than in ‘Lj’. For the fifth leaves, the contents of leucine and ornithine were higher in ‘Hj’ than in ‘Lj’. In the secondary metabolism of third leaves, the contents of flavan-3-ols and flavonols and their glycosides showed significant differences in ‘Hj’ compared to ‘Lj’ (Figure 6 and Table S4 of the Supporting Information). Flavan-3-ols, such as EGC, showed a higher content in ‘Hj’ than in ‘Lj’, while the contents of CG and EGCG were lower in ‘Hj’ than in ‘Lj’ (Figure 6 and Table S4 of the Supporting Information). For the fifth leaves, the contents of EGC and EGCG were higher in ‘Hj’ than in ‘Lj’, while quinic acid and succinyl-CoA were lower in ‘Hj’ than in ‘Lj’. Metabolite differences in the third leaves of ‘Hj’ from the third leaves of ‘Lj’ were mainly enriched in metabolic pathways, including biosynthesis of phenylpropanoids, phenylalanine metabolism, phenylpropanoid biosynthesis, valine, leucine, and isoleucine degradation, and biosynthesis of alkaloids derived from histidine and purine. Metabolite differences in the fifth leaves of ‘Hj’ from the fifth leaves of ‘Lj’ were mainly enriched in metabolic pathways, including galactose metabolism, phenylpropanoid biosynthesis, biosynthesis of phenylpropanoids, biosynthesis of alkaloids derived from histidine and purine, the citrate cycle (TCA cycle), and biosynthesis of plant hormones. Comparing Metabolomic Analyses of ‘Hj’ and ‘Fd’ Leaves at Different Stages. For the primary metabolism of the third leaves of ‘Hj’ and ‘Fd’, the contents of allose and fructose-6-phosphate related to carbon metabolism were higher in ‘Hj’ than in ‘Fd’, while panose and lactulose were lower in ‘Hj’ than in ‘Fd’ (Figure 2 and Table S3 of the Supporting Information). For the fifth leaves, the contents of sucrose, allose, galactose, and lactulose were higher in ‘Hj’ than in ‘Fd’, while the contents of erythrose, glucoheptose, and panose were lower in ‘Hj’ than in ‘Fd’. For the free amino acids in the third leaves, the contents of asparagine, proline, ornithine, and leucine were higher in ‘Hj’ than in ‘Fd’, while the content of lysine was lower in ‘Hj’ than in ‘Fd’. For the fifth leaves, the content of leucine was higher in ‘Hj’ than in ‘Fd’, while the contents of lysine, homoserine, and glutamic acid were lower in ‘Hj’ than in ‘Fd’. In the secondary metabolism of the third leaves, the contents of flavan-3-ols and flavonols and their glycosides showed significant differences in ‘Hj’ compared to ‘Fd’ (Figure 6 and Table S4 of the Supporting Information). Flavan-3-ols, such as C, EC, and GC, showed higher contents in ‘Hj’ than in ‘Fd’, while the contents of ECG and quercetin were lower in ‘Hj’ than in ‘Fd’ (Figure 6 and Tables S3 and S4 of the Supporting Information). For the fifth leaves, the content of GC was higher in ‘Hj’ than in ‘Fd’, while C, theophylline, hematoporphyrin IX, and proanthocyanidin were lower in ‘Hj’ than in ‘Fd’. Metabolite differences in the third leaves of ‘Hj’ and ‘Fd’ were mainly enriched in metabolic pathways, including biosynthesis of phenylpropanoids and aminoacyl-tRNA biosynthesis. Metabolite differences in the fifth leaves of ‘Hj’ and ‘Fd’ were mainly enriched in several metabolic pathways, including sulfur metabolism, biosynthesis of alkaloids derived from histidine and purine, and zeatin biosynthesis.
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DISCUSSION
Tea plant germplasms contain many genetically different cultivars;13,14 in particular, cultivars with unique leaf color have been developed during tea plant breeding and can be processed into unique, high-quality green tea. ‘Aj’ is an albino tea cultivar, with yellow or white young shoots when the environmental temperature is below 20 °C in early spring.2,3 Previous researchers found that the development of chloroplasts and accumulation of chlorophyll a and chlorophyll b were blocked in ‘Aj’ at the albinistic stage.5,12,15 Tea plant cultivars that exhibit unique colors have drawn increasing attention as a result of differences in the chemical composition of their leaves that can be processed into characteristic teas. In this research, we analyzed three albino tea cultivars (‘Aj’, ‘Bj’, and ‘Hj’) that present white or yellow leaves and two green tea cultivars (‘Lj’ and ‘Fd’) that present green leaves (Figure 1A). Analysis of chlorophyll a and chlorophyll b concentrations in the leaves of albino tea cultivars and green cultivars showed that the albino tea cultivars partially lack chlorophyll a and chlorophyll b (Figure 1B). This result was correlated with previous research, which reported that the arrested development of grana in the chloroplasts blocks chlorophyll a and chlorophyll b biosynthesis in albino cultivars.3,16 Previous researchers also found that changes in the expression of genes involved in chlorophyll a and chlorophyll b biosynthesis might result in the albino phenomenon in tea plant.11,15 Mg-chelatase, which consists of three subunits designated ChlD, ChlH, and ChlI, plays key roles in chlorophyll a and chlorophyll b biosynthesis. The genes encoding these subunits were highly expressed in albino tea plant, disrupt the balanced proportion, finally disturb the correct assembly of the enzyme complex, and reduced the Mg-chelatase activity.11,15 Along with lower chlorophyll a and chlorophyll b contents in albino tea cultivars, the chemical composition also changes in the leaf, altering the sensory quality and healthy effects of tea.2 In our study, differential metabolites between albino and green cultivars were identified on the basis of OPLS-DA models. These metabolites were enriched in some pathways, many of which were amino acid metabolic pathways, such as tryptophan metabolism, valine, leucine, and isoleucine biosynthesis, valine, leucine, and isoleucine degradation, and alanine, aspartate, and glutamate metabolism; these were strongly correlated with the significantly different concentrations of amino acids observed in albino cultivars and green cultivars (Figure 5). Previous studies found that decreased contents of chlorophyll a and chlorophyll b were correlated with an increase in the contents of total amino acids in albino tea plant cultivars.1,17 In accordance with this result, we found that most free amino acids were present in higher concentrations in albino tea cultivars than green tea cultivars. Our analysis also revealed that the concentrations of many carbohydrates, such as maltose, erythrose, lyxose, panose, lactulose, sorbitol, sucrose, glucose, allose, and galactose, significantly differed between albino tea cultivars and green cultivars (Figure 5). Through photosynthesis, plants use sunlight energy to convert carbon dioxide into organic compounds. Previous research found that proteins involved in carbon fixation varied significantly with leaf color in ‘Aj’.18 In our research, we found that the concentrations of carbohydrates in the albino tea cultivars significantly differed from green cultivars, possibly because carbon fixation was altered between the albino cultivars and green cultivars. G
DOI: 10.1021/acs.jafc.7b05623 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry The TCA cycle was significantly different in the fifth leaves of albino cultivars (‘Aj’, ‘Bj’, and ‘Hj’) compared to the normal green cultivars (‘Lj’ or ‘Fd’). The TCA cycle plays two critical functions in metabolism. First, under aerobic conditions, the TCA cycle is responsible for the total oxidation of acetyl-CoA, which is derived mainly from pyruvate produced by glycolysis. In addition, TCA cycle intermediates are involved in amino acid carbon skeleton biosynthesis.19 The significant difference in the TCA cycle between the albino cultivars and green cultivars might influence the metabolism and final content of amino acids. Previous research found that the plucking period influenced the amino acid contents in green tea; the levels of major free amino acids decreased at later plucking periods in spring.20 In our research, we also found that some free amino acids, such as isoleucine, threonine, and valine, were maintained at higher levels in the third leaves than in the fifth leaves. Flavonoids, which are synthesized in the general phenylpropanoid pathway, are carbon-based secondary metabolites; carbohydrates, such as glucose, fructose, and sucrose, can greatly stimulate flavonoid formation.21 Sugars can act as signaling molecules and can regulate many types of gene expression, including genes involved in the flavonoid biosynthesis pathway. 22,23 In our research, using differential metabolites mapped to KEGG pathways, we found that pathways, including flavonoid biosynthesis, phenylpropanoid biosynthesis, and flavone and flavonol biosynthesis, were significantly different between albino cultivars and green cultivars. The contents of flavonoids changed significantly in albino cultivars compared to green cultivars, as also found in previous research.1 With the growth of leaves, the concentrations of flavonoids, such as GC, EGC, and EGCG, were increased significantly in the fifth leaves of albino cultivars. The present results are in line with previous reports showing that the contents of EC, EGC, EGCG, GC, and CG increased in the young shoot at the later plucking periods in the spring season.20
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Funding
The work was supported by the Ministry of Agriculture of China through the Earmarked Fund for China Agriculture Research System (CARS-019), the Chinese Academy of Agricultural Sciences through the Agricultural Sciences Innovation Project (CAAS-ASTIP-2017-TRICAAS), the National Natural Science Foundation of China (31400581 and 31500568), and the Research Developmental Fund of Zhejiang Agriculture and Forestry University (2016FR032). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Shanghai Boyuan Biotechnology Co., Ltd. for assistance with GC−MS analysis.
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ABBREVIATIONS USED ‘Aj’, ‘Anji Baicha’; ‘Hj’, ‘Huangjinya’; ‘Bj’, ‘Baijiguan’; ‘Lj’, ‘Longjing 43’; ‘Fd’, ‘Fuding Dabaicha’; GC−MS, gas chromatography−mass spectrometry; UPLC−MS, ultraperformance liquid chromatography−mass spectrometry; QC, quality control; OPLS-DA, orthogonal partial least squares discriminant analysis; VIP, variable influence on projection; KEGG, Kyoto Encyclopedia of Genes and Genomes; PCA, principal component analysis; GC, galloylcatechin; C, catechin; EGCG, epigallocatechin gallate; EC, epicatechin; gly, glycine; val, valine; leu, leucine; ile, isoleucine; thr, threonine; lys, lysine; asp, aspartic acid; orn, ornornithine; glu, glutamic acid
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b05623. Typical fragment ions of authentic standards measured by GC−MS and HPLC−MS and OPLS-DA score scatter plots and permutation tests for each albino cultivar and green cultivar (Figures S1−S24) (PDF) Original HPLC−MS data used for PCA and OPLS-DA (Table S1) (XLSX) Original GC−MS data used for PCA and OPLS-DA (Table S2) (XLSX) Differential metabolites between each albino cultivar and green cultivar measured by GC × GC−TOF/MS (Table S3) (XLSX) Differential metabolites between each albino cultivar and green cultivar measured by UPLC−Q−TOF/MS (Table S4) (XLSX)
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REFERENCES
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AUTHOR INFORMATION
Corresponding Author
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[email protected]. ORCID
Liang Chen: 0000-0002-7507-3947 H
DOI: 10.1021/acs.jafc.7b05623 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.jafc.7b05623 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
