Accumulation of catechins and expression of catechin synthetic genes in Camellia sinensis at different developmental stages
© The Author(s) 2016
Received: 6 August 2016
Accepted: 4 October 2016
Published: 24 October 2016
Catechins are the main polyphenol compounds in tea (Camellia sinensis). To understand the relationship between gene expression and product accumulation, the levels of catechins and relative expressions of key genes in tea leaves of different developmental stages were analyzed.
The amounts of catechins differed significantly in leaves of different stages, except for gallocatechin gallate. Close correlations between the expression of synthesis genes and the accumulation of catechins were identified. Correlation analysis showed that the expressions of chalcone synthase 1, chalcone synthase 3, anthocyanidin reductase 1, anthocyanidin reductase 2 and leucoanthocyanidin reductase genes were significantly and positively correlated with total catechin contents, suggesting their expression may largely affect total catechin accumulation. Anthocyanidin synthase was significantly correlated with catechin. While both ANRs and LAR were significantly and positively correlated with the contents of (−)-epigallocatechin gallate and (−)-epicatechin gallate.
Our results suggest synergistic changes between the expression of synthetic genes and the accumulation of catechins. Based on our findings, anthocyanidin synthase may regulate earlier steps in the conversion of catechin, while the anthocyanidin reductase and leucoanthocyanidin reductase genes may both play important roles in the biosynthesis of galloylated catechins.
Polyphenolic compounds, which are widely distributed in plants, contribute to color (Tsuda et al. 2004) and flavor (Lea 1992) and are involved in pigment biosynthesis (Kurauchi et al. 2011), plant disease defense (Matern and Kneusel 1988) and damage caused by abiotic stress (Solovchenko and Schmitz-Eiberger 2003). Notably, polyphenols are responsible for the protective effects of plants against a variety of human diseases including cancer (Thomasset et al. 2007). Tea, which contains abundant polyphenols (Mukhtar and Ahmad 2000; Higdon and Frei 2003), is one of the most popular beverages in the world. Among the polyphenols, catechins are the major components, occupying more than 10 % of dry tea leaves (Wei et al. 2011). Catechins in tea can be separated into epicatechins (GTE) and GTE epimers. GTE include (−)-epigallocatechin gallate (EGCG), (−)-epicatechin gallate (ECG), (−)-epigallocatechin (EGC) and (−)-epicatechin (EC); while major GTE epimers include (−)-gallocatechin gallate (GCG), (−)-gallocatechin (GC) and (−)-catechin (C). The content and function vary with the individual catechin (Henning et al. 2003; Bigelow and Cardelli 2006).
In this study, quantitative real time PCR (qPCR) was performed to determine the relative expression of genes involved in catechin biosynthesis, including CHS1, CHS2, CHS3, ANS, ANR1, ANR2 and LAR in C. sinensis leaves at different developmental stages. Furthermore, the concentration of individual catechins was analyzed using HPLC. The aim of this study was to provide increased understanding of the regulation of catechin synthesis in the tea leaves by investigating the relationship among the accumulation of tea catechins, gene expression levels and leaf developmental stages.
Clonal tea (C. sinensis var. sinensis cultivar Longjing 43) was grown at the tea garden of the National Tea Research Institute of the Chinese Academy of Agricultural Sciences in Hangzhou, China. Leaf samples were collected in March 2013, when the forth leaves reached near final size. The buds, first leaves, second leaves, third leaves and completely mature leaves were collected from tea cultivar Longjing43 for subsequently analysis. Buds, the first, second and third leaves were defined as new shoots. Part of tea samples were microwaved at high power for 1 min and then dried in an oven at 60 °C for 24 h for catechin analysis (Gulati et al. 2003). The rest collected samples were immediately frozen in liquid nitrogen and stored at −70 °C for RNA isolation.
Catechin extraction and HPLC analysis
Catechins were extracted as described by Wang et al. (2014). Tea sample (0.2 g, dry weight) was extracted with 5 ml 70 % methanol in a water bath at 70 °C for 10 min with intermittent shaking. The supernatant was placed in a 10 ml volumetric flask, and the extraction step was repeated to reach the final volume of 10 ml. The extracts were filtered through a 0.45-μm Millipore filter before the injection was made. Each experiment was performed in triplicate.
The extracts were analyzed using an Agilent 1100 HPLC system. Separations were carried out using a Phenomenex RP-MAX 4 μm 250 mm × 4.6 mm i.d. C12 reverse phase column maintained at 40 °C, eluted at 1 ml min−1 with a 60 min gradient of 4–25 % gradient of acetonitrile in water containing 1 % formic acid and monitored at 280 nm (Del Rio et al. 2004).
The standard chemicals, including catechin (C), epicatchin (EC), gallocatechin (GC), epigallocatechin (EGC), Epicatechin gallate (ECG), gallocatechin gallate (GCG), epigallocatechin gallate (EGCG) were purchased from Sigma Chemical Company (St. Louis, MO, USA).
Quantitative real time PCR (qPCR)
Total RNA from the samples was extracted using the SV Total RNA Isolation System (Promega Corporation, USA) as described in the technical manual. The integrity and quality were checked by determining absorbance using the NanoDrop spectrophotometer ND-1000 (NanoDrop Technologies Inc., USA). First strand cDNA was synthesized from 320 ng of total RNA and oligo (dT) primer using the PrimeScript II 1st Strand cDNA Synthesis Kit (TAKARA, Japan) as described in the product manual. The cDNA was used immediately or stored at −20 °C until used for the PCR test.
Primers of catechin biosynthetic genes for real-time PCR analysis in Camellia sinensis
Primer sequence 5′−3′
Results were analyzed by one-way analysis of variance (ANOVA) and statistical differences were examined by the Fisher’s least significant difference (LSD) test. Differences at p < 0.05 were considered to be significant. Correlations analysis was determined using SAS 9.0 software, and charts were plotted using Sigma Plot 12 software.
Catechin analysis by HPLC
Expressions of catechin synthetic genes
The expression of LAR fluctuated dramatically during the developmental stage. Down by 20 % at the first leaf stage, LAR showed a sharp rise afterward, reaching its peak at the second leaf stage. Then it was followed by a significant fall in the third leaves. The expression of ANR1 and ANR2 showed a similar pattern to LAR during the developmental stages. Both of them reached their peaks at the second leaf stage while ANR2 showed larger variation. It is also worth noting that the expression pattern of ANS was largely different from the other genes. ANS showed a general downward trend during the developmental stage, with its peak at the bud stage and the bottom at the mature leaf stage.
Correlation analysis of catechin contents and relative gene expressions
The correlation between catechin contents and relative gene expressions
The concentration of 7 individual catechins in tea leaves, including C, GC, EC, EGC, ECG, EGCG and GCG were determined in the current study, among which EGCG and ECG were found to be the highest fractions and GC and GCG were the lowest. With the exception of GCG, which did not differ among developmental stages, the highest concentrations of individual catechins were in new shoots. Furthermore, for new shoots, a general increase in the levels of individual catechins and total catechins was observed with increasing age of the leaves with an exception of C, whose concentration is highest in the buds. These results are similar with the results of two other studies (Eungwanichayapant and Popluechai 2009; Wei et al. 2011). In contrast, the total catechins in C. sinensis var. sinensis cultivar Zhenong 139 were found to slightly decline with increasing age of the leaves (Mamati et al. 2006). These differences might be explained by genetic variations or differences in the methods of cultivation, post-harvest treatment, environment or agricultural practices (Eungwanichayapant and Popluechai 2009; Wei et al. 2011).
Many studies have shown that gene expression and product concentration are associated with phenotypic changes. Tea catechins are considered to be synthesized through flavonoid pathway and stored in the vacuole (Suzuki et al. 2003; Rani et al. 2009). As major enzymes involved in catechin oxidation such as polyphenol oxidase (PPO) and peroxidase (POD) are present in latent forms that require activation or in different subcellular compartments, catechin degradations only occur after senescence or stresses that disorganized the cell and initiated decompartmentalization (Pourcel et al. 2007). Therefore, the expressions of catechin synthetic genes are considered to be the major factors affecting catechin contents in tea (Saito et al. 1999; Punyasin et al. 2004; Xie et al. 2004; Wang et al. 2012). In this study, the expression levels of catechin synthetic pathway genes in tea leaves at different developmental stages were analyzed. General increases of both the catechin concentrations and the expression of synthetic genes were found in new shoots, indicating their potential correlations. High correlation coefficients were identified between total catechins and CHS1 (0.964, p < 0.01), CHS2 (0.854) and CHS3 (0.896, p < 0.05) (Table 2), indicating the importance of CHS in catechin biosynthesis. Furthermore, according to the changing pattern and correlation coefficients, CHS1 seems to play a more important role in affecting catechin accumulation. In fact, the expression of CHS1 was much higher than CHS2 and CHS3 in tea leaves based on RNA-seq analysis (data not shown). Therefore, CHS1 should be paid more attention to in future studies.
In terms of catechin individuals, C had a different expression profile than the other catechins, with peak concentration in the buds, which was consistent with our previous study (Wei et al. 2011). Moreover, ANS was also the highest in the bud stage, suggesting the possibility that C conversion is predominantly regulated by ANS. Previously, it was considered that C was converted from leucocyanidin by LAR (Ashihara et al. 2010). However, from the present results, no significant correlation between the expression of LAR and C content was found (Table 2). Besides, high expression of LAR seems to lead to a high accumulation of ECG and total catechins. This raised the question about the role of LAR in tea catechin biosynthesis. Stafford (1990) found LAR is able to convert leucocyanidin to C with an epimerase, and then convert C to EC. While, transformation of CsLAR into purple tobacco plants leads to a significant decrease in anthocyanin, with accumulation of 13.7- and 7.2- fold more EC and C, respectively (Pang et al. 2013). Our results are consistent with these studies and indicate that C might be involved in the conversion of leucocyanidin to EC as an intermediate. Moreover, according to Pang’s study, CsANR1 and CsANR2 were able to convert cyanidin to a mixture of EC and C. Therefore, it is hypothesized that compared to the conversion of leucocyanidin to C by LAR, the conversion of leucocyanidin to cyanidin catalyzed by ANS, and then to C by ANRs is more important for C accumulation (Fig. 1).
On the other hand, EC, EGC, EGC and EGCG are also known to be synthesized through enzymatic reactions catalyzed by ANS and ANR (Wang et al. 2012). While the accumulation patterns of these products had similar trends with ANRs. Furthermore, correlation analysis showed both ANR1 and ANR2 were significantly and positively correlated with ECG and EGCG (Table 2). Therefore, the expressions of ANRs may comprise a rate-limiting step in ECG and EGCG productions.
In conclusion, this study demonstrated that close correlations between the expressions of synthesis genes and the accumulation of catechins were present in tea plants. Based on our findings, CHS1 is more important in catechin biosynthesis than CHS2 and CHS3. ANS may regulate the conversion of C, while both ANRs and LAR may play important roles in the biosynthesis of EGCG and ECG.
flavan-3-ol gallate synthase
reverse transcriptase-polymerase chain reaction
LZ carried out the molecular studies and drafted the manuscript. KW participated in the design of the study and modified the manuscript. LW participated in catechin analysis. HC participated in the design of the study and performed the statistical analysis. CZ conceived of the study and participated in its design. All authors read and approved the final manuscript.
This work was supported by the National Natural Science Foundation of China (31470396), the Natural Science Foundation of Zhejiang Province (LY14C020001) and the Earmarked Fund for China Agriculture Research System (CARS-23).
The authors declare that they have no competing interests.
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