Xiaolan Jiang

Tissue-specific, development-dependent phenolic compounds accumulation profile and gene expression pattern in tea plant [Camellia sinensis].

Phenolic compounds in tea plant [Camellia sinensis (L.)] play a crucial role in dominating tea flavor and possess a number of key pharmacological benefits on human health. The present research aimed to study the profile of tissue-specific, development-dependent accumulation pattern of phenolic compounds in tea plant. A total of 50 phenolic compounds were identified qualitatively using liquid chromatography in tandem mass spectrometry technology. Of which 29 phenolic compounds were quantified based on their fragmentation behaviors. Most of the phenolic compounds were higher in the younger leaves than that in the stem and root, whereas the total amount of proanthocyanidins were unexpectedly higher in the root. The expression patterns of 63 structural and regulator genes involved in the shikimic acid, phenylpropanoid, and flavonoid pathways were analyzed by quantitative real-time polymerase chain reaction and cluster analysis. Based on the similarity of their expression patterns, the genes were classified into two main groups: C1 and C2; and the genes in group C1 had high relative expression level in the root or low in the bud and leaves. The expression patterns of genes in C2-2-1 and C2-2-2-1 groups were probably responsible for the development-dependent accumulation of phenolic compounds in the leaves. Enzymatic analysis suggested that the accumulation of catechins was influenced simultaneously by catabolism and anabolism. Further research is recommended to know the expression patterns of various genes and the reason for the variation in contents of different compounds in different growth stages and also in different organs.

Identification of UDP-glycosyltransferases involved in the biosynthesis of astringent taste compounds in tea (Camellia sinensis).

Highlight The identification of three UDP-glycosyltransferases involved in the biosynthesis of galloylated catechins and glycosylated flavonols which are astringent taste compounds in tea.

Analysis of accumulation patterns and preliminary study on the condensation mechanism of proanthocyanidins in the tea plant [ Camellia sinensis ]

In the present study, proanthocyanidins were qualitatively and quantitatively identified using hydrolysis and thiolysis assays, NP-HPLC, HPLC-ESI-MS, MALDI-TOF-MS, 1H-NMR, and 13C-NMR techniques in different organs of tea plants. The results showed that in leaves, the tri-hydroxyl, cis- and galloylated flavan-3-ols were the main monomeric catechins units, and (epi)catechin was found to be the major unit of polymeric flavan-3-ols when the degree of polymerization was greater than five. In roots, the PAs were found to be abundant, and epicatechin formed the predominant extension unit of oligomeric and polymeric PAs. In order to understand the mechanism of proanthocyanidins polymerization, auto-condensation of the flavan-3-ols was investigated. The results showed that the same trimers (m/z 865) were detected in the extracts of tea plants and in the non-enzymatic in vitro assay, in weak acid as well as weak alkaline solutions at room temperature, when the substrates used were either procyanidin B2 and monomeric flavan-3-ols (epicatechin or catechin), or only procyanidin B2. This suggested that procyanidin B2 not only released carbocation as electrophilic upper units, but also could be used as nucleophilic lower units directly itself, to form the procyanidin trimer in vitro or in vivo.

Functional analysis of Flavonoid 3′,5′-hydroxylase from Tea plant (Camellia sinensis): critical role in the accumulation of catechins

BackgroundFlavonoid 3′,5′-hydroxylase (F3′5′H), an important branch point enzyme in tea plant flavan-3-ol synthesis, belongs to the CYP75A subfamily and catalyzes the conversion of flavones, flavanones, dihydroflavonols and flavonols into 3′,4′,5′-hydroxylated derivatives. However, whether B-ring hydroxylation occurs at the level of flavanones and/or dihydroflavonols, in vivo remains unknown.ResultsThe Camellia sinensis F3′5′H (CsF3′5′H) gene was isolated from tea cDNA library. Expression pattern analysis revealed that CsF3′5′H expression was tissue specific, very high in the buds and extremely low in the roots. CsF3′5′H expression was enhanced by light and sucrose. Over-expression of CsF3′5′H produced new-delphinidin derivatives, and increased the cyanidin derivative content of corollas of transgenic tobacco plants, resulting in the deeper transgenic plant flower color. Heterologous expressions of CsF3′5′H in yeast were carried out to demonstrate the function of CsF3′5′H enzyme in vitro. Heterologous expression of the modified CsF3′5′H (CsF3′5′H gene fused with Vitis vinifera signal peptide, FSI) revealed that 4′-hydroxylated flavanone (naringenin, N) is the optimum substrate for CsF3′5′H, and was efficiently converted into both 3′4′- and 3′4′5′-forms. The ratio of 3′4′5′- to 3′4′-hydroxylated products in FSI transgenic cells was significantly higher than VvF3′5′H cells.ConclusionsCsF3′5′H is a key controller of tri-hydroxyl flavan-3-ol synthesis in tea plants, which can effectively convert 4′-hydroxylated flavanone into 3′4′5′- and/or 3′4′-hydroxylated products. These findings provide animportant basis for further studies of flavonoid biosynthesis in tea plants. Such studies would help accelerate flavonoid metabolic engineering in order to increase B-ring tri-hydroxyl product yields.

Quantification of flavonol glycosides in Camellia sinensis by MRM mode of UPLC-QQQ-MS/MS.

Phenolic compounds are major components of tea flavour, in which catechins and flavonol glycosides play important roles in the astringent taste of tea infusion. However, the flavonol glycosides are difficult to quantify because of the large variety, as well as the inefficient seperation on chromatography. In this paper, a total of 15 flavonol glycosides in the tea plant (Camellia sinensis) were identified by the high performance liquid chromatography (HPLC) coupled to a time-of-flight mass spectrometer (TOF-MS), and a quantitative method was established based on multiple reaction monitoring (MRM) mode of ultra-high performance liquid chromatography (UPLC) coupled to a triple quadrupole mass spectrometer (QQQ-MS/MS). It provided the limit of detection and quantification to the order of picogram, which was more sensitive than the HPLC detection of the order of nanogram. The relative standard deviations of the intra- and inter-day variations in retention time and signal intensity (peak area) of six analytes were less than 0.26% and 4%, respectively. The flavonol glycosides of four tea cultivars were relatively quantified using the signal intensity (peak area) of product ion, in which six flavonol glycosides were quantified by the authentic standards. The results showed that the flavonol mono-, di- and tri-glycoside mostly accumulated in young leaves of the four tea cultivars. Notably, the myricetin 3-O-galactoside was the major component among the six flavonol glycosides detected.

Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality

Significance A high-quality genome assembly of Camellia sinensis var. sinensis facilitates genomic, transcriptomic, and metabolomic analyses of the quality traits that make tea one of the world’s most-consumed beverages. The specific gene family members critical for biosynthesis of key tea metabolites, monomeric galloylated catechins and theanine, are indicated and found to have evolved specifically for these functions in the tea plant lineage. Two whole-genome duplications, critical to gene family evolution for these two metabolites, are identified and dated, but are shown to account for less amplification than subsequent paralogous duplications. These studies lay the foundation for future research to understand and utilize the genes that determine tea quality and its diversity within tea germplasm. Tea, one of the world’s most important beverage crops, provides numerous secondary metabolites that account for its rich taste and health benefits. Here we present a high-quality sequence of the genome of tea, Camellia sinensis var. sinensis (CSS), using both Illumina and PacBio sequencing technologies. At least 64% of the 3.1-Gb genome assembly consists of repetitive sequences, and the rest yields 33,932 high-confidence predictions of encoded proteins. Divergence between two major lineages, CSS and Camellia sinensis var. assamica (CSA), is calculated to ∼0.38 to 1.54 million years ago (Mya). Analysis of genic collinearity reveals that the tea genome is the product of two rounds of whole-genome duplications (WGDs) that occurred ∼30 to 40 and ∼90 to 100 Mya. We provide evidence that these WGD events, and subsequent paralogous duplications, had major impacts on the copy numbers of secondary metabolite genes, particularly genes critical to producing three key quality compounds: catechins, theanine, and caffeine. Analyses of transcriptome and phytochemistry data show that amplification and transcriptional divergence of genes encoding a large acyltransferase family and leucoanthocyanidin reductases are associated with the characteristic young leaf accumulation of monomeric galloylated catechins in tea, while functional divergence of a single member of the glutamine synthetase gene family yielded theanine synthetase. This genome sequence will facilitate understanding of tea genome evolution and tea metabolite pathways, and will promote germplasm utilization for breeding improved tea varieties.

Effect of low-intensity white light mediated de-etiolation on the biosynthesis of polyphenols in tea seedlings

Light is an important source of energy as well as environmental signal for the regulation of biosynthesis and accumulation of multiple secondary metabolites in plants. Polyphenols are the major class of secondary metabolites in tea, which possess potential antioxidant properties. In order to investigate the effect of light signal on the regulation of biosynthesis and accumulation of polyphenols in tea seedlings, a low-intensity white light was used and the change in trends of polyphenol contents, patterns of gene expression, and corresponding enzymatic activities were studied. LC-TOF/MS analysis revealed that light signal promoted the accumulation of hydroxycinnamic acid derivatives and nongalloylated catechin (EGC), while it restrained the accumulation of β-glucogallin and galloylated catechins. The quantitative reverse transcription-PCR analysis showed that the expression levels of the regulator genes and some structural genes involved in photomorphogenesis and biosynthetic pathway of nongalloylated catechins, respectively, were up-regulated. In contrast, the expression of DHD/SDH and UGT genes, which may be involved in biosynthetic pathway of βG, was down-regulated. The corresponding in vitro enzyme assays revealed decrease in the activity of ECGT (galloylates nongalloylated catechins) and an increase in activity of GCH (hydrolyzes galloylated catechins) during de-etiolation. The present study yielded inconsistent accumulation patterns of phenolic acids, flavan-3-ols, and flavonols in tea seedlings during de-etiolation. In addition, the accumulation of catechins was possibly jointly influenced by the biosynthesis, hydrolysis, glycosylation, and galloylation of polyphenols in tea plants.

Functional Characterization of Tea (Camellia sinensis) MYB4a Transcription Factor Using an Integrative Approach

Green tea (Camellia sinensis, Cs) abundantly produces a diverse array of phenylpropanoid compounds benefiting human health. To date, the regulation of the phenylpropanoid biosynthesis in tea remains to be investigated. Here, we report a cDNA isolated from leaf tissues, which encodes a R2R3-MYB transcription factor. Amino acid sequence alignment and phylogenetic analysis indicate that it is a member of the MYB4-subgroup and named as CsMYB4a. Transcriptional and metabolic analyses show that the expression profile of CsMYB4a is negatively correlated to the accumulation of six flavan-3-ols and other phenolic acids. GFP fusion analysis shows CsMYB4a’s localization in the nucleus. Promoters of five tea phenylpropanoid pathway genes are isolated and characterized to contain four types of AC-elements, which are targets of MYB4 members. Interaction of CsMYB4a and five promoters shows that CsMYB4a decreases all five promoters’ activity. To further characterize its function, CsMYB4a is overexpressed in tobacco plants. The resulting transgenic plants show dwarf, shrinking and yellowish leaf, and early senescence phenotypes. A further genome-wide transcriptomic analysis reveals that the expression levels of 20 tobacco genes involved in the shikimate and the phenylpropanoid pathways are significantly downregulated in transgenic tobacco plants. UPLC-MS and HPLC based metabolic profiling reveals significant reduction of total lignin content, rutin, chlorogenic acid, and phenylalanine in CsMYB4a transgenic tobacco plants. Promoter sequence analysis of the 20 tobacco genes characterizes four types of AC-elements. Further CsMYB4a-AC element and CsMYB4a-promoter interaction analyses indicate that the negative regulation of CsMYB4a on the shikimate and phenylpropanoid pathways in tobacco is via reducing promoter activity. Taken together, all data indicate that CsMYB4a negatively regulates the phenylpropanoid and shikimate pathways. Highlight: A tea (Camellia sinensis) MYB4a is characterized to encode a R2R3-MYB transcription factor. It is shown to repressively control the phenylpropanoid and shikimate pathway.

Identification of a Flavonoid Glucosyltransferase Involved in 7-OH Site Glycosylation in Tea plants ( Camellia sinensis )

Flavonol glycosides, which are often converted from aglycones in a process catalyzed by UDP-glycosyltransferases (UGTs), play an important role for the health of plants and animals. In the present study, a gene encoding a flavonoid 7-O-glycosyltransferase (CsUGT75L12) was identified in tea plants. Recombinant CsUGT75L12 protein displayed glycosyltransferase activity on the 7-OH position of multiple phenolic compounds. In relative comparison to wild-type seeds, the levels of flavonol-glucosides increased in Arabidopsis seeds overexpressing CsUGT75L12. In order to determine the key amino acid residues responsible for the catalytic activity of the protein, a series of site-directed mutagenesis and enzymatic assays were performed based on the 3D structural modeling and docking analyses. These results suggested that residue Q54 is a double binding site that functions as both a sugar receptor and donor. Residues H56 and T151, corresponding to the basic active residues H20 and D119 of VvGT1, were not irreplaceable for CsUGT75L12. In addition, residues Y182, S223, P238, T239, and F240 were demonstrated to be responsible for a ‘reversed’ sugar receptor binding model. The results of single and triple substitutions confirmed that the function of residues P238, T239, and F240 may substitute or compensate with each other for the flavonoid 7-O-glycosyltransferase activity.

Functional Characterization of a New Tea (Camellia sinensis) Flavonoid Glycosyltransferase

Tea (Camellia sinensis) is an important commercial crop, in which the high content of flavonoids provides health benefits. A flavonoid glycosyltransferase (CsUGT73A20), belonging to cluster IIIa, was isolated from tea plant. The recombinant CsUGT73A20 in Escherichia coli exhibited a broad substrate tolerance toward multiple flavonoids. Among them, kaempferol was the optimal substrate compared to quercetin, myricetin, naringenin, apigenin, and kaempferide. However, no product was detected when UDP-galactose was used as the sugar donor. The reaction assay indicated that rCsUGT73A20 performed multisite glycosidation toward flavonol compounds, mainly forming 3-O-glucoside and 7-O-glucoside in vitro. The biochemical characterization analysis of CsUGT73A20 showed more K7G product accumulated at pH 8.0, but K3G was the main product at pH 9.0. Kinetic analysis demonstrated that high pH repressed the glycosylation reaction at the 7-OH site in vitro. Besides, the content of five flavonol-glucosides was increased in CsUGT73A20-overexpressing tobaccos (Nicotiana tabacum).