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Nov 21, 2017 - ABSTRACT: Polyphenols are one of the largest groups of compounds that confer benefits to the health of plants and humans. Flavonol glyc...
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Functional Analysis of an Uridine Diphosphate Glycosyltransferase Involved in the Biosynthesis of Polyphenolic Glucoside in Tea Plants (Camellia sinensis) Xuecheng Zhao,#,† Xinlong Dai,#,‡ Liping Gao,#,† Lina Guo,† Juhua Zhuang,‡ Yajun Liu,† Xiubing Ma,† Rui Wang,† Tao Xia,*,‡ and Yunsheng Wang*,†,‡ †

School of Life Science and ‡State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, 130 West Changjiang Road, Hefei, Anhui 230036, People’s Republic of China S Supporting Information *

ABSTRACT: Polyphenols are one of the largest groups of compounds that confer benefits to the health of plants and humans. Flavonol glycosides are a major ingredient of polyphenols in Camellia sinensis. Flavonol-3-O-glycosides are characteristic astringent taste compounds in tea infusion. A polyphenolic glycosyltransferase (CsUGT72AM1) belonging to cluster IIIb was isolated from the tea plant. The full-length cDNA of CsUGT72AM1 is 1416 bp. It encodes 472 amino acids with a calculated molecular mass of 50.92 kDa and an isoelectric point of 5.21. The recombinant CsUGT72AM1 protein was expressed in Escherichia coli and exhibited catalytic activity toward multiple flavonoids and coniferyl aldehyde. The enzyme assay indicated that rCsUGT72AM1 could perform glycosidation of flavonols or coniferyl aldehyde in vitro to form 3-O-glucoside or 4-O-glucoside, respectively. Interestingly, this enzyme also had activities and performed multisite glycosidation toward flavanones. The consistent products were confirmed to be naringenin-7-O-glucoside and -4′-O-glucoside by the nuclear magnetism assay. In addition, in the enzyme assay with cyanidin as the substrate, the results suggested that the glycosylated activity of CsUGT72AM1 was remarkably inhibited by a high concentration of anthocyanins. The above results indicate that CsUGT72AM1 may be involved in the metabolism of flavonol, flavanone, anthocyanin, and lignin. KEYWORDS: flavonoid, UDP-glycosyltransferase, glycosidation, Camellia sinensis

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INTRODUCTION Polyphenols, including phenolic acids, flavones, flavanones, flavonols, flavan-3-ols, anthocyanins, and proanthocyanins, are one of the largest groups of compounds that confer health benefits to plants and animals.1,2 Different flavonoid subclasses possess protective roles in plant tissues, and the increases of flavonoid biosynthesis could often respond to external stress factors, such as excessive ultraviolet (UV) light.3 Multi-hydroxy groups attached to ring structures facilitate many potential pharmacological functions, including antioxidant,4 antimutagenic,5 anticarcinogenic,6 and antibacterial7 properties. A variety of modifications to the compound backbone, including glycosylation, acylation, and methylation, confer other unique health benefits for humans.8,9 Transformation of flavonoid aglycones to glycosides plays a pivotal role in flavonoid biosynthesis. Recent research showed that glycosylation of flavonoids can increase their antioxidant and other medicinal activities.8 Additionally, most clinical drugs contain glycosylated flavonoids, such as quercetin-3-O-rutinoside (rutin) and daidzein-8-C-glucoside (puerarin). Uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyze the biosynthesis of glycosylated polyphenolic compounds by transferring a sugar residue from UDP-glucose, UDP-galactose, UDP-glucuronic acid, UDP-xylose, UDPmannose, or UDP-rhamnose into low-molecular-weight lipophilic compounds (including polyphenols, hormones, and pesticides). Polyphenols are catalyzed by a series of glycosyltransferases into polyphenolic glucosides. These © 2017 American Chemical Society

enzymes belong to family I of the glycosyltransferases (GTs), which is the largest superfamily in plants (CAZy database, www.cazy.org).10 A substantial number of genes encoding UGTs were identified by searching sequence data of plants. More than 100 UGTs in Arabidopsis thaliana and 180 UGTs in Vitis vinifera were identified by the presence of the PSPG box.11 Additionally, the preferred bonding site of sugar is at the 3 position of the flavonoid backbone or less frequently at the 7 position and only rarely occurs in other positions.12 For example, Pang et al. cloned three flavonol glycosyltransferase genes of the UGT72 family, UGT72AD1, UGT72AH1, and UGT72A2 in Lotus japonicus seeds. The three genes can perform glycosidation toward flavonols, forming 3-O-glucoside in vitro.13 Several genes of flavonoid glucosyltransferase were cloned that are involved in glycosidation of flavonoids at the 7 position.14 Flavones and flavanones lack a 3-hydroxy, and the glucosides of these compounds can be found at the 7 or 4′ position. For instance, a UGT gene in Pueraria lobata led to a 4′-O-glucoside or 4′,7-O-diglucoside at the 4′ or 7-O position in vivo.15 In addition, some UGTs can catalyze monolignol, forming monolignol glucoside in plants. Generally, monolignol4-O-glucosides are potential intermediates in the lignin pathway. Several UGTs, such as AtUGT72E1 and AtUGT72E2, Received: Revised: Accepted: Published: 10993

October 25, 2017 November 20, 2017 November 21, 2017 November 21, 2017 DOI: 10.1021/acs.jafc.7b04969 J. Agric. Food Chem. 2017, 65, 10993−11001

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Journal of Agricultural and Food Chemistry

Sequence Alignment and Phylogenetic Analysis. In this study, the amino acid sequence alignment analysis of UGTs was conducted using the DNAMAN 7.0 software (Lynnon, Quebec, Canada). A phylogenetic analysis using the amino acid sequences of UGT members was performed using MEGA 6.0 software (http:// www.megasoftware.net/, Mega Software, State College, PA, U.S.A.),26 and a phylogenetic tree was constructed using neighbor-joining distance analysis.27 The tree nodes were evaluated with the Bootstrap method for 1000 replicates,28 and the evolutionary distances were computed using the p-distance method.29 Purification of Recombinant UGT Proteins in E. coli. The cDNA of CsUGT72AM1 was obtained by polymerase chain reaction (PCR), and then the gene was expressed in the pMAL-c2X vector (New England Biolabs, Ipswich, MA, U.S.A.). Recombinant pMALc2X-CsUGT72AM1 was transformed into E. coli NovaBlue (DE3) Competent Cells (Novagen, Schwalbach, Germany). The expression strain was grown at 37 °C in 200 mL of Luria−Bertani medium containing 0.100 g L−1 ampicillin and 2 g L−1 glucose. Later, 0.3 mM isopropyl-β-D-thioacetamide (IPTG) was added to induce protein expression when the optical density value of the cell culture reached 0.6. After 24 h of incubation at 28 °C with shaking, cells were harvested by centrifugation at 4 °C. Maltose-binding protein (MBP) fusion proteins were purified using maltose-binding resin according to the pMAL protein fusion and purification system (New England Biolabs). Enzyme Assay and Product Identification. To analyze recombinant UGT72AM1 protein activity, 10 μg of purified protein was incubated at 35 °C with 100 mM Tris−HCl (pH 7.0) and 10 mM substrates (including kaempferol, quercetin, myricetin, eriodictyol, dihydromyricetin, cyanidin, and coniferyl aldehyde). In addition, 2.5 mM UDP-glucose and UDP-galactose, which acted as sugar donors, were added to a final volume of 50 μL. Reactions were stopped by adding methanol after 30 min. Then, reaction compounds were analyzed by HPLC22,23 after centrifugation at 13 000 rpm for 12 min. All reactions were supplemented with 0.1% (v/v) β-mercaptoethanol. Additionally, the same proteins were reacted with 10 mM naringenin as a substrate at 35 °C with 100 mM Tris−HCl (pH 7.0) and 2.5 mM UDP-glucose as the sugar donor at a final volume of 50 mL. The reaction was stopped with the addition of methanol after 48 h. For kinetic analysis of the recombinant UGT72AM1 proteins, 10 μg enzymes were incubated in reaction mixtures, which included 100 mM Tris−HCl (pH 7.0) and 2.5 mM UDP-glucose in a final volume of 30 μL. The concentrations of the tested acceptor substrates ranged from 0 to 300 μM. The reactions were stopped by adding methanol after a 10 min incubation at 30 °C. The samples were centrifuged at 13 000 rpm for 12 min, and the reaction mixtures were analyzed by HPLC. The kinetic parameters Km and Kcat were calculated using the Hyper32 programs (http://hyper32.software.informer.com/). In addition, the flavonol glucosides and phenolic acid glucosides were further identified by LC−MS.22 Proton Nuclear Magnetic Resonance (1H NMR) Analysis. Naringenin glucosides were obtained by enzymatic reactions, and they were purified by ultrahigh-performance liquid chromatography (UPLC) Agilent InfinityLab Poroshell HPH-C18 (4.6 × 100 mm, 2.7 μm, Agilent, Santa Clara, CA, U.S.A.). The elution program was as follows: starting with 95% A (1% acetic acid) and 5% B (100% acetonitrile), a linear gradient from 5 to 10% B for 0−2 min, from 10 to 30% B for 2−4 min, from 30 to 60% B for 4−12 min, and from 60 to 5% B for 12−18 min was performed, followed by washing and equilibrating the column. Naringenin glucosides were collected with a Gilson FC 240 fraction collector. These glucosides were freeze-dried and resuspended in dimethyl sulfoxide (DMSO). The NMR spectra of the glucosides were acquired on a Bruker AMX 500 MHz NMR spectrometer at 22 °C. The data were processed using Bruker XWINNMR software, version 2.6.15

were confirmed to perform glycosylation at the 4 position of monolignols.16 Tea is one of the most popular non-alcoholic beverages and has been consumed by humans for thousands of years. It is rich in catechins (flavan-3-ol) and flavonoids, which account for approximately 12−24% of the dry mass of young leaves.17 In tea plants, more than 19 types of flavonol glycosides have been identified,18 and 14 of these compounds are responsible for the astringent taste in black tea.19 Of these compounds, flavonols and their derivatives are crucial components to the tea quality.20 In comparison to catechins or theaflavins, flavonol glycosides impart tea infusions with a velvety, mouth-coating, and mouthdrying sensation at very low threshold concentrations.21 To date, only a few UGTs involved in the biosynthesis of polyphenolic glycosides have been characterized at the molecular level in tea plants. In our previous study, both CsUGT78A14 and CsUGT78A15 were responsible for the biosynthesis of flavonol-3-O-glucoside and flavonol-3-O-galactoside in tea plants, respectively.22 Besides, another glycosyltransferase CsUGT73A20 had the activity of forming flavonol-3-O- and -7-O-glucoside.23 In this paper, a UGT gene (CsUGT72AM1) from the tea plant was isolated, and the expression of CsUGT72AM1 was analyzed by quantitative realtime polymerase chain reaction (qRT-PCR). In addition, its activities to flavonols, flavanones, anthocyanidins, and lignin were verified.

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MATERIALS AND METHODS

Plant Materials and Standard Chemicals. Plant Materials. Various organs (leaves and buds) of the tea varieties ‘Shuchazao’ and ‘Purple Tea’ were sampled from healthy plants grown in the tea garden of Anhui Agricultural University, China. These samples were randomly collected and frozen immediately in liquid nitrogen, and the samples were stored at −80 °C for a follow-up experiment. Branches of the tea plant grown in water were cultivated at a light intensity of 155 μmol m−2 s−1 at 25 °C, which was measured by a quantum light meter (3415F, Spectrum, Aurora, IL, U.S.A.). The tea branches were treated for 24 h under induction of white light, red light, blue light, and UV light. In addition, the same inductions lasted for 7 days as the above conditions but 1 day for UV induction,24 and the phenolic compounds of the leaves underwent relative quantitative analysis by a liquid chromatography−mass spectrometry (LC−MS) system20 (Agilent, Santa Clara, CA, U.S.A.). Moreover, the lignin content was determined according to the method of Moreiravilar et al.25 Chemicals. All biochemicals, including kaempferol (Ka), quercetin (Qu), myricetin (My), naringenin (Na), eriodictyol (Er), dihydromyricetin (DHM), cyanidin (Cya), coniferyl aldehyde (Ca), quercetin-3O-glucoside (Q3G), UDP-galactose, and UDP-glucose, were purchased from Sigma (St. Louis, MO, U.S.A.). Methanol, acetonitrile, and acetic acid for high-performance liquid chromatography (HPLC, LC10Avp system, Shimadzu, Kyoto, Japan) analysis were supplied from Tedia Co., Ltd. (Fairfield, OH, U.S.A.). RNA Isolation, cDNA Cloning, and Expression Analysis of CsUGT72AM1. Total RNA was isolated from the leaves, buds, and induction materials of the tea plant using RNAiso-mate (Takara, Dalian, China) and RNAiso Plus Kit (Takara, Dalian, China) according to the protocol of the manufacturer. RNAs were treated with DNase I (Takara, Japan) to remove any DNA contamination. cDNA was generated using the PrimeScript RT Master Mix (RR036A, Takara, Dalian, China) following the protocol of the manufacturer. Then, on the basis of the materials, an expression analysis by qRTPCR was performed. The GAPDH housekeeping gene was used as a control in qRT-PCR. Data were calculated from three biological replicates, and each biological replicate was examined in triplicate. All of the PCR primer sequences are shown in Table S1 of the Supporting Information.

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RESULTS cDNA Cloning of CsUGT72AM1. To obtain full-length cDNA of CsUGT72AM1, 3′ RACE and 5′ RACE clone assays 10994

DOI: 10.1021/acs.jafc.7b04969 J. Agric. Food Chem. 2017, 65, 10993−11001

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Journal of Agricultural and Food Chemistry were performed on the basis of the expressed sequence tag (EST) sequences of CsUGT72AM1. The full length of CsUGT72AM1 cDNA was 1416 bp (Figure S1 of the Supporting Information). It encoded polypeptides of 472 amino acid residues. The molecular mass of CsUGT72AM1 was predicted to be 50.92 kDa with an isoelectric point of 5.21 (ExPASy Tools Compute pI/Mw). This gene was designated as CsUGT72AM1 based on the nomenclature system agreement by a committee and submitted to the GenBank database (accession number KY399734).30 Phylogenetic and Sequence Analyses of CsUGT72AM1. Flavonoid glucosyltransferase can be classified into four functional groups (clusters I, II, IIIa, IIIb, and IV) using the Akio Noguchi classification.31 These groups were predicted to encode 3-O, 5-O, and 7-O-glucosyltransferases as well as a diglycoside/disaccharide chain glucosyltransferase. A phylogenetic tree of CsUGT72AM1 and 51 other flavonoid glucosyltransferase sequences were constructed (Figure 1). All

Figure 2. Amino acid sequence alignment of UGT72AM1 and four homologues. The identified UGTs in the multiple alignment are PGT1 (P. tomentosa HM776516), AtUGT72E1 (A. thaliana Al049862), LJUGT72Z2 (L. japonicas Kp410264), and Ct3GT2 (C. ternatea AB185904.1). The multiple alignment was performed using DNAMAN 7.0 software. The black rectangle indicates the domains of the PSPG box.

CsUGT72AM1 was significantly upregulated under blue light and UV light compared to white light and the dark control. However, the expression of the gene was unremarkably upregulated by red light compared to the control (Figure 3B). In addition, flavonoid and lignin compounds of various samples were detected. The content of lignin reached higher levels in old leaves, and flavonol-3-O-gulcosides (UDP-glucose) [F-3-O-glucosides (glc)] reached higher levels in third leaves (panels C and E of Figure 3). Moreover, the lignin content and F-3-O-glucoside (glc) compounds were increased under blue light treatment (panels D and F of Figure 3). Enzymatic Assays of Recombinant CsUGT72AM1 in Vitro. To investigate the enzymatic activity of CsUGT72AM1, we expressed the gene in E. coli with the constructed vector pMAL-c2X-CsUGT72AM1. The molecular weight of the recombinant protein was confirmed to be 93.42 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS−PAGE) analysis (Figure S2 of the Supporting Information). In addition, optimization conditions of CsUGT72AM1 were tested at different pH values and temperatures. Acid−sodium citrate buffer (pH 4.0−7.5), phosphate-buffered saline (PBS, pH 6.0−9.0), and Tris−HCl buffer (pH 7.0−10.0) were used for the reaction, and the reaction temperature tolerance ranged from 10 to 60 °C. The results showed that the optimal reaction pH was 7.5 in vitro. In addition, CsUGT72AM1 performed higher catalytic efficiency in PBS (pH 7.5) than that in Tris−HCl buffer (Figure S3A of the Supporting Information) and 40 °C (Figure S3B of the Supporting Information) with quercetin as the substrate. To test the substrate specificity of CsUGT72AM1, two sugar donors (UDP-glucose and UDP-galactose) and seven polyphenol compounds were tested as substrates, including flavonols (kaempferol, quercetin, and myricetin), flavanones (naringenin and eriodictyol), dihydroflavonol (dihydromyrice-

Figure 1. Phylogenetic tree of CsUGT72AM1 and UGT homologues from other plants. The phylogenetic tree was constructed using MEGA 6.0 with the neighbor-joining method. Clusters (I, II, IIIa, IIIb, and IV) are shown in the phylogenetic tree. CsUGT72AM1 is shown in bold.

of the GenBank accession numbers of the sequences used in the phylogenetic analysis are summarized in Table S2 of the Supporting Information. CsUGT72AM1 was clustered together with PGT1, AtUGT72E1, and LjUGT72Z2 in cluster IIIb. In addition, the alignment analysis indicated that CsUGT72AM1 had 47.74, 42.24, and 20.86% identity from Populus tomentosa, L. japonicas and Clitoria ternatea,32 respectively. The sequence alignment results showed a conserved PSPG box in the C terminus of CsUGT72AM1 (Figure 2). Expression Analysis of CsUGT72AM1 and Accumulation of Phenolic Compounds in Tea Plants. To further characterize the biological functions of CsUGT72AM1 in tea plants, the expression profiles of CsUGT72AM1 in various tissues and in different light quality inductions were analyzed by qRT-PCR. qRT-PCR results showed that expression of this gene is higher in third leaves, fourth leaves, and old leaves than buds. Moreover, this highest transcript level of the gene was found in old leaves, and a low expression of CsUGT72AM1 was found in the first leaves (Figure 3A). The expressional level of 10995

DOI: 10.1021/acs.jafc.7b04969 J. Agric. Food Chem. 2017, 65, 10993−11001

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Figure 3. Expression patterns of CsUGT72AM1 and the accumulation profiles of the lignin content and flavonoid compounds in various organs and light induction conditions. (A and B) Expression patterns of CsUGT72AM1 in various organs and different wavelengths of light inductions. (C and D) Lignin contents in various organs and different wavelengths of light inductions. (E and F) Relative quantitative analysis of F-3-O-glucoside in various organs and different wavelengths of light inductions. F-3-O-glucosides include kaempferol-3-O-glucoside, quercetin-3-O-glucoside, and myricetin-3-O-glucoside. Values represent the means ± standard deviation (SD) of triplicate analytical replicates from data presented as the means of three independent assays. Data were statistically evaluated using Student’s test (∗∗, p < 0.01; ∗, p < 0.05).

Figure 4. HPLC analyses of the enzymatic products of CsUGT72AM1. HPLC chromatograms for the enzymatic products of the CsUGT72AM1 protein with (A) Ka, (B) Qu, (C) My, (D) Na, (E) Er, (F) DHM, and (G) Ca as polyphenolic acceptors and UDP-glucose as the sugar donor.

tin), and phenolic acid (coniferyl aldehyde). The enzyme assay results showed that rCsUGT72AM1 can catalyze all of the above polyphenol compounds to form their own glucosides with UDP-glucose as the sugar donor in vitro (Figure 4). In

addition, the enzymatic products were confirmed (Figure S4 of the Supporting Information). enzymatic products were confirmed by analysis to authentic quercetin-3-O-glycosides (Figure 10996

by LC−MS Then, the comparison S5 of the

DOI: 10.1021/acs.jafc.7b04969 J. Agric. Food Chem. 2017, 65, 10993−11001

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Figure 5. Initial reaction rate to concentration plots and reciprocal plots showing kinetics of rCsUGT72AM1 to different substrates. (A−F) Kinetics of rCsUGT72AM1 to substrates, including kaempferol (Ka), quercetin (Qu), myricetin (My), naringenin (Na), enodictyol (Er), and coniferyl aldehyde (Ca), at pH 7.0 and 30 °C.

Figure 6. Enzyme kinetics of recombinant CsUGT72AM1 proteins. (A−C) Chemical structures of polyphenol substrates. (D) Kinetic parameters of the recombinant CsUGT72AM1 proteins with polyphenolic aglycones as acceptor substrates and UDP-glucose as the donor substrate. Values represent the means ± SD from triplicate enzymatic assays.

catalytic efficiencies (Kcat/Km) of rCsUGT72AM1 for kaempferol, quercetin, myricetin, naringenin, eriodictyol, and coniferyl aldehyde were 20.70, 43.26, 64.10, 67.9, 548, and 224 M−1 s−1, respectively, with Vmax values of 17.92, 49.26, 86.96, 33.56, 200, and 312 pkat mg−1, respectively (Figure 6). The enzyme kinetic results showed that catalytic efficiency of rCsUGT72AM1 was low for flavonols. However, the catalytic efficiency of rCsUGT72AM1 was high for eriodyctyol and coniferyl aldehyde, with Kcat/Km values of 548 and 224 M−1 s−1, respectively. Overall, the results suggested that rCsUGT72AM1 may exhibit a higher affinity for flavanone and/or monolignol in vivo.

Supporting Information). The above enzymatic assay results indicated that the rCsUGT72AM1 protein exhibited a broad substrate preference toward multiple polyphenolic compounds. Enzyme Kinetics of CsUGT72AM1. To investigate the substrate affinity of rCsUGT72AM1, the kinetic parameters toward UDP-glucose sugar donors and polyphenolic acceptors were determined in Tris−HCl buffer at pH 7.0 and 30 °C for 10 min. The results based on hyperbolic Michaelis−Menten saturation curves, indicated that the corresponding Km values for kaempferol, quercetin, myricetin, naringenin, eriodictyol, and coniferyl aldehyde were 71.81, 94.99, 113.15, 28.68, 30.42, and 116.47 μM, respectively (Figure 5). In addition, the 10997

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spectra were nearly identical to the spectra reported for naringenin-7-O-glucoside and naringenin-4′-O-glucoside (Figure 8). This result indicated that rCsUGT72AM1 possesses multi-site catalytical activities against naringenin.

In addition, in the enzyme assay with cyanidin as the substrate, the concentration of cyanidin was varied from 0 to 100 μM in Tris−HCl buffer at pH 7.0 and 30 °C for 10 min with 2.5 mM UDP-glucose as the sugar donor. The enzyme assay indicated that rCsUGT72AM1 could perform glycoxidation of cyanidin, forming cyanidin-3-O-glucosides (Cya3GTs) in vitro; in addition, the Cya3GTs were further identified by LC−MS (Figure 7A). The reaction velocity increased with

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DISCUSSION Multiple Functions of CsUGT72AM1. UGTs exist widely and perform various functions in plants. In Zea mays, the first isolated UGT encoded a flavonol glucosyltransferase using UDP-glucose as the sugar donor.34 Since then, many UGTs have been identified from different plant species. For instance, anthocyanidin 3-O-glucosyltransferases were isolated from Gentiana triflora35 and Iris hollandica,36 respectively. Several flavonol-3-O-glucosyltransferase genes were isolated from Glycine max.37 In our previous research, 132 UGTs of tea plant were screened.22 Among them, CsUGT84A22, CsUGT78A14, CsUGT78A15, CsUGT75L12,38 and CsUGT73A2023 were isolated and functionally verified. However, the function of most CsUGTs has not been revealed. In this study, a new UGT gene (CsUGT72AM1) was isolated and identified by transcriptome sequencing. The phylogenetic tree results showed that CsUGT72AM1, AtUGT72E1, PGT1, and LJUGT72Z2 were on the same branch. According to the functional analysis, AtUGT72E1 can catalyze monolignol into monolignol glucosides,16 and PGT1 is involved in the synthesis of lignin.39 The Km and Kcat/Km of AtUGT72E1 for coniferyl aldehyde were 270 μM and 452 M−1 s−1, respectively. However, LJUGT72Z2 is a flavonol glycosyltransferase.13 The Km and Kcat/Km of LJUGT72Z2 for kaempferol were 40 μM and 1000 M−1 s−1, respectively, in vitro. To the best of our knowledge, few UGTs in the UGT72 family have the function of catalyzing the biosynthesis of both phenolic acid glucosides and flavonoid glucosides in plants. Interestingly, our research showed that expression of CsUGT72AM1 is the highest in old leaves and the expressional level of this gene was significantly upregulated under blue and UV light. In addition, the enzymatic assay results showed that rCsUGT72AM1 can catalyze not only monolignol but also flavonol, flavonone, dihydroflavonol, and anthocyanin in vitro. The enzyme kinetic results show that the catalytic efficiency of rCsUGT72AM1 was high for eriodyctyol and coniferyl aldehyde, with Kcat/Km values of 548 and 224 M−1 s−1, respectively. Thus, CsUGT72AM1 may exhibit a higher affinity for flavanone and/or monolignol in vivo. Therefore, the results of this study suggest that CsUGT72AM1 may have multiple functions in plants (Figure 9). In future studies, we will overexpress CsUGT72AM1 in the model plant, which will

Figure 7. HPLC and LC−MS analyses of the enzymatic products of CsUGT72AM1. (A) HPLC and LC−MS chromatograms of the enzymatic product of the CsUGT72AM1 protein with cyanidin. (B) UDP-glucose as the sugar donor. Points represent the mean of three assays ± SD.

cyanidin concentrations up to 20 μM and progressively decreased with increasing cyanidin concentrations. The results indicated that the enzymatic activity was inhibited by a high concentration of cyanidin (Figure 7B), which were the same results as found by Kovinich et al.33 1 H NMR Analysis of Reaction Products with Naringenin as the Substrate. Interestingly, in the enzyme assay with naringenin as the substrate, two products were detected (Figure 4D). These two products were concentrated in vitro, purified by UPLC, and analyzed by NMR subsequently. In comparison to aglycone, the NMR spectrum of naringenin showed a significant upshift at the neighboring proton attached to the C6, C8, C3′, and C5′ positions in Table 1, which indicated that neighboring C7-OH and C4′-OH were conjugated. The NMR

Table 1. 1H NMR Spectral Data of Naringenin and the Corresponding Glucosides position C2 C3 C6 C8 C2′ C3′ C5′ C6′ 5-OH 7-OH 4′-OH

naringenin 5.44 2.67 3.27 5.87 5.87 7.31 6.78 6.78 7.31 12.15 10.79 9.59

(1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H,

naringenin-7-O-glucoside

dd, J = 12.6 and 3.0 Hz) dd, J = 16.8 and 3.0 Hz) dd, J = 16.8 and 13.2 Hz) s) s) d, J = 8.4 Hz) d, J = 8.4 Hz) d, J = 8.4 Hz) d, J = 8.4 Hz) s) s) s)

6.14 6.14 7.32 6.79 6.79 7.32 12.05

(1H, (1H, (1H, (1H, (1H, (1H, (1H,

d) d) d, J d, J d, J d, J s)

= = = =

8.4 8.4 8.4 8.4

Hz) Hz) Hz) Hz)

naringenin-4′-O-glucoside

7.42 7.05 7.05 7.42 12.28

(1H, (1H, (1H, (1H, (1H,

d, J d, J d, J d, J s)

= = = =

8.4 8.4 8.4 8.4

Hz) Hz) Hz) Hz)

9.63 (1H, s) 10998

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Figure 8. 1H NMR spectra of (A) NG1, (B) stands, and (C) NG2 in acetone-d6/D2O.

Figure 9. Proposed pathways for polyphenol glycosylations catalyzed by CsUGT72AM1 in the tea plant. PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumaroyl-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3′-hydroxylase; FLS, flavonol synthase; DFR, dihyhroflvonol 4-reductase; LAR, leucoanthocyanidin reductase; ANS, anthocyanidin synthase; and ANR, anthocyanidin reductase.

example, CsUGT78A14 encodes flavonoid 3-O-glycosyltransferases in Camellia sinensis.22 Additionally, PlUGT2, a UGT identified and characterized in P. lobata, was active with various flavonoid acceptors and catalyzes consecutive glycosylation activities at the O-4′ and O-7 positions.15 However, a few UGTs can catalyze multiple hydroxyl glycosylations. For example, AtUGT88A1 had simultaneous catalytic activity at 3-, 7-, 3′-, and 4′-OH of quercetin.22,40 A recent study showed

help us understand the catalytic mechanism of CsUGT72AM1 in the tea plant. Regioselectivity of CsUGT72AM1. In plants, glycosylation is a very universal phenomenon and many glycosylation reactions occur on individual hydroxyl groups of polyphenol at 3-, 5-, 7-, 3′-, and 4′-OH or multiple hydroxyl groups simultaneously.23 Several UGTs involved in flavonoid biosynthesis have been characterized at the molecular level. For 10999

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Journal of Agricultural and Food Chemistry that CsUGT73A20 can catalyze kaempferol into kaempferol-3O-glucoside (K3G) and kaempferol-7-O-glucoside (K7G).23 In this study, CsUGT72AM1 was involved in the biosynthesis of monolignol glucosides and exhibited catalytic activity toward lignin monolignol at the 4 position. Furthermore, rCsUGT72AM1 catalyzed flavonol compounds, which mainly formed 3-O-glucoside in vitro. However, flavones and flavanones are special in the glucoside position of these compounds, which can be found at the 7 or 4′ positions. 41 For example, rCsUGT72AM1 performed multi-site glycosidation toward naringenin and mainly formed 7-O-glucoside, followed by 4′O-glucoside. Biochemical characterization analysis of CsUGT72AM1 showed that rCsUGT72AM1 has multi-site characteristics. In the future, point mutation analysis of the key amino acid residues in CsUGT72AM1 will help us further understand the catalyzing mechanism of CsUGT72AM1 on different hydroxyl positions.

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the Natural Science Foundation of Anhui Province (1708085QC51), the Collegiate Natural Science Foundation of Anhui Province (KJ2016A223), and the Special Foundation for Independent Innovation of Anhui Province, China (13Z03012). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Huarong Tan, Tiejun Ling, and Jingwei Hu for assistance with the LC/MS and 1H NMR analyses.

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ABBREVIATIONS USED UDP, uridine diphosphate; UGT, UDP-glycosyltransferase; rCsUGT72AM1, recombinant CsUGT72AM1 protein; K3G, kaempferol-3-O-glucoside; K7G, kaempferol-7-O-glucoside; Q3G, quercetin-3-O-glucoside; Cya3GT, cyanidin-3-O-glucoside; F-3-O-glucosides (glc), flavonol-3-O-gulcosides (UDPglucose)

ASSOCIATED CONTENT

S Supporting Information *

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b04969. Gel electrophoresis of high-fidelity PCR products (Figure S1), SDS−PAGE of purified recombinant protein PMAL-CsUGT72AM1 (Figure S2), optimization of reaction pH and temperature (Figure S3), LC−MS analyses of the enzymatic products of CsUGT72AM1 (Figure S4), HPLC analysis of putative glucoside from the products of recombinant CsUGT72AM1 compared to authentic Q3G (Figure S5), PCR primer sequences (Table S1), and GenBank accession numbers of the sequences used in the phylogenetic analysis (Table S2) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Telephone: 86-551-65786003. Fax: 86-551-65785833. E-mail: [email protected]. *Telephone: 86-551-65786232. Fax: 86-551-65785729. E-mail: [email protected]. ORCID

Tao Xia: 0000-0003-0814-2567 Yunsheng Wang: 0000-0002-0348-4608 Author Contributions #

Xuecheng Zhao, Xinlong Dai, and Liping Gao contributed equally to this work. Author Contributions

Yunsheng Wang, Tao Xia, and Xuecheng Zhao conceived and designed the study. Yunsheng Wang and Xuecheng Zhao drafted the manuscript. Xuecheng Zhao, Xinlong Dai, Lina Guo, Yajun Liu, and Xiubing Ma performed the experiments. Liping Gao, Xiujuan He, Xuecheng Zhao, and Yajun Liu analyzed the data. Liping Gao, Xiaolan Jiang, Xiujuan He, Xingxing Shi, Xinlong Dai, and Yajun Liu contributed reagents/ materials/analysis tools. All authors read and approved the final manuscript. Funding

This work was supported by the Natural Science Foundation of China (31770729, 31570694, 31470689, 31300577, 31270730, and 31200229), the Postgraduate Foundation of Anhui Agricultural University, Anhui Province, China (2017yjs-36), 11000

DOI: 10.1021/acs.jafc.7b04969 J. Agric. Food Chem. 2017, 65, 10993−11001

Article

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DOI: 10.1021/acs.jafc.7b04969 J. Agric. Food Chem. 2017, 65, 10993−11001