Functional Characterization of a New Tea (Camellia sinensis

Article pubs.acs.org/JAFC

Functional Characterization of a New Tea (Camellia sinensis) Flavonoid Glycosyltransferase Xianqian Zhao,†,‡,§ Peiqiang Wang,†,§ Mingzhuo Li,† Yeru Wang,‡ Xiaolan Jiang,† Lilan Cui,† Yumei Qian,† Juhua Zhuang,† Liping Gao,*,‡ and Tao Xia*,† †

State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui 230036, China School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, China

‡

S Supporting Information *

ABSTRACT: 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). KEYWORDS: flavonoids glycosyltransferase, multisite glycosidation, Camellia sinensis

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INTRODUCTION As essential metabolites, flavonoids play important roles in UV protection and plant defense and also are responsible for pigmentation in some plants.1−4 For human beings, flavonoids are both healthy and medicinal compounds, for which consumption of flavonoids can decrease body fat accumulation and reduce the incidence of cancer.5−8 In plants, the flavonoids consist of several subgroups such as flavonols, flavones, flavanones, flavanols, anthocyanins, proanthocyanidin, and isoflavones.9,10 All of these compounds possess a basic benzopyran ring nucleus structure, synthesized from L-phenylalanine, and one or more glycosylated hydroxyl points.11 Glycosylation is a widespread and significant modification process in plants. After glycosylation, the physicochemical stability, solubility, cellular localization, bioactivity, and pharmacokinetics of many compounds (such as flavonoids) would be changed.12,13 In plants, the glycosylation reactions are catalyzed by a series of glycosyltransferases, of which one type is the flavonoids UDP-glycosyltransferase (UFGT). UFGTs transfer the saccharide groups from activated glycosyl donors to a nucleophilic glycosyl acceptor (including oxygen-, carbon-, nitrogen-, and sulfur-based) on flavonoid compounds.14,15 Phenolic compounds are the main substrates of UFGTs, such as phenolic acids, flavonols, flavones, and isoflavones.16,17 UFGTs can perform glucosylation at the 3, 5, 7, 3′, and 4′ hydroxyl group sites on the benzopyran ring nucleus of various substrates.18−22 In the UFGT super family, some members showed strict regiospecificity, performing glucosylation reactions at 3-, 7-, and 4′-OH positions, respectively.23,24 AtUGT78D1 and AtUGT78D2 encoded two flavonol 3-Oglycosyltransferases in Arabidopsis thaliana.25,26 A flavonoid glycosyltransferase (WsGT) from Withania somnifera catalyzed © 2017 American Chemical Society

luteolin, apigenin, and naringenin at the 7-OH site in Withania somnifera, and AtUGT73J1 only glycosylated isoquercitrin and genistein at their C-7 hydroxyl sites.20,21,23 In our previous study, two 3-OH site UFGTs (CsUGT78A14 and CsUGT78A15) were identified from tea plant, which were responsible for the biosynthesis of flavonol 3-O-glucoside and flavonol 3-O-galactoside, respectively.27 5-OH glycosylation commonly occurs toward anthocyanin substrates but not flavonols. AtUGT75C1 from Arabidopsis encoded a glucosyltransferase which catalyzed the glycosylation reaction to anthocyanins.28 A UDP-glucose:anthocyanin 5-O-glucosyltransferase responsible for the modification of anthocyanins to more stable molecules in Perilla f rutescens was well characterized.4 In contrast, some UFGTs can simultaneously glycosylate substrates at multiple hydroxyl positions. A flavonoid glucosyltransferase, identified in Dianthus caryophyllus, catalyzed naringenin producing naringenin-7-O-glucoside and naringenin-4′-O-glucoside.29 Few glycosylation events can occur simultaneously at more than two of above-mentioned positions. For example, AtUGT74F1 can glycosylate 3′-OH, 4′OH, and 7- OH positions of quercetin.30 Quercetin 7-, 3′-, and 4′-glucoside could be formed by CsGT45 from Crocus sativus,31 and in Arabidopsis thaliana, C-3, C-7, and C-4′ hydroxyl groups could be glycosylated simultaneously by AtUGT73C6.32 As the potential utilization of glycosyltransferases in chemical and food industries increases, the search on specific substrate recognition Received: Revised: Accepted: Published: 2074

December 15, 2016 February 16, 2017 February 21, 2017 February 21, 2017 DOI: 10.1021/acs.jafc.6b05619 J. Agric. Food Chem. 2017, 65, 2074−2083

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Journal of Agricultural and Food Chemistry

Figure 1. Substrate structures. (A) Flavanones; (B) flavone; (C) flavonol. PCR program 1 was as follows: 94 °C for 3 min, 7 cycles of 94 °C for 25 s, 72 °C for 3 min, 32 cycles of 94 °C for 25 s, 67 °C for 3 min, followed by a 7 min extension at 67 and 16 °C forever. PCR program 2 was as follows: 94 °C for 3 min, 5 cycles of 94 °C for 25 s, 72 °C for 3 min, 20 cycles of 94 °C for 25 s, 67 °C for 3 min, followed by a final extension at 67 °C for 7 min. Both programs 1 and 2 worked with the positive control 5′- and 3′-RACE TFR and UPM Primers. The PCR products were cloned into PMD19-T vectors (Takara, DaLian, China) and sequenced at BGI (http://www.genomics.cn/index). The ORF (open reading frame) of CsUGT73A20 was cloned by using Phusion High-Fidelity DNA Polymerase (Thermo Scientific, Lithuania, EU), and the conditions for this PCR were 98 °C for 30 s, 30 cycles of 98 °C for 10 s, 60 °C for 30 s, 72 °C for 30 s, followed by a final extension at 72 °C for 10 min. Sequence Alignment and Phylogenetic Analysis. The amino acid sequence alignment analysis of UFGTs was conducted by using the DNAMAN software. A phylogenetic analysis using amino acid sequences of UFGT members was performed using MEGA6.1 software. Evolutionary distances were estimated by a p-distance method, and the tree nodes were evaluated with a 1000 bootstrap value. Heterologous Expression in E. coli and Purification. The ORF of CsUGT73A20 was cloned into expression vector pMAL-c2X (New England Biolabs, MA, USA) with a maltose-tag. The forward and reverse primers were designed with a BamHI and SalI restriction site, respectively, and are shown in Table S2. The ligation of CsUGT73A20 and pMAL-C2X vector was conducted by a T4-ligase system and confirmed by sequencing in BGI (www.genomics.cn). The recombinant plasmid of CsUGT73A20-pMAL was transferred into the E. coli Novablue (DE3) competent cells (Novagen, Schwalbach, Germany) using the manufacturer’s protocol. The positive clones were chosen and shaken in 5 mL of Luria−Bertani medium with 50 mg/L ampicillin for 8 h at 37 °C. Then 2 mL of E. coli culture was added into 200 mL of LB medium containing 50 mg/L ampicillin with shaking at 250 rpm. Cells were grown to an optical density of 0.6 at 600 nm, and isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM to induce the expression of CsUGT73A20. After inducing at 28 °C for 24 h, the cells were harvested by centrifugation at 4 °C (6000 rpm for 15 min) and then lysed by sonication for 25 min at 4 °C (SCIENTZ-IID, NingBo, China). The CsUGT73A20 recombinant protein was purified by an amylose resin affinity chromatography system (New England Biolabs, E8201S) by following the manufacturer’s protocol. The purified CsUGT73A20 recombinant protein was analyzed by a 12% SDS polyacrylamide gel. Protein concentration was determined by spectrometric analysis using Coomassie Brilliant Blue G-250. Enzymatic Assay and Optimization of Reaction Conditions. The CsUGT73A20 activity assay was conducted using 10 μg of protein in 50 μL of reaction buffer (100 mM Tris-HCl pH 7.5) containing 0.1% (v/v) β-mercaptoethanol, 2.5 mM UDP-glucose or UDP-galactose, and 1 μL of 10 mM substrates (including naringenin,

of flavonoid glycosyltransferases and sugar-donor selectivity is imperative to improve metabolic engineering.33 Tea is one of the most widely consumed beverages in the world, and flavonoid compounds are the predominant secondary metabolites in tea plants, mainly in the form of flavonoid glycosides including glucoside, galactoside, galactosylrutinoside, glucosylrutinoside, rhamnosylgalactoside, rhamnosylglucoside of kaempferol (K), quercetin (Q), and myricetin (M) at 3-OH groups.34−36 132 UGTs in tea plant were screened based on a transcriptome database, and three of them (CsUGT78A14, CsUGT78A15, and CsUGT82A22) were identified to be involved in the biosynthesis of astringent taste compounds.27 Here, a new UFGT, CsUGT73A20, was isolated from mature leaves of the tea plant. Heterologous expression of CsUGT73A20 in tobacco (Nicotiana tabacum) was used to detect the gene function in vivo. The recombinant CsUGT73A20 (rCsUGT73A20) protein expressed in Escherichia coli (E. coli) showed a rather broad substrate tolerance and performed multisite glycosidation toward many flavonoids forming different products mainly containing 3-O-glucoside and 7-O-glucoside in vitro (Figure 1). In addition, optimization of reaction pH, temperature, and time course was conducted to identify the biochemical characterization of CsUGT73A20.

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MATERIALS AND METHODS

Plant Materials. Tea plants (Camellia sinensis cv. shuchazao) were grown in the tea garden of Anhui Agricultural University, and the young buds, leaves (first, second, third, fourth, fifth leaf), stem, and roots were selected, immediately frozen in liguid nitrogen, and then stored at −80 °C. The tobacco (Nicotiana tabacum) plants used for transgenic assays were grown in a growth chamber with constant temperature of 28 ± 3 °C, as well as a 12/12 h (light/dark) photoperiod with a constant light intensity (150−200 μmol m−2 s−1). Chemicals. All biochemicals including kaempferol (K), quercetin (Q), myricetin (M), naringenin (N), apigenin (A), kaempferide (KC), K3G, K7G, and UDP-glucose were purchased from Sigma (St. Louis, MO, USA). The methanol, acetonitrile, and acetic acid for HPLC analysis were supplied from Tedia Co. Ltd. (Fairfield, OH, USA). RNA Isolation, Cloning of CsUGT73A20. Total RNA was isolated from the different tea organs (bud, young stem, and tender root) using the RNAiso-mate (Takara, DaLian, China) and RNAiso Plus Kit (Takara, DaLian, China) according to the manufacturer’s protocol. The cDNA was generated by using the PrimeScript RT Reagent Kit (Takara, Dalian, China), following the manufacturer’s protocol. The 5′-RACE (rapid amplification of cDNA ends) and 3′RACE cDNA library was constructed by using a SMARTer RACE cDNA Amplification Kit. The PCR primers are shown in Table S2. 2075

DOI: 10.1021/acs.jafc.6b05619 J. Agric. Food Chem. 2017, 65, 2074−2083

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Journal of Agricultural and Food Chemistry apigenin, kaempferol, kaempferide, quercetin, myricetin, epicatechin, and cyanidin). The reaction was maintained at 30 °C for 35 min, and the reaction was stopped by adding 50 μL of chromatographic pure methanol. Optimal reaction condition analysis was performed using kaempferol and UDP-glucose as substrates at varying pH and temperatures. The pH (from pH 4.0 to 11.0) was controlled by acid−sodium citrate buffer (pH 4.0−7.5), Tris-HCl buffer (pH 7.0− 10.0), and NaHCO3−Na2CO3 buffer (pH 9.0−11.0). The reaction temperatures were from 0 to 70 °C with 5 °C increments. Analysis of Enzymatic Products. Products from CsUGT73A20 activity assay were analyzed by a reverse-phase high performance liquid chromatography (HPLC) LC10Avp system (Shimadzu, Kyoto, Japan). The column was eluted with the mobile phase consisting of eluent A (1% acetic acid) and eluent B (100% acetonitrile) at room temperature according to the detection method described previously.27 The elution program was as follows: starting with 10% B, a linear gradient from 10 to 25% B for 0−5 min, 25−50% B for 5−10 min, 50−80% B for 10− 17 min, 80−30% B for 17−20 min, 30−10% B for 20−21 min was performed, followed by washing and equilibrating of the column. A UPLC-MS/MS system was used to identify the products. The system contains a quaternary pump with a vacuum degasser, thermostated column compartment, autosampler, DAD, and a triple quadrupole mass spectrometer (QQQ) purchased from Agilent Technologies (Palo Alto, CA, USA). Samples were analyzed using UPLC-QQQMS/MS with an Agilent 20RBAX RRHD Eclipse Plus C18 column (particle size, 1.8 mm; length, 100 mm; and internal diameter, 2.1 mm) with a flow rate of 0.4 mL·min−1 following previously published protocols.27,37 Accumulation Trend of Products and Kinetic Parameter Analysis. The time course reactions were performed at 35 °C from 0 to 300 min in 100 mM Tris-HCl (pH 8.0) and NaHCO3−Na2CO3 (pH 9.0) buffer. Furthermore, rCsUGT73A20 was investigated at pH 8.0 using naringenin, apigenin, kaempferol, kaempferide, and kaempferol monoglucosides as acceptor substrates and UDP-glucose as the donor substrate. Kaempferol, K3G, and K7G were chosen to study the kinetic parameters of rCsUGT73A20 at pH 9.0, in order to characterize the different regioselective substrate recognition of rCsUGT73A20. The reaction conditions were assayed in a volume of 50 μL (incubated at 35 °C for 10 min) containing 2.5 mM UDPglucose and varying concentrations (0 μM to 300 μM) of substrates, 100 mM Tris-HCl (pH 8.0) or NaHCO3−Na2CO3 (pH 9.0), 10 μg of recombinant protein, and 0.1% (v/v) β-mercaptoethanol. All kinetic assays were repeated in triplicate, along with reactions without recombinant protein as controls. Products from activity assay were stored at −20 °C for HPLC analysis. The kinetic parameter analysis was performed as previously described in pH 8.0, and the naringenin, apigenin, kaempferol, kaempferide, kaempferol monoglucosides, and UDP-glucose were the substrates. Development of Binary Construct and Transformation of CsUGT73A20 into Tobacco. Using the ORF of CsUGT73A20 as a template, the BP primers (Table S2) were used to add a BP adaptor. The PCR products were purified and cloned into the entry vector pDONR207 by Gateway BP Clonase Enzyme mix according to the manufacturer's instructions (Invitrogen, USA). Subsequently, the CsUGT73A20 was transfered into the expression vector PCB2004 with the Gateway LR Clonase (Invitrogen, USA) system. At last the recombinant pCB2004-CsUGT73A20 plasmid was transferred into the Agrobacterium tumefaciens EHA105-competent through electroporation at 2500 V for about 5.5 ms. The positive clones were screened for tobacco transformation. A positive pCB2004-CsUGT73A20 EHA105 colony was selected on agar-solidified medium containing 50 mg/L kanamycin and 50 mg/L spectinomycin for the subsequent genetic transformation of tobacco plants via a leaf disk transformation method.38 The identification and selection of potential transgenic plants for regeneration utilized MS medium supplemented with 25 mg/L of phosphinothricin. Multiple transgenic plants were identified using both genomic DNA-based PCR and RT-PCR. The phenotypes of the transgenic plants growing in an environmental chamber were recorded in order to characterize the effect of transgene overexpression

on growth. Three lines with high transcript levels were used for metabolic profiling analysis as described below. Flavonoid Analysis for Transgenic versus Wild-Type Tobacco Plants. Flavonoid compounds were extracted using the following procedure. The fresh young leaves of two-month-old tobaccos were ground to a fine powder in liquid nitrogen. Then powdered samples (0.2 g) were extracted with extraction solution (80% methanol, 19% water, 1% hydrochloric acid) by vortexing for 5 s followed by water bath sonication for 15 min. After centrifugation at 6000 rpm in low temperature for 10 min, the supernatant was collected and placed in a new 2 mL tube. The residue was extracted three times following the method mentioned above, and the final volume was then adjusted to 2 mL. Pooled supernatants were extracted twice with chloroform to remove chlorophyll and then filtered through a 0.45 μm polytetrafluoroethylene (PTFE) syringe filter (Millipore, Billerica, MA, USA). Flavonoids were analyzed using ultrahigh performance liquid chromatography (UPLC)-QQQ-MS/MS on an Agilent LC-MS system (Palo Alto, CA, USA). Phenolics were separated in an Agilent 20RBAX RRHD Eclipse Plus C18 column (particle size, 1.8 μm; length, 100 mm; and internal diameter, 2.1 mm; Palo Alto, CA, USA). The column oven, mobile gradient, and electrospray ionization technique were utilized as previously described.37

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RESULTS

cDNA Cloning, Sequence Comparison, and Phylogenetic Analysis. In our previous study, 132 UGTs were screened from tea transcriptome databases.27 One of them, the CsUGTD15 located in group D according to the phylogenetic tree, is predicted to encode a flavonoid glycosyltransferase. In order to get the full length cDNA of this gene, 3′ RACE and 5′ RACE clone assays were performed based on the EST sequences of CsUGTD15 (Figure S1). After sequencing of the RACE PCR products, it was shown that the full-length cDNA of the gene is 1789 bp long containing a 3′ untranslated region (UTR) of 236 bp, a 5′ UTR sequence of 128 bp, and an open reading frame of 1428 bp, encoding a protein of 475 amino acid residues. This putative protein has a theoretical molecular mass of 53.72 kDa and an isoelectric point of 5.62. Then this gene was designated as CsUGT73A20 based on the nomenclature system agreed upon by a committee and submitted to the GenBank database (accession number: KP682358).39 A blastp procedure was performed to find homologous genes with CsUGT73A20, and an alignment of these UGTs was constructed using DNAMAN software. CsUGT73A20 showed 65% identity to a scopoletin glucosyltransferase (TOGT) from Nicotiana tabacum, that could be induced by salicylic acid and pathogens.40,41 CsUGT73A20 also shared 61% identity to a flavonoid 7-O-glucosyltransferase UGT73A10 from Lycium barbarum, and 53% identity to FaGT7, from Fragaria ananassa, which catalyzed and formed quercetin 3-O-glucoside and 4′-O-glucoside and also participated in xenobiotic metabolism.42,43 Our gene shared only 30% identity to AtUGT89C1, which transfers rhamnose from UDPrhamnose to the 7-OH site of Q3G, and 21% identity with VvGT1, a UDP-glucose:flavonoid 3-O-glycosyltransferase.44,45 According to the Akio Noguchi43 classification, flavonoid glycosyltransferases can be classified into four functional groups (clusters I, II, IIIa, IIIb, and IV). The four groups are predicted to encode 3-O, 5-O, and 7-O glycosyltransferases and diglycoside/disaccharide chain glycosyltransferases, respectively. We constructed a phylogenetic tree of CsUGT73A20 and 47 other flavonoid glycosyltransferases using MEGA6 by neighbor-joining distance analysis (Figure 2). Tree nodes were evaluated by the Bootstrap method for 1000 replicates, and the 2076


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donor (Figure S3I,J). The single reaction product was detected when using naringenin and apigenin as substrates in the enzymatic activity assay (Figure 4A,B). Interestingly, at least two product peaks were detected when using flavonol, its methylate derivatives, and glycoside as substrates (Figure 4C− H). This indicated that CsUGT73A20 performed multiposition glycosidation toward flavonol compounds. The above-mentioned products were verified using UPLC-QQQ-MS/MS analysis by retention time (time), UV (λmax), parent ions ([M − H]−), and daughter ion spectra (MS/MS) of standards (Table 1). Product peak 1 was confirmed as naringenin glucoside, and product peak 2 was apigenin glucoside. Product peaks 3−5 were confirmed as kaempferol disaccharides, of which peak 3 was identified as kaempferol 3,7-O-diglucoside according to the standard. Product peaks 6−8, peaks 9−11, and peaks 12−15 were confirmed as kaempferol, quercetin, and myricetin monoglucosides, respectively. Peaks 16 and 17 were identified as kaempferide disaccharide and monoglucosides, respectively. The major products of the above-mentioned flavonol monoglucoside were identified as 3-O-glucoside and 7O-glucoside according to corresponding standards (Table 1). The other products were not verified accurately due to unavailable standards. In all, CsUGT73A20 showed a broad substrate tolerance and performed multisite glycosidation toward flavonoid compounds forming products mainly containing 3-O-glucoside and 7-O-glucoside in vitro. Optimization of Reaction pH, Temperature, and Time Values of rCsUGT73A20. In order to identify the biochemical characterization of CsUGT73A20, the kaempferol and UDPglucose were used as substrates to determine the optimal reaction conditions. The reaction buffer was designed from pH 4.0 to pH 11.0, and the reaction temperature ranged from 10 to 70 °C. It was found that more K7G product accumulated at pH 8.0 at 55 °C, but K3G was the main product at pH 9.0 at 35 °C (Figure 5A−D). From the reaction time course, K7G was the first product formed whether in pH 8.0 or 9.0. K-3,7-di-Oglucoside (K37G) and K3G accumulated later than K7G, suggesting that a 3-OH site glycosylation on K and K7G took place after 7-OH site glycosylation (Figure 5E,F). More K37G accumulated at pH 8.0 than pH 9.0, although the first product, K7G, was not found at pH 9.0. It was possible that the higher pH, especially pH 9.0, repressed the productivity of K7G, thereby resulting in a decrease of K37G compared to pH 8.0 (Figure 5E,F). Due to the lack of K7G, more K3G accumulated at pH 9.0 than pH 8.0. K M Values for Each Substrate. The kinetics of CsUGT73A20 was further investigated on flavonoid substrates at pH 8.0 and 35 °C. CsUGT73A20 showed the highest affinity to K substrate (KM = 4.9 μM) (Figure 6C) compared to the other substrates: N (KM = 13.6 μM), A (KM = 18.2 μM), Q (KM = 37.7 μM), K3G (KM = 27.5 μM), K7G (KM = 13.1 μM), and KC (KM = 19 μM) (Figure 6A,B,D,E,F,G). Interestingly, CsUGT73A20 showed higher affinities toward K7G (KM = 13.1 μM) rather than K3G (KM = 27.5 μM). To verify if the glycosylation was repressed in high pH, the kinetics of CsUGT73A20 were investigated at pH 9.0 and 35 °C (Figure 6H,I). CsUGT73A20 still exhibited strong activity to K and K7G at pH 9.0, with higher affinity to K7G (KM = 32 μM) than to K (KM = 46.7 μM). No product was found when using K3G as substrate at pH 9.0 (data not shown). This kinetic analysis demonstrated that high pH repressed the glycosylation reaction at the 7-OH site in vitro.

Figure 2. Phylogenetic tree of CsUGT73A20 and UGT homologues from other plants. The phylogenetic tree was constructed using MEGA5 with the neighbor-joining method. Clusters (I, II, IIIa, IIIb, and IV) are shown with gray background. CsUGT73A20 are shown in boldface letters. All the GenBank accession numbers of the sequences used in the phylogenetic analysis are summarized in Table S1.

evolutionary distances were computed using the p-distance method. All the GenBank accession numbers of the sequences used in the phylogenetic analysis are summarized in Table S1. CsUGT73A20 was clustered together with UGT73A10, TOGT1, and TOGT2 in cluster IIIa. The enzymes in this cluster were predicted to be involved in transferring glycosyl group to the 7-OH position at the A ring of flavonoid compound. An alignment of the CsUGT73A20 with VvGT1 and other UGTs grouped in cluster IIIa showed a conserved PSPG box in the C-terminus (Figure 3). Key amino acid residues are described with red dots (involved in ligands), blue asterisks (UDP-sugar donor active sites), green asterisks (sugar acceptor active sites), red asterisks (the key residues determining the GalT and GlcT activity specificity), and black rectangles (PSPG box) compared with VvGT1.45 Heterologous Expression in E. coli and Product Identification. The ORF of CsUGT73A20 was cloned into the pMAL-C2X expression vector with a maltose-binding protein (MBP) tag and expressed in E. coli. The recombinant CsUGT73A20 (rCsUGT73A20) protein was purified followed by a MBP tag purification system (Figure S2). Enzymatic reaction analysis indicated that rCsUGT73A20 protein exhibited a broad substrate preference toward multiple flavonoids including naringenin (N), apigenin (A), quercetin (Q), myricetin (M), kaempferol (K), K3G, K7G, and kaempferide (KC) when UDP-glucose was used as the sugar donor (Figure 4), but no product was detected when UDPgalactose was used as the sugar donor (Figure S3A−H). The transferase activity assay of rCsUGT73A20 toward rhamnose was not performed due to unavailable commercial UDPrhamnose donor. Cyanidin and epicatechin were also chosen as substrates, but rCsUGT73A20 protein showed no enzyme activity to these two flavonoid compounds, no matter whether UDP-glucose or UDP-galactose was supplied as the sugar 2077


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Figure 3. Amino acid sequence alignment of UGT73A20 and five homologies. The identified UGTs in multiple alignment are VvGT1 (AAB81683.1), FaGT6 (Q2 V6K0), FaGT7 (Q2 V6J9), NtTOGT1 (AAK28303), and UGT73A10 (BAG80536). The multiple alignment was performed using DNAMAN software. Red dots indicate amino acid residues that are involved in ligand binding. Blue asterisks indicate UDP-sugar donor active sites. Green asterisks indicate sugar acceptor active sites. Red asterisks indicate the key residues determining the GalT and GlcT activity specificity. Black rectangles indicate PSPG box.

Heterologous Expression in Tobacco. The ORF of CsUGT73A20 was overexpressed in tobacco, and the positive transgenic plants were obtained using antibiotic selection. There was no phenotypic difference between CsUGT73A20 transgenic and wild-type tobacco plants (data not shown). The total flavonoid compounds in CsUGT73A20 transgenic leaves and WT tobacco leaves were extracted and measured through a UPLC-QQQ-MS/MS system. Compared with WT tobacco, the contents of five flavonol-glucosides were found to be increased (Figure 7, peaks 1, 2, 3, 4, 5). Three kinds of new flavonol-glucosides (peaks 1, 2, 4) were produced in transgenic tobacco. According to the UPLC-QQQ-MS/MS results and previous reports,37 the peak 1 product was identified to be Q3G7Rha, peak 2 was Q3Rha7Rha, peak 3 was K3G7Rha, and peak 4 was K3G. CsUGT73A20 transgenic tobacco accumulated more Q3Rha7Rha (peak 2) and Q3Rha (peak 5), indicating that CsUGT73A20 could function as a 3-O- and 7-Oglucosyltransferase, and it likely used UDP-rhamnose as a sugar donor with flavonol and flavonol monoglucoside as substrates in vivo, similar to UGT72AD1 and UGT72Z2 in A. thaliana.46 Since UDP-rhamnose was not available, we cannot determine whether CsUGT73A20 has rhamnosyltransferase function in vitro.

can occur on individual hydroxyl groups of the aglycon at 3-, 5-, 7-, 3′-, 4′-OH, or multiple hydroxyl groups simultaneously. But only a few UGTs were demonstrated to catalyze multiple hydroxyl glycosylations. In Arabidopsis, 91 UGTs were isolated, and 29 of them showed activity to quercetin, but only one, AtUGT88A1, showed catalyzing activities to 3-, 7-, 3′-, and 4′OH of quercetin simultaneously.11 FaGT6 from strawberry can catalyze quercetin to form Q3G, Q7G, Q4′G, Q3′G, and a Qdiglucoside. Similarly, FaGT7 catalyzed quercetin to form Q3G, Q4′G, Q7G, and Q3′G, but no Q-diglucoside.42 Here, a new UGT (CsUGT73A20) was isolated in tea plants. Phylogenetic analysis predicted it had a putative 7-hydroxy position catalyzing activities to flavonoid substrates (Figure 2). The rCsUGT73A20 protein in E. coli could catalyze multiple flavonoids, including N, A, flavonol, and flavonol monoglucoside at 3- and 7-OH and other hydroxy positions. This indicated that CsUGT73A20 would be powerful enough to transfer different kinds of compounds to meet the needs of plants (Figure 5). CsUGT73A20 also showed changeable activity that was more similar to FaGT6 than FaGT7 from strawberry.42 Another interesting phenomenon was that CsUGT73A20 catalyzed kaempferol to form three diglucoside products simultaneously. In contrast, only one diglucoside product was detected from AtUGT88A1.11 In the future, point mutation analysis of the key amino acid residues in CsUGT73A20 will help us further understand the catalyzing mechanism of CsUGT73A20 on different hydroxyl positions.

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DISCUSSION Multisite Glycosidation Is Catalyzed by CsUGT73A20. In plants, the glycosylation process to flavonoid compounds 2078


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Figure 4. Analysis of recombinant CsUGT73A20 enzymatic reaction products. Panels A−H indicate the HPLC chromatograms of the enzymatic reaction (top panel) and control (bottom panel) with substrates including naringenin (N), apigenin (A), kaempferol (K), quercetin (Q), myricetin (M), kaempferide (KC), kaempferol 3-O-glucoside (K3G), and kaempferol 7-O-glucoside (K7G), respectively. Arabic numbers 1−17 indicate enzymatic products listed in Table 1.

Heterologous Expression in Tobacco. It had been reported that gene transformation, both overexpression and knockout, of some UGTs would increase or decrease the plant’s defense to external pathogens and endogenous toxins. Overexpression of AtUGT73C5 enhanced resistance to deoxynivalenol in Arabidopsis thaliana.47 AtUGT76B1 knockout and overexpression lines showed accelerated and delayed senescence in growth period, respectively, and altering pathogen susceptibility simultaneously.48 To determine if it could catalyze the relevant reaction in vivo, the ORF of CsUGT73A20 was transferred into tobacco plants. The metabolite analysis showed that some Q3G7Rha, Q3Rha7Rha, K3G, Q3Rha, flavonol glucosides, and flavonol rhamnosides accumulated in CsUGT73A20 transgenic tobacco plants. This suggested that CsUGT73A20 might function as a 3-O- and 7O-rhamnosyl and glucosyltransferase in vivo, similar to UGT72AD1 and UGT72Z2 in A. thaliana. We did not test the direct effects of CsUGT73A20 on senescence, toxin and pathogen resistance, and hormone response of transgenic tobacco, but it was reported that the accumulation of flavonol glucosides and flavonol rhamnosides would improve the ability

of plants to adapt adverse environmental conditions (source).1,49 pH Regulates the Catalyzed Activities of rCsUGT73A20. The biochemical characteristic analysis can provide the basic characteristics of UGTs in vivo and are also significant approaches in metabolic engineering. Here, the biochemical characteristic analysis of CsUGT73A20 was performed, and the results indicated that K was the optimal substrate compared to Q, N, and K7G substrates (Figure 6). Most glycosyltransferase activity could be affected by pH, but their catalytic sites would not be changed.23 It was found that more K7G product was accumulated at pH 8.0, but K3G was the main product in pH 9.0. More importantly, CsUGT73A20 catalyzed to form more K37G in a pH 8.0 reaction system, but to form more K3G in a pH 9.0 reaction system, indicating that the pH could regulate the activity of CsUGT73A20 and higher pH repressed the 7OH site glucosylation (Figure 5D,E). The effect of pH on the activity of CsUGT73A20 may be due to the change of the regioselectivity of enzyme, or the changes of the structure of the substrate. In addition, Tris buffer also had an inhibitory effect on the activity of recombinant UGTs in a prokaryotic 2079


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Journal of Agricultural and Food Chemistry Table 1. MS Analyses of Products in Figure 5a

a

peak/no.

tR1 (min)

tR2 (min)

[M − H] (m/z)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

8.474 8.391 5.943 6.547 6.781 8.240 8.374 8.609 13.025 13.301 15.097 10.29 11.446 13.554 15.805 5.439 7.423

13.57 13.58 10.51 11.26 11.7 13.56 13.7 13.95 12.70 12.88 13.61 11.49 12.12 12.98 13.88 11.76 14.61

433 431 609 609 609 447 447 447 463 463 463 479 479 479 479 623 461

MS/MS (m/z) 433, 431, 609, 609, 609, 447, 447, 447, 463, 463, 463, 479, 479, 479, 479, 623, 461,

271 269 447, 285 447, 285 447, 285 285 285 285 301 301 301 317 317 317 317 461, 298 298.9

UV λmax (nm)

identification

287 335 346 366 340 347 366 366 370 365 365 372 356 362 365 346 366

naringenin 7-O-glucoside apigenin 7-O-glucoside kaempferol 3,7-O- diglucoside kaempferol diglucoside kaempferol diglucoside kaempferol 3-O-glucoside kaempferol 7-O-glucoside kaempferol glucoside quercetin 7-O- glucoside quercetin 3-O-glucoside quercetin glucoside myricetin 7-O-glucoside myricetin 3-O-glucoside myricetin glucoside myricetin glucoside kaempferide diglucoside kaempferide 7-O- glucoside

* * *

* * * * * *

*

The asterisks represent the substance that we can identify based on the standards or our previous experimental results.

Figure 5. Optimization of reaction pH, temperature, and time values of rCsUGT7A20. (A) Product K7G accumulation profile at different pH conditions (pH 4.0−11.5). (B) Product K3G accumulation profile at different pH conditions (pH 4.0−11.5). (C) Product accumulation profile under various temperatures (10−70 °C) at pH 8.0. (D) Product accumulation profile under different temperatureS 10−70 °C at pH 9.0. (E) A time course of product accumulation from 0 to 7 h at pH 8.0 at 55 °C. (F) A time course of product accumulation from 0 to 7 h at pH 9.0 at 35 °C. Buffer A: acid−sodium citrate buffer (pH 4.0−7.5). Buffer B: Tris-HCl buffer (pH 7.0−10.0). Buffer C: NaHCO3−Na2CO3 buffer (pH 9.0−11.0).

system.17,50 These problems need to be further studied. The characterization of multisite glucosylation of CsUGT73A20

contributes that the tea plant is more adaptable to many environments. 2080


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Figure 6. Initial reaction rate to concentration plots and reciprocal plots showing kinetics of rCsUGT73A20 to different substrates. (A−G) Kinetics of rCsUGT73A20 to substrates including naringenin (N), apigenin (A), kaempferol (K), quercetin (Q), kaempferol 3-O-glucoside (K3G), kaempferol 7-O-glucoside (K7G), and kaempferide (KC) at pH 8.0 at 35 °C. (H) Kinetics of rCsUGT73A20 to K and K7G at pH 9.0 at 35 °C.

Figure 7. LC-MS profiles showing products from transgenic CsUGT73A20 tobacco. (A−D) The QQQ-MS/MS chromatogram of total ions and separated product molecular ions of flavonol glycosides extracted from transgenic tobacco (A, m/z of 609; B, m/z of 593; C, m/z of 447; D, m/z of 463). The black line represents the transgenic tobacco sample, and the red line represents the control sample.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b05619. Race and high-fidelity PCR electrophoresis diagram, SDS−PAGE of protein fractions, and analysis of the control of recombinant CsUGT73A20 enzymatic reaction products (PDF) Sequence information used in the phylogenetic tree (PDF) Primer sequences (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*T.X.: State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui 230036, China. Tel: 86-551-65786003. Fax: 86-551-65785833. E-mail: [email protected]. *L.G.: School of Life Science, Anhui Agricultural University, 130 West Changjiang Rd, Hefei, Anhui 230036, China. Tel: 86551-65786232. Fax: 86-551-65785729. E-mail: gaolp62@126. com ORCID

Tao Xia: 0000-0003-0814-2567 Author Contributions §

X.Z. and P.W. contributed equally to this work.

Funding

This work was supported by the Natural Science Foundation of China (31470689, 31270730, 31200229, and 31570694), the Specialized Research Fund for the Doctoral Program of Higher Education (20133418130001), the Natural Science Foundation of Anhui Province, China (1708085MC58), the Special Foundation for Independent Innovation of Anhui Province, China (13Z03012), and the Biology Key Subject Construction of Anhui. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Huarong Tan for assistance with LC/MS analysis. ABBREVIATIONS USED UGT, UDP-glycosyltransferase; UDP, uridine diphosphate; rCsUGT73A20, recombinant CsUGT73A20 protein; UPLCQQQ-MS/MS, ultrahigh performance liquid chromatography coupled to a triple quadrupole mass spectrometer; Q3Rha, quercetin 3-O-rhamnoside; K3G, kaempferol 3-O-glucoside; Q3Rha7Rha, quercetin 3-O-rhamnoside-7-O-rhamnoside; Q3G7Rha, quercetin 3-O-glucoside-7-O-rhamnoside; K3G7G, kaempferol 3-O,7-O-diglucoside; Q7G, quercetin 7-O-glucoside; Q3G, quercetin 3-O-glucoside; Q4′G, quercetin 4′-Oglucoside; Q3′G, quercetin 3′-O-glucoside

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Functional Characterization of a New Tea (Camellia sinensis

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