Characterization of Three Glucosyltransferase Genes in Tartary

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Characterization of Three Glucosyltransferase Genes in Tartary Buckwheat and Their Expression after Cold Stress Jing Zhou,†,∥ Cheng-Lei Li,†,∥ Fei Gao,† Xiao-Peng Luo,† Qing-Qing Li,† Hai-Xia Zhao,† Hui-Peng Yao,† Hui Chen,† An-Hu Wang,§ and Qi Wu*,† †

College of Life Science, Sichuan Agricultural University, 46 Xinkang Road, Ya’an, Sichuan 625014, China Xichang College, Xichang, Sichuan 615000, China

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S Supporting Information *

ABSTRACT: Anthocyanins confer the red color in the hypocotyl of tartary buckwheat sprouts. Uridine diphosphate (UDP)glucose:flavonoid 3-O-glycosyltransferase (UFGT) stabilizes anthocyanin by attaching the glucosyl moiety from UDP-glucose to the C3 hydroxyl of anthocyanin. In this study, we characterized three UFGT-like genes, designated FtUFGT1, 2, and 3 from tartary buckwheat. The results revealed that FtUFGT1, FtUFGT2, and FtUFGT3 can convert cyanidin to cyanidin 3-Oglucoside, with specific activities of 20.01 × 10−3, 8.93 × 10−3, and 20.24 × 10−3 IU/mg, respectively. The active-site residues of the C-terminal domains and the N-terminal domains are important for the donor and acceptor recognition of these proteins. The expression of the three FtUFGTs paralleled the tissue-specific anthocyanin accumulation. After cold treatment, the increased content of anthocyanin was accompanied by the up-regulated expression of the three FtUFGTs. Among these three UGFT gene members, FtUFGT3 showed the highest expression level and the highest specific activity, suggesting that FtUFGT3 might be the major gene involved in anthocyanin biosynthesis. These results suggested that the FtUFGT genes, FtUFGT3 in particular, might be important candidates for anthocyanin formation in tartary buckwheat sprouts. KEYWORDS: UDP-glucose:flavonoid 3-O-glycosyltransferase, anthocyanins, cold treatment, specific activity, tartary buckwheat sprouts

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INTRODUCTION Tartary buckwheat (Fagopyrum tataricum), a member of the Polygonaceae family, is a traditional medicinal and edible cereal crop.1 Tartary buckwheat sprouts have been recognized for their high nutritive value, including minerals, crude fiber, amino acids, and protein.2,3 In recent years, there have been some coherent reports about the pigments of tartary buckwheat sprouts. The red color of the tartary buckwheat sprouts has been attributed to the accumulation of anthocyanins, including cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside.4−6 Anthocyanins, a subclass of plant flavonoids, are the major watersoluble pigments in fruits and vegetables.7 Many studies have indicated that moderate consumption of anthocyanins is beneficial to health because of their antioxidative, antitumor, anti-inflammatory, and antiatherosclerotic properties.8,9 As a functional vegetable, tartary buckwheat sprouts have become increasingly popular.3 Anthocyanin is biosynthesized via the flavonoid biosynthetic pathway. The main steps of the pathway are well-known.7,10,11 Chalcone synthase (CHS), chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose:flavonoid 3-O-glycosyltransferase (UFGT) are involved in the formation of colored and stable anthocyanidins. Most anthocyanidins can be further modified by glycosylation, acylation, and methylation, leading to the structural diversity of secondary metabolites. In particular, glycosylation occurs at the final step in anthocyanin biosynthesis and plays a significant role in modulating the solubility, stability, and bioactivity of anthocyanins.12 Glycosylation reactions are generally catalyzed by uridine diphosphate © XXXX American Chemical Society

glycosyltransferases (UGTs) belonging to family-1 glycosyltransferases, of which the best studied is UFGT (EC 2.4.1.91). The stability of anthocyanin is increased by UFGT by the transfer of the glucosyl moiety from uridine diphosphate (UDP)-glucose to the C3 hydroxyl to produce the first stable pigment (Figure 1).13,14 One of the most regulated steps in the

Figure 1. Glycosylation at the 3-hydroxyl of anthocyanin. UDP, uridine diphosphate; Glc, glucose.

anthocyanin biosynthetic pathway is the expression of the UFGT genes.15−17 In particular, two famous mutants of bronze1 and ANL1 accumulate less anthocyanin than the corresponding nonmutant plants.18,19 UFGT is a multigene family, with more than 120 UFGTs identified in Arabidopsis thaliana20 and 165 in Medicago truncatula.21 Phylogenetic analyses based on the amino acid sequences of the UFGTs indicate that these Received: May 6, 2016 Revised: July 31, 2016 Accepted: August 29, 2016

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DOI: 10.1021/acs.jafc.6b02064 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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uk/chebi/) with ChEBI ID 17659 and 71682, respectively. All simulations were performed using Accelrys Discovery Studio 2.5 (DS) software (http://www.accelrys.com/). Molecule docking was performed using the receptor−ligand docking tool (LibDock) in DS. MEGA 5.0 was used to construct the phylogenetic tree (http://www. megasoftware.net/). Expression and Purification of the Three Recombinant FtUFGTs in E. coli. The three plasmids were constructed by inserting each of the three full-length FtUFGT genes into the pGEX 4T-1 GST fusion expression vector. The primers designed for amplifying the coding region of three FtUFGTs are listed in Table S1 (primers for expression). Each construct was transformed into E. coli BL21 (DE3) cells (Novagen, Germany). Cells were grown at 37 °C overnight in LB medium containing ampicillin (50 μg/mL) until the optical density at 600 nm (OD600) reached 0.6. The soluble recombinant protein was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM and incubating the culture at 25 °C for 10 h. A control culture containing an empty pGEX 4T-1 GST vector was grown under the same conditions. The cells were harvested after 10 h of induction. To assay the activities of the crude enzymes, 30 μL of crude extract was added to a 200 μL reaction mixture of the crude UFGT assay described by Ford et al.23 The reaction mixture was extracted twice by the addition of equal volumes of ethyl acetate. The ethyl acetate extracts (40 μL) were analyzed by thin-layer chromatography (TLC) on silica gel G in a solvent composed of 4 parts n-butanol, 2 parts acetic acid, and 4 parts water. The standards cyanidin and cyanidin 3O-glucoside were used for external standards. The retention factor (Rf) values of the two standards were used to identify reaction products. Protein purification was performed at 4 °C. The recombinant proteins were purified by glutathione−agarose affinity chromatography using a GST-Sefinose Kit (Sangon Biotech, China) according to the manufacturer’s instructions. All samples were analyzed by 12.5% SDSPAGE. Enzyme Assays. The activities of the enzymes were assayed as previously described;23 100 mM Tris-HCl (pH 8.0), 14 mM βmercaptoethanol, 9 mM UDP-glucose (RYON, RZ1070), and 100 μM cyanidin were added to the reaction mixture at 30 °C. After preincubation for 5 min, the reaction was started by adding 5 μg of purified enzyme, and then it was incubated at 30 °C for 30 min. The reaction was terminated and extracted twice with ethyl acetate and then evaporated to dryness. The dried reaction products were redissolved in methanol, and 10 μL of the solution was infused into the triple-quadrupole mass spectrometer (TSQ Quantum Ultra; Thermo Fisher Scientific, USA). The chromatographic separation was performed on a Hypersil Gold C18 column (150 mm × 2.1 mm, 5 μm) with a mobile phase of 0.1% aqueous formic acid/acetonitrile (95:5, v/v) for 5 min. The flow rate was 0.2 mL/min. The column temperature was maintained at 30 °C. The mass spectrometer was operated in the negative ion electrospray mode, and the spray voltage was 3000 V. The sheath gas pressure was set at 35 psi and the auxiliary gas pressure at 5 psi. The MS/MS transition for cyanidin was m/z 174.93 → 240.95, with m/z 191.00 → 283.96 monitored for cyanidin 3-O-glucoside. The calibration curve was established by measuring different concentrations of reaction products (0.08, 0.16, 0.4, 0.8, and 4 μg/mL standard cyanidin 3-O-glucoside) by LC-MS/MS. The calibration curve in the range 0.08−4 μg/mL had good linearity with the regression equation Y = 188.4X (Y = peak area; X = concentration in μg/mL), R2 = 0.999. The peak area of cyanidin 3-O-glucoside was used for the quantification of reaction products. There were two replicates for every sample. One unit (IU) was defined as the quantity of UFGT that catalyzed the formation of 1 μmol of reaction product per minute at 30 °C. Quantitative Real-time PCR Analysis. Quantitative real-time PCR (qRT-PCR) was carried out using SYBR Premix Ex Taq II (TaKaRa, Japan) and the CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Each reaction (final volume, 15 μL) contained 7.5 μL of SYBR Green Mix, 2.5 μL of each the forward and reverse primers (10 mM), 1 μL of the cDNA template (corresponding to 50 ng of total RNA), and 4 μL of RNase-free water. The thermal

enzymes can be classified into three different groups, UF3GT, UF5GT, and UF7GT, according to the regioselectivity of flavonoid glycosylation.17,22 UF3GT enzymes, which glycosylate the anthocyanins at the 3-O-position, have been identified and functionally characterized in several plant species, including Litchi chinensis,17 Vitis vinifera,23 and Fragaria × ananassa.24 Anthocyanins are important bioactive substances that serve multiple ecological and physiological functions in plant.25 Besides the great contribution to pollination and seed propagation, anthocyanins play important roles in protection against cold temperature, ultraviolet B radiation, and other abiotic stresses.26 Cold temperature can induce anthocyanin accumulation in various plants, such as Sorghum, Arabidopsis, Cotinus, and Poncirus.27 McKown et al.28 suggested that there was a close correlation between anthocyanin synthesis and cold tolerance through both synthetic and regulatory pathways. Many previous studies demonstrated that the cold-induced anthocyanin accumulation was accompanied by the expression of anthocyanin biosynthetic genes, including CHS, DFR, ANS, and UFGT.29−31 For example, Crifò et al.30 observed that UFGT was strongly induced by cold treatment and associated with anthocyanin accumulation in blood oranges at 4 °C. In this study, we characterized three UFGT genes from tartary buckwheat. The catalytic activities of three recombinant proteins were assayed in the presence of UDP-glucose and cyanidin, and the substrate-binding mechanism of these proteins was proposed. In addition, we analyzed their association with anthocyanin accumulation and cold tolerance in tartary buckwheat sprouts by expression analysis.

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MATERIALS AND METHODS

Plant Materials and Treatments. Tartary buckwheat was grown under field conditions (sown in May) in the experimental farm of Sichuan Agricultural University, Ya’an, Sichuan, China (102.98° E, 29.98° N). Flowers, stems, leaves, and roots were sampled during florescence (in July) and stored at −80 °C for RNA, DNA, and anthocyanin extraction. To detect FtUFGT expression under cold treatment, tartary buckwheat seeds were germinated at 30 °C for 2 days in the dark and then were grown in 1/4 Hoagland’s solution with a 14 h photoperiod at 25 °C for 10 days.32 Then, 10-day-old tartary buckwheat seedlings were transferred to a growth chamber for 24 h at 4 °C with a 14 h photoperiod. The treated seedlings were sampled at 0, 3, 6, 12, and 24 h after treatment. The samples were stored at −80 °C for RNA and anthocyanin extraction. Cloning of the Full-Length cDNA and Genomic DNA of the Three FtUFGTs. Plant Genomic DNA Kits (TIANGEN, China) were used to extract genomic DNA from tartary buckwheat leaves. RNAout kits (TIANGEN, China) were used to extract total RNA. PrimeScript first-strand cDNA Synthesis Kits (TaKaRa, Japan) were subsequently used to synthesize the cDNA according to the manufacturer’s instructions. The DNA and cDNA sequences of the three FtUFGTs were amplified by PCR with PrimeSTAR Max DNA polymerase (TaKaRa, Japan) using three pairs of gene-specific primers (Table S1, primers for cloning). The thermal cycling conditions were 30 cycles of PCR (one cycle consisted of 98 °C for 10 s, 62 °C for 5 s, and 72 °C for 30 s), followed by an extension step at 72 °C for 10 min. The PCR products were gel purified and subcloned into the pMD19-T vector (TaKaRa, Japan) and sequenced. Sequence Analysis. The amino acid sequences of the three proteins were deduced using DNAMAN and analyzed using the NCBI Blast server (http://www.ncbi.nlm.nih.gov/BLAST/). The threedimensional (3D) structures of these proteins were built using the SWISS-MODEL program (http://www.swissmodel.expasy.org/). The 3D structures of UDP and cyanidin were obtained from the ChEBI database (Chemical Entities of Biological Interest, https://www.ebi.ac. B

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Journal of Agricultural and Food Chemistry cycling conditions were 95 °C for 5 min, then 39 cycles of 95 °C for 10 s, 58 °C for 5 s, and 72 °C for 30 s, followed by a melting temperature cycle, with constant fluorescence data acquisition from 65 to 95 °C. Three pairs of specific primers for these genes were used for qRTPCR (Table S1, primers for real-time PCR assay). A housekeeping gene, H3 (histone 3, GenBank accession no. HM628903),33 was used as the reference gene for the expression analysis by qRT-PCR, using the specific H3 primers (Table S1). The gene expression data were normalized using the Ct value corresponding to the H3 gene. The expression levels of the genes were calculated using the 2−ΔΔCt method.34 There were three replicates for each gene. Quantification of the Anthocyanin Content. The anthocyanin content was quantified according to the UV spectrophotometry method described by Rabino and Mancinelli35 The pigments were extracted by shaking the samples in acidic (1% HCl, w/v) methanol overnight at 4 °C. The extracts were diluted 2-fold with distilled water and centrifuged at 13000 rpm for 3 min. The absorbance of the supernatants was measured at 530 and 657 nm. The concentration of total anthocyanin was calculated using the following formula: Q = (A530 − 0.25A657) g−1 fresh weight (FW). There were three replicates for each sample. Statistical Analysis. The statistical significance of the difference between the treatments was measured by SPSS 13.0 software using Student’s t tests.

The putative amino acid sequences of the three FtUFGT proteins showed low identity with those of other plant UGTs. FtUFGT1, FtUFGT2, and FtUFGT3 showed 37, 31, and 40% identity with VvGT1 from Vitis vinifera,14 which has been functionally characterized as a UDP-glucose:flavonoid 3-Oglycosyltransferase and shows activity with cyanidin. For understanding the catalytic mechanism of the three FtUFGT proteins, their 3D structures were predicted with the SWISS-MODEL program. Structural homology searches revealed that FtUFGT1, FtUFGT2, and FtUFGT3 are 42, 33, and 44% identical to Medicago truncatula UGT78G1 (PDB ID 3hbf), respectively. Structural comparisons indicated that the structure of UGT78G1 and our target proteins shared a common backbone architecture. The 3D structures of the three FtUFGT proteins were constructed using the crystal structure of M. truncatula UGT78G1 as a template.36 For example, molecular docking of FtUFGT3 with UDP and cyanidin is shown in Figure 3A. FtUFGT3 contains two “Rossmann-like” (β/α/β) domains, which belong to a typical GT-B fold structure.14 The seven-stranded twisted parallel β-sheet is surrounded by ten α-helices at the N-terminal β/α/β domain. Eight α-helices are arranged around the six-stranded twisted parallel β-sheet at the C-terminal β/α/β domain. The two domains form a deep narrow cleft in which cyanidin and UDP are located. Furthermore, the interactions of the two molecules (cyanidin and UDP) with the active site of FtUFGT3 protein are shown in Figure 3B. The uracil ring of UDP interacts with the indole ring of Trp348 through a π-stacking interaction. The α-phosphate of UDP forms hydrogen bonds with Asn370 and Ser371. The β-phosphate of UDP forms hydrogen bonds with Ser25 and His366. The simulation of UDP recognition is consistent with the complexes of flavonoid UGTs and sugar donors previously described.14,36,37 The acceptor-binding pocket is mainly composed of several loops and helices and is mostly hydrophobic with many aromatic residues (e.g., Phe21, Phe23, Phe127, and Trp147) and some hydrophobic residues (e.g., Leu90, Val389, and Ala390). Met93 and His157 interact with cyanidin through hydrogen bonds. His26 and Glu391 also participate in the interaction with cyanidin. The molecular docking simulations of the two molecules (cyanidin and UDP) to the 3D structures of FtUFGT1 and FtUFGT2 are shown in Figures S1 and S2, respectively. A phylogenetic tree of UGTs from different plants indicated that the three FtUFGT proteins belong to the UF3GT cluster, which is divided into monocot and dicot subclusters (Figure 4). P. frutescens and P. hybrida UF3GT did not cluster with P. frutescens and P. hybrida UF5GT, although they derive from the same species. H. vulgare and Z. mays both belong to the monocot subcluster of the UF3GT cluster, but the three FtUFGT proteins classified into the dicot subcluster of the UF3GT cluster. The results showed that the three UDP glycosyltransferase clusters diverged before the speciation of monocot and dicot plants as reported by Imayama et al.13 Expression and Purification of FtUFGTs. The three FtUFGT proteins were expressed in E. coli BL21 (DE3) to study the enzymatic properties of the gene products. Analysis by SDS-PAGE indicated that the three recombinant FtUFGT proteins were accumulated at high levels in the total expressed proteins (Figure S3). The FtUFGT3 protein showed an approximate molecular mass of 76 kDa with a GST-tag, which was in good agreement with the sum of the molecular weight predicted for FtUFGT3 and that of the GST. The molecular masses of FtUFGT1 and FtUFGT2 were ∼75 and

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RESULTS Cloning and Sequence Analysis of FtUFGTs. To search for UFGTs in the anthocyanin biosynthetic pathway, we screened the tartary buckwheat flower transcriptome database focusing on glycosyltransferase genes that were annotated as UDP-glucose:flavonoid 3-O-glucosyltransferase (data not shown). Three full-length UFGT-like cDNA sequences were obtained and designated FtUFGT1, FtUFGT2, and FtUFGT3 (GenBank IDs KX216512, KX216513, and KX216514). The open reading frames of these genes were 1341, 1500, and 1413 bp, respectively. Genomic DNA sequences containing some structural information for the three FtUFGTs are shown in Figure 2. Comparisons between the full-length cDNA and the

Figure 2. Gene structures of the three FtUFGTs of tartary buckwheat. Solid boxes and lines represent exons and introns, respectively. The start codons and stop codons are represented by ATG and TAA, respectively. The splice junction sites are represented with the base pair numbers on both ends of the lines.

genomic DNA sequences of the three FtUFGTs indicated that FtUFGT1 and FtUFGT2 consisted of two exons and one intron, whereas FtUFGT3 consisted of three exons and two introns. The splice sites were consistent with the GT-AG rule. The putative proteins, FtUFGT1, FtUFGT2, and FtUFGT3 had molecular masses of 48.61, 54.90, and 50.25 kDa, respectively, and consisted of 446, 499, and 470 amino acids. C

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Figure 3. Molecular docking of FtUFGT3 with UDP and cyanidin. (A) Ribbon diagram of the 3D structure of FtUFGT3 with UDP and cyanidin. The N-terminal and C-terminal domains are shown in blue and orange, respectively. The UDP and cyanidin molecules are shown as stick models. (B) Stereoview showing interactions of cyanidin and UDP with the active site of FtUFGT3 protein. The UDP and cyanidin molecules are shown as stick models. Residues interacting with UDP and cyanidin are shown as yellow lines and labeled. Hydrogen bonds are indicated by green dotted lines.

Figure 4. Phylogenetic analysis of the deduced amino acid sequences of flavonoid UGTs from different plant species. Three groups are UF3GTs (squares), UF5GTs (triangles), and UF7GTs (circles). The numbers on the branches indicate bootstrap values. Accession numbers are placed after the plant names.

∼81 kDa (Figure S4). Soluble crude protein extracts were assayed for the activities of the three FtUFGT proteins in the presence of the substrates UDP-glucose and cyanidin, and the products were analyzed by TLC. As shown in Figure 5, the three recombinant FtUFGTs catalyzed the formation of cyanidin 3-O-glucoside, and the Rf value of the reaction product was the same as that of the standard cyanidin 3-Oglucoside. The expressed recombinant FtUFGT proteins were purified using glutathione agarose affinity chromatography. The molecular masses of the purified FtUFGT1, FtUFGT2, and FtUFGT3 were estimated to be ∼49, ∼55, and ∼50 kDa, respectively (Figure S4). The purified yields of FtUFGT1, FtUFGT2, and FtUFGT3 were 0.314, 0.383, and 0.369 g/L of culture, respectively.

Enzyme Assays in Vitro. The activities of three FtUFGT proteins were assayed in the presence of UDP-glucose and cyanidin, and the reaction products were analyzed by LC-MS/ MS. As shown in Figure 6, the retention times of cyanidin and cyanidin 3-O-glucoside were 2.64 and 2.61 min, respectively. The mass spectra revealed molecular ions at m/z 285 [M − H]− for cyanidin and m/z 447 [M − H]− for cyanidin 3-Oglucoside. To increase the specificity, cyanidin and cyanidin 3O-glucoside were detected in the MS/MS mode. The parent ions m/z 285 and 447 were fragmented, and the transitions of m/z 174.93 → 240.95 for the cyanidin and m/z 191.00 → 283.96 for the cyanidin 3-O-glucoside were monitored. The results showed that the purified proteins, FtUFGT1, FtUFGT2, and FtUFGT3 catalyzed the conversion of cyanidin to cyanidin 3-O-glucoside in the presence of UDP-glucose. The specific D

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carried out. All three FtUFGT genes were up-regulated after cold treatment (Figure 8A). FtUFGT1 was quickly induced and peaked at 6 h after cold treatment with 1.7-fold more mRNA than the control (P < 0.05). The expression level of FtUFGT2 fluctuated, with the overall trend of the increase after cold treatment reaching a peak of 3-fold more mRNA than the control (P < 0.05) at 24 h. FtUFGT3 increased slowly at early times after cold treatment and peaked at 12 h with 2-fold more mRNA than the control (P < 0.05). Although all three FtUFGT genes were induced by cold treatment, the expression levels varied among the three genes. The expression levels of FtUFGT3 were much higher than those of FtUFGT1 and FtUFGT2. For example, at 0 h, the expression level of FtUFGT3 was 1527- and 16.8-fold higher than the levels of FtUFGT1 and FtUFGT2, respectively. Taken together, these results showed that the increased anthocyanin content accompanied the up-regulation of the three FtUFGT genes, suggesting that the three FtUFGTs, particularly FtUFGT3, were probably involved in cold protective anthocyanin formation.

Figure 5. TLC analyses of the reaction products catalyzed by the three FtUFGT proteins. By comparing the Rf values of the reaction products to those of the standard samples, the reaction products were identified as cyanidin 3-O-glucoside. Lanes: 1, cyanidin standard (Rf = 0.88); 2, cyanidin 3-O-glucoside standard (Rf = 0.72); 3, control (Rf = 0.88); 4, reaction products of FtUFGT1 (Rf = 0.88, 0.72); 5, reaction products of FtUFGT2 (Rf = 0.88, 0.72); 6, reaction products of FtUFGT3 (Rf = 0.88, 0.72).

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DISCUSSION In this study, three UFGTs were cloned from tartary buckwheat. Although their encoded proteins exhibit low sequence identity with other plant UGTs, their 3D structures are highly conserved. The structures are mainly composed of N- and C-terminal domains. The sugar acceptor interacts with the N-terminal domain, whereas the sugar donor interacts with the C-terminal domain.39 The acceptor-binding pocket is generally composed of residues in the loop regions, which are less conserved. The His26-Asp125 dyad of FtUFGT3 was identified as the catalytic site, which is commonly conserved among the flavonoid UGTs.14,37 The catalytic dyads are His17Asp109 for FtUFGT1 and His28-Asp137 for FtUFGT2. The two conserved residues, Glu381 and Gln382 of UGT71G1 from M. truncatula (Asp374 and Gln375 for VvGT1), form hydrogen bonds with the sugar moiety of the sugar donor.14,39,40 The glucosyl moiety of UDP-glucose may interact with Glu391 and Gln392 in FtUFGT3. For FtUFGT1, the two corresponding residues are Glu370 and Gln371, and for FtUFGT2, they are Asp424 and Gln425. It is worth mentioning that the Q382H UBGT mutant of Scutellaria baicalensis showed significantly decreased glucosyltransferase activity, suggesting that glutamine (Q) determined the sugar specificity of the glucosyl transfer activity.41 In our study, the conserved residue of the three FtUFGT proteins, which are able to utilize UDPglucose as the sugar donor, is Q (Gln371 for FtUFGT1, Gln425 for FtUFGT2, and Gln392 in FtUFGT3). In the previous study, cyanidin 3-O-rutinoside and cyanidin 3-O-glucoside were identified as the only two anthocyanins in tartary buckwheat sprouts, with more of the former than of the latter.3,42 Cyanidin 3-O-rutinoside is synthesized by the 3glycosylation of cyanidin following the rhamnosylation of cyanidin 3-O-glucoside. The first reaction is catalyzed by UDPglucose:flavonoid 3-O-glycosyltransferase.43 In this study, we demonstrated that the three FtUFGTs encode functional proteins, which can convert cyanidin to cyanidin 3-O-glucoside, and that FtUFGT3 has a higher specific activity than FtUFGT1 and FtUFGT2. The UFGT activity is closely linked to the formation of the two anthocyanins, which are the main anthocyanins in tartary buckwheat sprouts. Many previous studies indicated that cold stress induced anthocyanin synthesis in seedlings in various plants, including quinoa,44 tartary buckwheat,38 and Arabidopsis.45 In this study,

activities of the purified FtUFGT1, FtUFGT2, and FtUFGT3 were 20.01 × 10−3, 8.93 × 10−3, and 20.24 × 10−3 IU/mg, respectively. Tissue-Specific Expression Profiles of the FtUFGTs and Anthocyanin Accumulation. To determine where the three FtUFGTs were expressed, qRT-PCR was carried out to detect the expression levels in the root, stem, leaf, and flower of tartary buckwheat during florescence. As shown in Figure 7A, the results showed that the expression of all three FtUFGT genes could be detected in all tissues, with the highest expression level in the flower. The FtUFGT3 gene was expressed at relatively high levels in all tissues, especially in the flower, where the expression level was 23.4-, 4.7-, and 4.5-fold higher than in the root, stem, and leaf, respectively. In contrast, the expression level of FtUFGT1 was the lowest in all tissues and was 2-, 1.1-, and 4.4-fold higher in the flower than in the root, stem, and leaf, respectively. The highest level of FtUFGT2 expression was observed in the flower, with moderate levels in the leaf and root and the lowest level in the stem. The expression level of FtUFGT2 was 143.0-, 185.8-, and 3.2-fold higher in the flower than in the root, stem, and leaf, respectively. The anthocyanin content was also analyzed in various tissues. The highest level of anthocyanin was observed in the flower, moderate levels were present in the leaf and root, and the lowest level was in the stem (Figure 7B). All three FtUFGTs were strongly expressed in the flower, where relatively high levels of anthocyanin accumulated, indicating subfunctionalization in expression patterns. Expression of FtUFGTs and Anthocyanin Accumulation in Tartary Buckwheat Sprouts after Cold Treatment. Exposure to low temperature induced anthocyanin accumulation by activating genes involved in anthocyanin synthesis.31,38 To determine the total anthocyanin content in tartary buckwheat sprouts after cold treatment, anthocyanins were extracted using acidic methanol. As shown in Figure 8B, the results indicated that the total anthocyanin content increased after cold treatment and peaked at 6 h. The peak level of anthocyanin in the cold-treated sprouts was about 2.3fold higher than that of the control (P < 0.01). This result was consistent with the conclusion of the previous study.38 To analyze the expression patterns of the three FtUFGTs in tartary buckwheat sprouts after cold treatment, qRT-PCR was E

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Figure 6. Liquid chromatography−mass spectrometry/mass spectrometry analyses of reaction products catalyzed by the three FtUFGT proteins: (A) cyanidin standard; (B) cyanidin 3-O-glucoside standard; (C) control; (D) reaction product of purified FtUFGT1; (E) reaction product of purified FtUFGT2; (F) reaction product of purified FtUFGT3.

than nonmutant plants. Chen et al.16 observed a significant decrease in the anthocyanin content of Phalaenopsis flowers when a virus induced gene silencing of PeUFGT3, which suggested that UFGT plays a key role in anthocyanin biosynthesis. In our study, although similarly up-regulated expression of the three FtUFGTs was observed in tartary buckwheat sprouts after cold treatment, FtUFGT3 was

anthocyanin accumulation was found in tartary buckwheat sprouts after cold treatment, which was similar to the response described by Li et al.38 In a study of maize, nearly all of the anthocyanin synthesis genes were found to be up-regulated in response to cold treatment.46 Kubo et al.19 obtained a mutated ANL1, which encodes a UFGT in Arabidopsis thaliana, and found that the mutant plants accumulated less anthocyanin F

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Figure 7. Anthocyanin accumulation and expression of three FtUFGTs in various tissues of tartary buckwheat during florescence: (A) expression profiles of three FtUFGTs in various tissues by qRT-PCR; (B) quantification of the anthocyanin content of various tissues. FtH3 was used as the housekeeping gene. The expression level of FtUFGT1 in roots was set to “1”. Data are mean values of triplicate tests ± SD.

Figure 8. Anthocyanin content and expression of three FtUFGTs of tartary buckwheat sprouts after cold treatment: (A) expression analyses by qRTPCR of three FtUFGTs after cold treatment; (B) quantification of anthocyanin content after cold treatment. The controls are represented as 0 h of treatment. FtH3 was used as housekeeping gene. The expression level of FtUFGT1 at 0 h was set to “1”. Data are mean values of triplicate tests ± SD. Asterisks represent significant differences between the control and cold-treated tartary buckwheat sprouts (∗, P < 0.05; ∗∗, P < 0.01).

expressed at higher levels than FtUFGT1 or FtUFGT2. Similar results were observed in the red orange; that is, the transcript level of UFGT was increased by cold treatment.31 On the basis of the results of specific activities and gene expression levels, FtUFGT3 was considered to be an important gene in anthocyanin biosynthesis. Thus, the anthocyanin content of tartary buckwheat sprouts can be increased by cold stress, which might attract more consumers. In conclusion, the results suggested that the anthocyanin accumulation in tartary buckwheat sprouts was rapidly induced in response to cold treatment and was correlated with the expression of the three FtUFGT genes. Among the three FtUGFT proteins, FtUFGT3 was inferred to be the major protein catalyzing the glycosylation at the 3-hydroxyl group of cyanidin in the synthesis of anthocyanin. Furthermore, the three FtUFGT proteins play regulatory roles in anthocyanin biosynthesis and may provide a new approach to improving the anthocyanin content of tartary buckwheat sprouts.

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and cyanidin; Figure S3, expression of three recombinant proteins in E. coli BL21 (DE3); Figure S4, SDS-PAGE analyses of the recombinant proteins purification (PDF)

AUTHOR INFORMATION

Corresponding Author

*(Q.W.) Phone: +86-835-2886126. E-mail: [email protected]. Author Contributions ∥

J.Z. and C.-L.L. contributed equally to this work.

Funding

This study was supported by the National Natural Science Foundation of China (NSFC), project 31500289. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank ACS ChemWorx Authoring Services for a critical reading of the manuscript.

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.6b02064. Table S1, primers used in this study; Figure S1, molecular docking of FtUFGT1 with UDP and cyanidin; Figure S2, molecular docking of FtUFGT2 with UDP

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