Functional Characterization of Salicylic Acid Carboxyl

Nov 21, 2017 - The split ratio was 5:1, and the column (HP-5MS, 30 m × 0.25 mm × 0.25 μm, Agilent, U.S.A.) temperature ranged from 50 to 250 °C. M...
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Cite This: J. Agric. Food Chem. 2017, 65, 11036−11045

Functional Characterization of Salicylic Acid Carboxyl Methyltransferase from Camellia sinensis, Providing the Aroma Compound of Methyl Salicylate during the Withering Process of White Tea Wei-Wei Deng,† Rongxiu Wang,† Tianyuan Yang, Li’na Jiang, and Zheng-Zhu Zhang* State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, 130 Changjiang West Road, Hefei, Anhui 230036, China S Supporting Information *

ABSTRACT: Methyl salicylate (MeSA) is one of the volatile organic compounds (VOCs) that releases floral scent and plays an important role in the sweet flowery aroma of tea. During the withering process for white tea producing, MeSA was generated by salicylic acid carboxyl methyltransferase (SAMT) with salicylic acid (SA), and the specific floral scent was formed. In this study, we first cloned a CsSAMT from tea leaves (GenBank accession no. MG459470) and used Escherichia coli and Saccharomyces cerevisiae to express the recombinant CsSAMT. The enzyme activity in prokaryotic and eukaryotic expression systems was identified, and the protein purification, substrate specificity, pH, and temperature optima were investigated. It was shown that CsSAMT located in the chloroplast, and the gene expression profiles were quite different in tea organs. The obtained results might give a new understanding for tea aroma formation, optimization, and regulation and have great significance for improving the specific quality of white tea. KEYWORDS: CsSAMT, characterization, white tea, withering, MeSA

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INTRODUCTION

catalyzed the formation of amino acids in leaves; and unsaturated fatty acids such as linoleic acid and linolenic acid are enzymatically degraded to small molecule compounds of alcohol, aldehydes, ketones, and acids.4 MeSA, as an important and specific floral scent, can be preserved and enhanced in the final tea product, especially in white tea with a long time withering. However, the biosynthesis of MeSA from salicylic acid (SA) in the withering process of white tea was still not reported. SA, a substance of a plant hormone, is one of the necessary endogenous signal molecules. From the published reports, salicylic acid carboxyl methyltransferase (SAMT) catalyzes the production of MeSA from SA (Figure 1).5−7 The methylation reaction catalyzed by S-adenosyl-L-methionine-dependent methyltransferase is a ubiquitous reaction that occurs in plants.8 Recently, SAMTs in plants have been studied gradually, but SAMTs in tea plants and MeSA formation in white tea have not been well studied. To elucidate the enzymatic function of SAMT from C. sinensis (CsSAMT) in generating floral aroma, the variation of MeSA in tea leaves during the withering for white tea processing was investigated in the present study. A CsSAMT was isolated from tea leaves, and the encoded protein was characterized using Escherichia coli and Saccharomyces cerevisiae individually. This study provides the first evidence of CsSAMT in tea leaves and the amount of MeSA in white tea during the withering process and advances a better

Methyl salicylate (MeSA) is synthetically used as a specific fragrance in foods, beverages, and liniments nowadays, which is one of the organic esters naturally produced by many species of plants, especially wintergreens. Together with methyl benzoate (MeBA), MeSA is one of the volatile organic compounds (VOCs) that releases the floral scent, which plays an important role in plant defense mechanisms.1,2 Tea is the most popular beverage in the world and processed from the leaves of tea plants (Camellia sinensis). There are six types of teas (green tea, black tea, yellow tea, dark tea, oolong tea, and white tea) in China, based on their specific processing methods. The aroma of different teas is quite discriminative because they could be defined by the tea plant variety itself, withering duration, and rolling phase. In tea, even though the VOCs existed in a very limited amount (i.e., 0.01% of the total dry weight), they have a very high impact on tea aroma.3 Among the VOCs in tea, MeSA is one of the important compounds that gives a high quality level of sweet flowery and floral aroma to tea. After comparing the content of MeSA in the six types of teas made from the same tea leaves, the highest content of MeSA was shown in the white tea sample (white tea > black tea > oolong tea > yellow tea > green tea > dark tea, unpublished data). The traditional manufacturing process of white tea is only two stages of withering and drying. During these two stages, many compounds are generated to express the specific characters of smell and taste for white tea. The prolonged withering stage of white tea causes complex internal physical and chemical changes in leaves: the loss of water results in an increase of volatile compounds in leaf cells and also changes the permeability of cell membranes;4 hydrolases are activated and © 2017 American Chemical Society

Received: Revised: Accepted: Published: 11036

October 2, 2017 November 20, 2017 November 21, 2017 November 21, 2017 DOI: 10.1021/acs.jafc.7b04575 J. Agric. Food Chem. 2017, 65, 11036−11045

Article

Journal of Agricultural and Food Chemistry

Figure 1. Partial pathway for the biosynthesis of phenylpropanoid/benzenoids. Red box indicates the biosynthesis of methyl salicylic from salicylic acid. 2 °C/min and maintained for 1 min, and then increased to 250 °C at a rate of 10 °C/min and maintained for 2 min.10 Cloning of CsSAMT from Tea Leaves. The annotated sequence of CsSAMT was screened out from a transcriptome database of tea leaves (unpublished data). Total RNA was isolated from the tea leaves of C. sinensis Shuchazao by using an RNA prep pure plant kit (Axygen, Tewksbury, MA), and the first-strand cDNA was synthesized using a PrimeScript RT Reagent Kit (Takara Bio, Dalian, China). The polymerase chain reaction (PCR)-specific primers TYF and TYR with BamH I and Sac I restriction enzyme sites used for cloning were designed using Primer Premier 5.0 software after bioinformatics analysis (primer sequences are shown in the Supporting Information, Table 1); the primers were then synthesized by Sangon (Shanghai, China). The PCR product (the open reading frame, ORF) was purified from agarose gels by using an AxyPrep DNA Gel Extraction Kit (Axygen) and was ligated into the pEASY-Blunt vector (TransGen Biotech, China) after the gene sequence was confirmed through sequencing (Sangon). Construction of CsSAMT Expression Vector, Prokaryotic Expression, and Enzyme Assay. The plasmid containing recombinant CsSAMT and the pET-32a(+) vector was digested using the respective restriction enzymes (BamH I and Sac I), and the ORF of CsSAMT was ligated into pET-32a(+) to obtain the recombinant pET-32a(+)−CsSAMT expression vector. After the ligation of the cloned fragment was confirmed, the recombinant expression vector was transformed into Escherichia coli (BL21; TransGen Biotech) for inducible His-tagged protein expression. For comparison, the pET-32a(+) vector served as a control. Transformed cells were cultured overnight at 37 °C in LB media (Lysogeny broth) containing ampicillin (100 μg/mL).11 The supernatants from the disrupted cells were used to measure the activity of recombinant pET-32a(+)−CsSAMT. The reaction mixture for the enzyme activity determination was prepared using the following components: MgCl2 (200 mM), 20 μL; SAM (S-adenosylL-methionine, 5 mM), 50 μL; adenosine triphosphate (1 mM), 10 μL; dithiothreitol (400 mM), 10 μL; one of the substrates (SA, benzoic acid, cinnamic acid, vanillic acid, caffeic acid, and jasmonic acid), 100 mM, 100 μL; and crude proteins, 810 μL. The mixture was adjusted to a final volume of 1.0 mL and then incubated at 30 °C for 1 h.

understanding and regulation of aroma formation in tea and especially in white tea.

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MATERIALS AND METHODS

Materials. Different tea organs (bud, first leaf, second leaf, old leaf, stem, and root) were collected from C. sinensis Shuchazao in the Tea Garden of Anhui Agricultural University, China. These organs were frozen in liquid nitrogen and stored at −80 °C until use. One bud with two tea leaves was also collected and was used in indoor withering of white tea. pET-32a(+), pESC-His, pDONR207, and pK7GWF2.0 vectors were obtained from the State Key Laboratory of Tea Plant Biology and Utilization in Anhui Agricultural University. A BD GenomeWalker Kit was obtained from Clontech (Mountain View, CA). Benzoic acid (BA) and methyl benzoate (MeBA) were purchased from Aladin (Shanghai, China). Cinnamic acid, vanillic acid, caffeic acid, and jasmonic acid were obtained from Yuanye Bio-Technology (Shanghai, China). Salicylic acid (SA) and methyl salicylate (MeSA) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). MeSA Content in White Tea after Indoor Withering. Two tea leaves with a bud were withered in a wilting sifter and then dried immediately at 80 °C for 30 min after 6, 12, 24, 36, and 72 h of withering. As a control, fresh leaves were directly steamed for 2−3 min (to deactivate the enzymes) and then dried at 80 °C for 30 min. MeSA was extracted from dry tea leaves (20 g) with 30 mL of anhydrous diethyl ether by using simultaneous distillation−extraction, and the anhydrous diethyl ether was concentrated to 1 mL by nitrogen blow controller and was analyzed using gas chromatography−mass spectrometry (GC−MS).9 The retention index was calculated using a mixture of n-paraffin C7−C25 as standards. The sample was injected into an Agilent 7890A/5975C GC−MS instrument. The injection temperature and injected volume were set at 250 °C and 1 μL, respectively. The split ratio was 5:1, and the column (HP-5MS, 30 m × 0.25 mm × 0.25 μm, Agilent, U.S.A.) temperature ranged from 50 to 250 °C. MS was performed in the selected-ion monitoring mode after electron ionization (70 eV). The following GC−MS temperature program was applied: the initial column temperature was set at 50 °C for 5 min, increased to 180 °C at a rate of 11037

DOI: 10.1021/acs.jafc.7b04575 J. Agric. Food Chem. 2017, 65, 11036−11045

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Journal of Agricultural and Food Chemistry The activity of recombinant pET−32a(+)-CsSAMT was assayed and analyzed using GC−MS. The extract was injected into an Agilent 7890A/5975C GC−MS instrument. When SA and benzoic acid were used as substrates, the following GC−MS temperature program was applied: the initial column temperature was set at 50 °C for 5 min, increased to 180 °C at a rate of 2 °C/min and maintained for 1 min, and then increased to 250 °C at a rate of 10 °C/min and maintained for 2 min.10 When cinnamic acid, vanillic acid, caffeic acid, and jasmonic acid were used as substrates, the initial column temperature was set at 60 °C for 3 min. Purification of the Recombinant Protein and Activity Assay. The protein was purified using the method reported previously.8 The recombinant histidine-tagged protein was purified using the HisTrap HP (GE Healthcare Life Sciences, Marlborough, MA) according to the manufacturer’s instructions. The purified protein was used for further enzymatic assays. For the later optimum pH and temperature determination, reactions were carried out according to a previous report.8 For the pH optima, assays were performed at 30 °C for 30 min using 100 mM PBS buffer with pH levels ranging from 6.0 to 8.0. For the temperature optima, reactions were performed at pH 7.5 for 30 min, with the temperature ranging from 10 to 60 °C. The enzyme assay was performed according to the same procedure in the former section. Construction of CsSAMT Expression Vector, Eukaryotic Expression, and Enzyme Assay. The PCR specific primers TZF and TZR with EcoR I and Spe I restriction enzyme sites were designed based on the sequence of CsSAMT by using Primer Premier 5.0. One pair of primers GALI-F and GALI-R was used to identify the positive clones of the pESC-His vector. Primer sequences are shown in the Supporting Information, Table 1. The plasmid containing the sequence of CsSAMT and the pESC-His vector were digested using the respective restriction enzymes (EcoR I and Spe I), and the ORF of CsSAMT was inserted into the pESC-His vector to obtain the recombinant pESC-His−CsSAMT expression vector. After the confirmation of the ligated target fragment, the recombinant expression vector was transformed into the competent yeast strain InVscI for expression. For comparison, the pESC-His vector was used as a control. Transformed cells were cultured overnight at 30 °C in SD (Synthetic Dropout Medium)-His medium containing 2% glucose, and the cells were further cultured at 30 °C for 24 h.12 The enzyme assay was performed according to the same procedure used for the enzyme assay of CsSAMT in E. coli, except that the only substrate used was SA. The GC−MS condition was the same as that applied when SA served as the substrate. Construction of Transformation Vectors and Transient Expression in Tobacco. Specific Gateway primers GTF1 and GTP2 based on the sequence of the amplified cDNA fragment of CsSAMT were designed using Primer Premier 5.0 and were then synthesized by Sangon (primer sequences were shown in the Supporting Information, Table 1). The mixture for the PCR was prepared using the following components: ddH2O, 16 μL; Prime STAR Max Premix, 30 μL; GTF primer (10 μM), 1.0 μL; GTR primer (10 μM), 1.0 μL; and the plasmids containing the cDNA sequence of CsSAMT, 2.0 μL. The specific fragments were purified from agarose gels by using the AxyPrep DNA Gel Extraction Kit (Axygen). The purified cDNA was cloned into the entry vector pDONR207 using the Gateway method. After the confirmation, the entry vector was carried out to introduce the ORF into the destination vector pK7GWF2.0 containing the enhanced green fluorescent protein (EGFP) gene. The recombinant expression vector and only the pK7GWF2.0 vector used as the control were separately transferred to the Agrobacterium tumefaciens strain EHA105 through electroporation. The tobacco leaf disks were cut into 0.5 cm × 0.5 cm pieces by using a blade, and the expression of EGFP was then observed under a confocal laser scanning microscope (Olympus, Japan).13 DNA Sequence of CsSAMT. Total genomic DNA was isolated from two leaves with one bud of the tea plant C. sinensis Shuchazao according to the SDS method.14 Based on the cDNA sequence of CsSAMT, pairs of specific primers were designed using Primer Premier 5.0.

Figure 2. Variation of methyl salicylic obtained in tea leaves during the withering for white tea processing. Content of the target analyte at different wilting times (6, 12, 24, 36, and 72 h) was calculated based on the standard curve (Supporting Information, Figure 2A). “0 h” corresponded to the sample directly fixed using steam (the control). Data represented the mean ± standard deviation of three independent experiments. The primer sequences are shown in the Supporting Information, Table 1. The mixture for the PCR was prepared using the following components: ddH2O, 10 μL; dNTP (2 mM), 10 μL; KOD FX, 1.0 μL; 2× PCR buffer, 25 μL; primers (10 μM), 1.5 μL each; and DNA, 1.0 μL. The PCR was run using the following program: denaturing at 94 °C for 2 min, 30 cycles of denaturing at 94 °C for 10 s, annealing at Tm, extension at 68 °C for 1 min/kb, and final extension at 72 °C for 10 min. Each reaction tube was run on a 1.0% agarose gel, and the specific bands were sequenced subsequently. Ligation of Genomic DNA to GenomeWalker Adaptors. The total genomic DNA isolated from the tea leaves, as described in the preceding section, was applied to amplify the promoter of CsSAMT by using genome walking with the BD GenomeWalker Kit of Clontech. The total genomic DNA was digested by the restriction enzymes Stu I, Pvu II, EcoR V, and Dra I separately.15 Each reaction tube was run on a 1.0% agarose gel to determine whether digestion was complete. A total of four ligation reactions were performed to set up four libraries: DNA library-Stu I, DNA library-Pvu II, DNA library-EcoR V, and DNA library-Dra I. Cloning the Promoter of CsSAMT. One pair of specific primers (GSP1 and GSP2) was designed using Primer Premier 5.0 after bioinformatics analysis of the DNA sequence of CsSAMT, and the primers were then synthesized by Sangon. The adaptor primers were AP1 and AP2 provided by BD GenomeWalker Kit. The 5′ region flanking the DNA sequence of CsSAMT was amplified using nestedPCR with the primers of GSP1/AP1 followed by GSP2/AP2.16 The primer sequences are shown in the Supporting Information, Table 1. The primary PCR was performed separately using 2 μL of DNA library-Stu I, DNA library-Pvu II, DNA library-EcoR V, and DNA library-Dra I as templates and 10 μM primer GSP1/AP1 in a 25 μL reaction volume. After primary PCR, the second nested-PCR was performed using the same procedure as that for the first PCR. However, different templates and primers were used: 2.0 μL of the first PCR product diluted 50 times and 10 μM primers GSP2/AP2 were added to the second reaction mixture. Each reaction tube was run on a 1.2% agarose gel to determine whether specific bands were present. Next, the specific fragments were purified from agarose gels by using an AxyPrep DNA Gel Extraction Kit (Axygen). The purified amplified fragment was cloned into the pEASY-Blunt vector, and the positive clone was obtained through sequencing and identifying. Quantitative Analysis Using Real-Time Quantitative PCR. Total RNA was extracted from different tea organs (bud, first leaf, second leaf, old leaf, stem, and root) by using the RNA Prep Pure Plant Kit (Axygen). cDNA was transcribed using the PrimeScript RT Reagent Kit (Takara, Japan). Real-time quantitative PCR (qRT-PCR) was performed using 2 μL of cDNA and 0.4 μM of each primer in a 20 μL reaction volume with SYBR Premix Ex TaqII (Takara, Japan). GAPDH (GAPDHF and GAPDHR) was used as an internal control, and the specific primers (QTF and QTR) used for qRT-PCR analysis 11038

DOI: 10.1021/acs.jafc.7b04575 J. Agric. Food Chem. 2017, 65, 11036−11045

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Article

RESULTS AND DISCUSSION

Variation of MeSA in Tea Leaves during the Withering for White Tea Processing. White tea has a unique processing method: fresh tea leaves → withering (sun withering or indoor withering) → drying (air drying, solar drying, or machine drying) → white tea.17 The processing steps of panning, rolling, and shaking are not required for white tea, and thus the withering process exerts an important effect on aroma formation for white tea. In this study, the retention index of MeSA was 1191, which was calculated against those of n-paraffin. The identification of the MeSA in tea samples was confirmed by the reported retention index (1190),18 the retention time of the standard of MeSA, the mass spectrum, and the total ion chromatogram of the MeSA standard. We found that the MeSA content increased almost linearly with the withering time (Figure 2), and the MeSA content was 2.5-fold higher in tea samples withered for 72 h than withered for 36 h. After 72 h withering, MeSA content in the samples was approximately 1.08 μg/g (calculated from the standard curve of MeSA, Supporting Information Figure 2A). However, MeSA was not detected in the control tea samples, whose fresh leaves were directly treated by steam fixation but could be detected in fresh tea leaves (trace amount, data not shown). The hardly detectable amount of MeSA in the directly fixed tea samples was probably due to the high temperature during steam fixation

Figure 3. Agarose gel electrophoresis of PCR amplification product. M: Marker; l−5: PCR amplification product obtained using cDNA as a template. were designed based on the ORF of CsSAMT. The primer sequences are shown in the Supporting Information, Table 1. Each PCR was performed in three replicates, and the expression level in the third leaf was used as a control. According to the threshold cycle (Ct), the changes in gene expression were quantified using the 2−ΔΔCt method.11

Figure 4. Phylogenetic tree of amino acid sequences of SAMT in different plant species. Phylogenetic analysis of CsSAMT and related, functionally characterized SAMTs in the following plants: Medicago truncatula, Antirrhinum majus, Camellia japonica, Populus x beijingensis, Populus tomentosa, Clarkia breweri, Hoya carnosa, Stephanotis f loribunda, Atropa belladonna, Datura wrightii, Solanum lycopersicum, Nicotiana alata, Nicotiana benthamiana, Nicotiana suaveolens, and Oryza sativa. The neighbor-joining phylogenetic tree was generated using MEGA 6.0 software. The proteins included in the tree are represented by their GenBank accession number. 11039

DOI: 10.1021/acs.jafc.7b04575 J. Agric. Food Chem. 2017, 65, 11036−11045

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Journal of Agricultural and Food Chemistry which might evaporate the trace amount of MeSA. MeSA is an important component of white tea aroma; the mechanism underlying the formation of MeSA should be elaborated.4 During the withering process, the formation of MeSA can be catalyzed by SAMT. In this study, a CsSAMT isolated from tea leaves and the encoded protein was also characterized. cDNA Sequence of CsSAMT and Phylogenetic Relationship Analysis. After the bioinformatic analysis from our tea transcriptome database, a 1104-bp fragment of CsSAMT was amplified from tea leaves by using the specific primers (Figure 3). The fragment with the initiation codon ATG and the termination codon TAG was acquired after PCR amplification and sequencing. The sequence was submitted to NCBI, and GenBank accession no. MG459470 was assigned afterward. The deduced translation product of CsSAMT consisted of 367 amino acids, with a molecular weight of 41297.38 Da, a GC content of 43.03%, and a theoretical isoelectric point of 5.224. Regarding phylogenetic analysis, the sequence alignment of presently known SAMTs by ClustalW revealed 42%−100% homology to other SAMTs at the amino acid level (Figure 4). The amino acid sequence of CsSAMT showed the highest homology with SAMTs from C. japonica. The SAMT from C. breweri was shown to have a crystal structure, which led to the influence of the dimer formation by its N-terminal sequence. Many conserved residues were found at the N- and C-termini, and the C-terminal domain was demonstrated to be primarily involved in substrate binding.19,20 The S-22, D-57, SF, and DL residues are SAM/SAH-binding residues in CsSAMT, because of SAM-binding sites are highly conserved, while the Q-25, SSYSLMW, MRAV, and IW residues are amino acid side chains in contact with SA in SAMT and substrates in other members of this methyltransferase family.19,21 Expression of CsSAMT in E. coli and GC-MS Analysis of Enzyme Products. To confirm the enzyme activity of CsSAMT encoded by the cDNA sequence of CsSAMT, recombinant pET-32a(+)−CsSAMT was expressed in E. coli and was analyzed using an in vitro functional enzyme assay; the pET-32a(+) vector was used as the control. The putative ORF of CsSAMT in E. coli was induced by adding IPTG (1.0 mM) and incubating at various temperatures (16, 28, and 37 °C). The analysis showed that inducing the expression of recombinant pET-32a(+)−CsSAMT resulted in the production of a protein with a molecular weight of approximately 60 kDa, which was consistent with the molecular weight predicted by DNAStar software (Figure 5). In the reaction of MeSA formation by SMAT, SA was used as the substrate, and SMAT was derived from bacterial lysates containing the recombinant protein pET-32a(+)−CsSAMT after induction. After that, GC−MS was used to identify the metabolites produced upon incubation of substrates with pET-32a(+)−CsSAMT. The identification of the MeSA was confirmed by the reported retention index,18 the retention time of the standard of MeSA, the mass spectrum, and the total ion chromatogram of the MeSA standard. In our research, volatile products catalyzed by CsSAMT were verified to MeSA and methyl benzoate by GC−MS determination (Figure 6A,B). For investigating substrate specificity, SA was replaced with the following substrates: benzoic acid, cinnamic acid, vanillic acid, caffeic acid, and jasmonic acid. However, the enzyme activity of CsSAMT was only detected when benzoic acid as substrate and generated a small amount product of methyl benzoate (Figure 6C). Methyltransferases can transfer a methyl group from a methyl donor, S-adenosyl-L-methionine, to the carboxyl group or ring nitrogen of a substrate, forming

Figure 5. SDS-PAGE analysis of recombinant CsSAMT expressed in E. coli. CsSAMT was ligated into the pET-32a(+) vector and expressed in E. coli [BL21 (DE3)]. After IPTG-induced protein expression, all cells or cell lysates after sonication were subjected to SDS-PAGE. The gel was stained using Coomassie blue R-250. M: protein molecular mass marker; 1: total crude protein from uninduced cells was transformed using the pET-32a(+) vector alone; 2: total crude protein from induced cells was transformed using the pET-32a(+) vector alone; 3: total crude protein from uninduced cells was transformed using pET-32a(+)−CsSAMT; 4: total crude protein from induced cells was transformed using pET-32a(+)−CsSAMT; 5: suspension of total crude protein from induced cells was transformed using pET-32a(+)− CsSAMT after sonication; and 6: precipitation of total crude protein from induced cells was transformed using pET-32a(+)−CsSAMT after sonication. The bands of pET-32a(+)-CsSAMT are indicated by arrows in lanes 4−6.

S-adenosyl-L-homocysteine and methyl esters or N-methylated compounds.22 The family of SAMTs has a preference for SA,23 and the enzyme activity of CsSAMT was verified by the substrates of SA and BA, with SAM as methyl donor in this research. Enzyme Assay of Purified Protein, pH, and Temperature Optima. After the expression of CsSAMT in E. coli, protein expressed in supernatant was purified by HisTrap HP. The purified CsSAMT was found in the SDS-PAGE analysis (Figure 7A), and the purified protein from the supernatant was subsequently used for the enzyme assay. After the GC-MS analysis, the purified CsSAMT could catalyze the biosynthesis of MeSA but display a lower activity level compared to the crude protein (Figure 7C). CsSAMT had a pH optimum of 7.5. At pH 6.0−6.5, activity of CsSAMT was not detected, while at pH 8.0 it was about 70% of optimal activity, and at pH 7.0, it was less than 50% of the optimal activity. CsSAMT was almost 100% stable for 30 min at 20−30 °C and 50% stable for 30 min at 10 °C, but after 30 min incubation at higher than 40 °C it completely lost 70% activity (Supporting Information, Figure 2B,C). Similar patterns of pH and temperature optima were found in a SAMT from the annual California plant Clarkia breweri.8 It also showed a pH optimum of 7.5 and 75% at pH 8.0, while at pH 6.0, activity was still 80% of the optimal activity. This SAMT was 100% stable for 30 min at 20 °C and 80% stable for 30 min at 30 °C, and after 30 min incubation at 40 °C it completely lost activity.8 Expression of CsSAMT in Yeast and GC-MS Analysis of Enzyme Products. Since the eukaryotic expression system has not been employed to express SAMTs up to now, the expression of the recombinant CsSAMT in yeast was performed, together with a control of the pESC-His vector in this study. The pESC vectors containing the GAL1 and GAL10 yeast promoters in opposing orientation are a series of epitope-tagging vectors designed for the expression and functional analysis of 11040

DOI: 10.1021/acs.jafc.7b04575 J. Agric. Food Chem. 2017, 65, 11036−11045

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Journal of Agricultural and Food Chemistry

Figure 6. GC-MS analysis of the volatile product of recombinant CsSAMT in E. coli. A: Aa, pET-32a(+)−CsSAMT: Partial GC chromatogram from a reaction with salicylic acid as the substrate and lysates of cells with the recombinant pET-32a(+)−CsSAMT after IPTG induction; Ab, pET-32a(+): Partial GC chromatogram from a reaction with salicylic acid as the substrate and lysates of cells with the recombinant pET-32a(+) after IPTG induction; Ac, standard: GC chromatogram of MeSA. GC mass spectrum of the characteristic peak was identified as that of MeSA, as indicated by the arrow (retention time = 26.7 min); Ad, pET-32a(+)−CsSAMT: GC mass spectrum of enzymatic product of recombinant pET-32a(+)− CsSAMT; Ae, methyl salicylic standard, the authentic standard spectrum of methyl salicylic. B: Ba, pET-32a(+)−CsSAMT: Partial GC chromatogram from a reaction with salicylic acid as the substrate and lysates of cells with the recombinant pET-32a(+)−CsSAMT after IPTG induction; Bb, pET32a(+): Partial GC chromatogram from a reaction with salicylic acid as the substrate and lysates of cells with the recombinant pET-32a(+) after IPTG induction; Bc, standard: GC chromatogram of methyl benzoate. GC mass spectrum of the characteristic peak was identified as that of methyl benzoate, as indicated by the arrow (retention time = 20 min); Bd, pET-32a(+)−CsSAMT: GC mass spectrum of enzymatic product of recombinant pET32a(+)−CsSAMT; Be, methyl benzoate standard, the authentic standard spectrum of methyl benzoate. C: Ca, pET-32a(+)−CsSAMT: Partial GC chromatogram from a reaction with benzoate acid as the substrate and lysates of cells with the recombinant pET-32a(+)−CsSAMT after IPTG induction; Cb, pET-32a(+): Partial GC chromatogram from a reaction with salicylic acid as the substrate and lysates of cells with the recombinant pET-32a(+) after IPTG induction; Cc, standard: GC chromatogram of methyl benzoate. GC mass spectrum of the characteristic peak was identified as that of methyl benzoate, as indicated by the arrow (retention time = 20 min); Cd, pET-32a(+)−CsSAMT: GC mass spectrum of enzymatic product of recombinant pET-32a(+)−CsSAMT; Ce, methyl benzoate standard, the authentic standard spectrum of methyl benzoate. 11041

DOI: 10.1021/acs.jafc.7b04575 J. Agric. Food Chem. 2017, 65, 11036−11045

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Journal of Agricultural and Food Chemistry

Figure 7. SDS-PAGE analysis of the purified CsSAMT and activity assay. A, SDS-PAGE analysis of the recombinant CsSAMT expressed in E. coli and the purified CsSAMT: lane 1, total crude protein from uninduced cells was transformed using pET-32a(+)−CsSAMT; 2, total crude protein from induced cells was transformed using pET-32a(+)−CsSAMT; 3, precipitation of total crude protein from induced cells was transformed using pET32a(+)−CsSAMT after sonication; 4, suspension of total crude protein from induced cells was transformed using pET-32a(+)−CsSAMT after sonication; 5, the purified CsSAMT from suspension of total crude protein. The bands of pET-32a(+)-CsSAMT are indicated by arrows in lanes 3−5. B, Partial GC chromatogram from a reaction with salicylic acid as the substrate catalyzed by the purified-pET-32a(+). C, Partial GC chromatogram from a reaction with salicylic acid as the substrate catalyzed by the purified-CsSAMT. D, Partial GC chromatogram of the MeSA standard. GC mass spectrum of the characteristic peak was identified as MeSA, indicated by arrows (retention time = 26.7 min). E, GC mass spectrum of enzymatic product of the purified-CsSAMT. F, Methyl salicylic standard, the authentic standard spectrum of methyl salicylic.

Figure 8. Subcellular localization of CsSAMT in tobacco leaf disks. Subcellular localization analysis of (a) pK7GWF2.0 and (b) pK7GWF2.0− CsSAMT; (c) leaf containing pK7GWF2.0−CsSAMT was in a bright field; (d) leaf containing pK7GWF2.0−CsSAMT was merged. eGFP: enhanced green fluorescent protein.

eukaryotic genes in S. cerevisiae. The gene of interest can be inserted in front of the epitope sequence to generate C-terminal tagging or can be inserted after the epitope sequence for N-terminal tagging.24 In our established yeast system using S. cerevisiae to validate the in vitro function of CsSAMT involved in aroma compound formation, SA was used as a substrate in this enzymatic reaction.

The identification of MeSA formation was confirmed by the reported retention index,18 the retention time of the standard of MeSA, the mass spectrum, and the total ion chromatogram of the MeSA standard. Compared with CsSAMT expressed in E. coli, only a smaller amount of MeSA with no methyl benzoate was generated by the recombinant CsSAMT in yeast (Supporting Information, Figure 1). 11042

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Journal of Agricultural and Food Chemistry Table 1. Length of Intron/Exon in the Genomic Sequence of CsSAMT q-name Exon Exon Exon Exon

1 2 3 4

s-name

alignment

mismatch

q-start

q-end

s-start

s-end

DNA DNA DNA DNA

75 411 262 368

0 0 3 0

1 73 478 737

75 484 739 1104

1 996 1951 5975

75 1406 2212 6342

Subcellular Localization of CsSAMT. In order to investigate the subcellular localization of CsSAMT, the fragment of CsSAMT was transferred into vectors containing compatible recombination sites (attB × attP or attL × attR) in reactions mediated by Gateway.13 Subsequently, the recombination vector and the empty vector pK7GWF2.0 used as a control were transformed into tobacco leaf disks through Agrobacteriummediated transformation. EGFP-fused CsSAMT was observed in the chloroplast of the tobacco leaf cell. However, EGFPfused pK7GWF2.0 was observed at the tobacco leaf cell edge (Figure 8). The immunogold localization of the BAMT protein in Snapdragon petals provided in vivo evidence for its cytosolic location,25 suggesting that carboxyl methyltransferases are cytoplasmic enzymes, while our subcellular localization investigations revealed that CsSAMT was located in the chloroplast in the tobacco leaf cell. The different subcellular localization was probably due to distinct functions of the enzymes in different species. DNA and Promoter Sequences of CsSAMT. The DNA sequence of CsSAMT was amplified and was found to comprise four exons and three introns (Table 1). The identified DNA sequence is similar to that of N-methyltransferase gene families.26 For obtaining the promoter sequence, genome walking is a widely used method.15 It is crucial to use an appropriate genome walker and design appropriate nested gene-specific primers. For a more effective nested-PCR, the primers GSP1 and GSP2 used in our cloning procedure were designed based on the DNA sequence of CsSAMT, and finally a 2675-bp fragment was targeted after the nested-PCR amplification.16 The DNA and promoter sequences were submitted to NCBI, and GenBank accession no. MG459471 was assigned afterward. Bioinformatics analysis of this 2675-bp sequence through PlantCARE (http:// bioinformatics.psb.ugent.be/webtools/plantcare/html/) revealed that several cis-acting enhancers were associated with various functional elements. The cis-acting element conferred high transcription ability; moreover, the 5UTR Py-rich stretch, CAAT box, and TATA (TATAAAT) box, in addition to other special motifs or elements, conferred functions such as SA responsiveness, MeJA responsiveness, light responsiveness, defense, and stress responsiveness. These elements are listed in the Supporting Information, Table 2. Some previous reports have shown that SAMT genes are involved in defense and biotic or abiotic stresses,27−29 and the expression of SAMT genes is thought to be induced by mechanical wounding and treatment with SA and methyl jasmonate.30,31 In C. sinensis, the expression of SAMT could be induced by the damage of Ectropis oblique.32 In addition, the expression and enzyme activity of SAMTs from Antirrhinum majus, Stephanotis f loribunda, and Nicotiana suaveolens have been shown to oscillate during a light/dark cycle.23,25,33,34 Analysis of CsSAMT Expression in Different Tea Organs Using qRT-PCR. The expression levels of CsSAMT in different tea organs (bud, first leaf, second leaf, old leaf, stem, and root) were detected using qRT-PCR, and the expression level in the old leaf was used as a control (Figure 9). The results showed that the maximum expression levels of CsSAMT were

Figure 9. Relative expression levels of CsSAMT in different tea organs, as determined through qRT-PCR. GAPDH was used as an internal control. The expression levels of CsSAMT are shown in the bud, first leaf, second leaf, old leaf, stem, and root. The expression level in old leaf was used as the control. Data represent the means ± standard deviation (n = 3).

found in the second leaf, whereas the expression in old leaf was the lowest, together with the expression levels of CsSAMT in the bud and first leaf were almost similar. The high expression levels of CsSAMT in young tea leaves, might lead to the high content of MeSA, which can protect tender tissues from insects.35 The traditional manufacturing process of white tea is only two stages of withering and drying. After these two stages, the specific flavor and aroma were generated to express the characteristic properties for white tea. As for a specific aroma compound for white tea, the MeSA content was almost in a linear growth phenomenon during the withering process. In order to investigate the enzymatic reaction for producing MeSA by SAMT with SA in tea, a 1104-bp fragment of CsSAMT was amplified from tea leaves and its enzyme activity of CsSAMT in prokaryotic and eukaryotic expression systems was identified. The full length and promoter fragment of CsSAMT was obtained after genome walking and nested-PCR amplification. The obtained results might give a new understanding for tea aroma formation, provide a further study for tea aroma optimization and regulation, and have great significance for improving the specific quality for tea, especially for white tea. For the further investigation, it would play an important role in plant defense mechanisms, because MeSA is one of the volatile organic compounds and releases the floral scent.

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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.7b04575. Supplemental Table 1: The primer sequences for PCR amplification in this study. All the primers used in this research were shown as the functions, the primer names, and the sequences (5′→ 3′). Supplemental Table 2: Regulatory elements in the cloned CsSAMT promoter sequence. The site name, the organism, sequence, and the function were shown. Supplemental Figure 1: GC-MS analysis of the volatile product of recombinant CsSAMT in yeast 11043

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(Saccharomyces cerevisiae). The established yeast system using S. cerevisiae to validate the in vitro function of CsSAMT involved in aroma compound formation, SA was used as a substrate in this enzymatic reaction. A, Partial GC chromatogram from a reaction with salicylic acid as the substrate and lysates of cells with the recombinant pESC-His−CsSAMT after galactose induction; B, partial GC chromatogram from a reaction with salicylic acid as the substrate and lysates of cells with the recombinant pESC-His after galactose induction; C, GC chromatogram of the MeSA standard. GC mass spectrum of the characteristic peak was identified as that of MeSA, as indicated by the arrow (retention time = 26.7 min); D, GC mass spectrum of enzymatic product of recombinant pESC-His−CsSAMT; E, methyl salicylic standard, the authentic standard spectrum of methyl salicylic. Supplemental Figure 2: The standard curve of methyl salicylate and the detection of the optima pH and temperature. For the pH optima, assays were performed at 30 °C for 30 min using 100 mM PBS buffer with pH levels ranging from 6.0 to 8.0. For the temperature optima, reactions were performed at pH 7.5 for 30 min, with the temperature ranging from 10 to 60 °C. Data represent the means ± standard deviation (n = 3). A, The standard curve of methyl salicylate established by GC-MS; B, the enzyme assays performed at 30 °C with different pH ranging from 6.0−8.0 for 30 min and determined by GC-MS; C, the enzyme assays performed at pH 7.5 with different temperatures ranging from 10−60 °C for 30 min and determined by GC-MS. (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel/fax: +86 551 65785471. E-mail: [email protected]. ORCID

Zheng-Zhu Zhang: 0000-0003-1564-1428 Author Contributions †

W.-W.D. and R.W. contributed equally to this work.

Author Contributions

W.-W.D. and Z.-Z.Z. designed the research, W.-W.D. and R.W. performed the experiments and wrote the manuscript, T.Y. revised the figures, and L.J. helped to do some experiments for this research. Funding

This study was supported by the Natural Science Foundation of Anhui Province Project 1608085QC60, the National Natural Science Foundation of China (NSFC) Projects 31300576 and 31570692, and the Changjiang Scholars and Innovative Research Team in University (IRT_15R01). Notes

The authors declare no competing financial interest.

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