Occurrence of Glycosidically Conjugated 1-Phenylethanol and Its

Jul 28, 2014 - Guangdong Food and Drug Vocational College, Longdongbei Road 321, ... Graduate School of Science and Technology, Shizuoka University, ...
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Occurrence of Glycosidically Conjugated 1‑Phenylethanol and Its Hydrolase β‑Primeverosidase in Tea (Camellia sinensis) Flowers Ying Zhou,†,∥ Fang Dong,‡,∥ Aiko Kunimasa,§ Yuqian Zhang,† Sihua Cheng,† Jiamin Lu,† Ling Zhang,† Ariaki Murata,⊗ Frank Mayer,⊥ Peter Fleischmann,⊥ Naoharu Watanabe,# and Ziyin Yang*,† †

Key Laboratory of Plant Resource Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Xingke Road 723, Tianhe District, Guangzhou 510650, China ‡ Guangdong Food and Drug Vocational College, Longdongbei Road 321, Tianhe District, Guangzhou 510520, China § Graduate School of Agriculture and ⊗Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan ⊥ Institute of Food Chemistry, Technische Universität Braunschweig, Schleinitzstrasse 20, DE-38106 Braunschweig, Germany # Graduate School of Science and Technology, Shizuoka University, 3-5-1 Johoku, Naka-ku, Hamamatsu 432-8561, Japan S Supporting Information *

ABSTRACT: A previous study found that 1-phenylethanol (1PE) was a major endogenous volatile compound in tea (Camellia sinensis) flowers and can be transformed to glycosically conjugated 1PE (1PE-Gly). However, occurrences of 1PE-Gly in plants remain unknown. In this study, four 1PE-Glys have been isolated from tea flowers. Three of them were determined as (R)-1PE βD-glucopyranoside ((R)-1PE-Glu), (S)-1PE-Glu, and (S)-1PE β-primeveroside ((S)-1PE-Pri), respectively, on the basis of NMR, MS, LC-MS, and GC-MS evidence. The other one was identified as (R)-1PE-Pri on the basis of LC-MS and GC-MS data. Moreover, these 1PE-Glys were chemically synthesized as the authentic standards to further confirm their occurrences in tea flowers. 1PE-Glu had a higher molar concentration than 1PE-Pri in each floral stage and organ. The ratio of (R) to (S) differed between 1PE-Glu and 1PE-Pri. In addition, a 1PE-Gly hydrolase β-primeverosidase recombinant protein produced in Escherichia coli exhibited high hydrolysis activity toward (R)-1PE-Pri. However, β-primeverosidase transcript level was not highly expressed in the anther part, which accumulated the highest contents of 1PE-Gly and 1PE. This suggests that 1PE-Gly may not be easily hydrolyzed to liberate 1PE in tea flowers. This study provides evidence of occurrences of 1PE-Glys in plants for the first time. KEYWORDS: glucopyranoside, 1-phenylethanol, primeveroside, primeverosidase, stereochemistry, tea flower

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INTRODUCTION Plants synthesize and emit a large variety of volatile organic compounds, which possess extremely important ecological functions. The volatile compounds can also be converted from free forms to glycosidically bound forms, which are more watersoluble and less reactive than their free aglycone counterparts.1 Many glycosylated volatile compounds have been isolated and identified in various plant species or plant-derived foods. In many cases, alcoholic volatiles such as benzyl alcohol, 2phenylethanol, methyl salicylate, (Z)-3-hexenol, linalool, linalool oxides, and geraniol can be transformed to glycosidically bound form.2−6 Besides alcoholic volatile compounds, some nonalcoholic volatile compounds such as benzaldehyde, coumarin, and damascenone were found to occur as glycosidically bound form, and these glycosidically bound volatile compounds take more steps than the glycosidically bound alcoholic volatiles to release free volatile compounds.7−9 The glycosylated volatile compounds are involved in diverse events in plants. For example, when plants are exposed to stresses or affected by environmental factors, the glycosylated volatile compounds can be hydrolyzed to release free volatile compounds, which are involved in the plant defense system.10 Another example is that the glycosylated volatile compounds are related to regulation of emission of floral volatiles.11 In our previous study, we found that 1-phenylethanol (1PE) was a © 2014 American Chemical Society

major endogenous volatile compound in tea (Camellia sinensis) flowers, whereas only a little 1PE was emitted from the flowers.12 This may be because 1PE was transformed to glycosically conjugated 1PE (1PE-Gly). However, occurrences of 1PE-Gly in plants remain unknown. There is extensive literature on human health benefits of tea leaves and their bioactive compounds. Recently, tea flowers have attracted increasing interest of researchers. One reason is that tea flowers are an abundant and nonutilized resource; for example, over 1.8 billion kilograms of tea flowers are available annually in China.13 Another reason is that tea flowers contain many bioactive compounds including catechins, caffeine,14 floratheasaponins,15−17 flavonol glycosides,18 polysaccharides,19 amino acids,20 spermidine derivatives,21 and volatile compounds.12,22 In the present study, four glycosylated volatile compounds including (R)-1PE-β-D-glucopyranoside ((R)-1PEGlu), (S)-1PE-Glu, (R)-1PE-β-primeveroside ((R)-1PE-Pri), and (S)-1PE-Pri were first identified in tea flowers. Moreover, the four 1PE-Glys were chemically synthesized for the first time to further confirm their occurrences in tea flowers, although the Received: Revised: Accepted: Published: 8042

May 13, 2014 July 10, 2014 July 26, 2014 July 28, 2014 dx.doi.org/10.1021/jf5022658 | J. Agric. Food Chem. 2014, 62, 8042−8050

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16% MeCN as mobile phase, at 254 nm and a flow rate of 10 mL/min. The injection volume was 100 μL/isolation. Each collection volume was 10 mL/fraction. The obtained fractions 14 and 15 (RT 18−20 min) were subjected to the second preparative HPLC isolation, for which conditions were the same as for the first preparative HPLC isolation. The obtained fraction 6 (RT 17−18 min) was subjected to the third preparative HPLC isolation. The third preparative HPLC used a Capcell Pak C18 reversed-phase column (10 mm × 250 mm, 5 μm particle size, Shiseido Co. Ltd., Japan) with 16% MeCN as mobile phase, at 254 nm and a flow rate of 2.5 mL/min. The injection volume was 50 μL/isolation. The collection volume was 1.25 mL/fraction. The obtained fractions 4−8 were identified to have 1PE-Gly (Figure S2). Fractions 4 and 5 (RT 18.0−19.0 min) and fraction 6 (RT 19.0−19.5 min) were subjected to the recycle HPLC (LC pump, Jasco PU-9986; detector, Jasco 875-UV; Jasco) isolation, respectively. The recycle HPLC used a Capcell Pak C18 reversed-phase column (10 mm × 250 mm, 5 μm particle size, Shiseido Co. Ltd., Japan) with 18% MeCN as mobile phase, at 210 nm and a flow rate of 4.0 mL/min. The injection volume was 50 μL/isolation. Purified disaccharide of (S)-1PE and monosaccharide of (S)-1PE were obtained from fractions 4 and 5. The disaccharide of (S)-1PE and monosaccharide of (R)-1PE were obtained from fraction 6. Fractions 7 and 8 (RT 19.5−20.5 min) were subjected to the further preparative HPLC isolation to obtain the purified monosaccharide of (R)-1PE. The HPLC used a Capcell Pak C18 reversed-phase column (10 mm × 250 mm, 5 μm particle size, Shiseido Co. Ltd., Japan) with 18% MeCN as mobile phase, at 210 nm and a flow rate of 2.5 mL/min. The injection volume was 50 μL/ isolation. Determination of 1PE-Gly Isolated from Tea Flowers. Determination of 1PE-Gly by Enzymatic Hydrolysis Combined with GC-MS Analysis during the Isolation. The enzymatic hydrolysis combined with GC-MS analysis method is the same as described by Dong et al.12 The isolated fraction (50 μL) as shown above was concentrated in a vacuum, dried under nitrogen gas, redissolved in 0.4 mL of 50 mM citric acid buffer (pH 6.0) containing 10 mg of βprimeverosidase (1.12 unit) and 0.6 mg β-glucosidase (from almonds, 3.12 unit), and allowed to react at 37 °C for 14 h. Sodium chloride (144 mg) was added to the reaction solution, which was allowed to stand for 15 min. Afterward, 5 nmol of ethyl n-decanoate as an internal standard was added. The solution was extracted with 0.4 mL of hexane/ethyl acetate (1:1) and centrifuged (16400g, 4 °C, 3 min), and the supernatant was dried over anhydrous sodium sulfate. The samples were then analyzed by GC-MS QP5000 (Shimadzu Corp.), which was controlled by a Class-5000 workstation. The temperature of the injector was 230 °C. The GC was equipped with a capillary SUPELCOWAX 10 column (30 m × 0.25 mm i.d., and 0.25 μm film thickness, Supelco Inc., Bellefonte, PA, USA). Helium was used as the carrier gas at a flow rate of 1.6 mL/min. The GC temperature was 60 °C for 3 min, ramped at 40 °C/min to 180 °C followed by 10 °C/ min to 240 °C, and then held at 240 °C for 3 min. MS was performed in full scan mode (mass range m/z 70−200). Determination of 1PE-Gly by LC-MS Analysis during the Isolation. To further investigate the molecular weight of the isolated fractions containing 1PE-Gly, the isolated fractions were analyzed by a LCMS-2010 A system (Shimadzu Corp.) equipped with a UG120 C18 reversed-phase column (2.0 mm × 150 mm i.d., 5 μm particle size, Shiseido Co. Ltd., Japan) with solvent A (HCOOH/H2O, 0.05:99.95, v/v) and solvent B (MeCN), at a flow rate of 0.2 mL/min at 40 °C. A total of 5 μL of the sample solutions was analyzed using a gradient elution. Elution was started under isocratic conditions of 16% of solvent B for 5 min and followed by a linear increase of solvent B to 90% at 10 min and subsequently brought back within 0.5 min to 16% of solvent B and held for another 7.5 min to allow for column equilibration. UV−vis spectra were recorded between 190 and 370 nm for each chromatographic peak. Optimized electrospray operating conditions were as follows: dry gas, 1.5 L/min; capillary voltage, 1.5 kV; dry temperature, 250 °C. MS was performed in full scan mode (mass range m/z 100−500) and SIM mode (m/z 283, 329, 415, and 461).

enzymatic syntheses of 1PE-Gly by cultured plant cells have been reported previously.23−25 In addition, β-primeverosidase, one of 1PE-Gly hydrolases, transcript level was not highly expressed in the anther part, which accumulated the highest contents of 1PE-Gly and 1PE. This suggests that 1PE-Gly may not be easily hydrolyzed to liberate 1PE in tea flowers.

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MATERIALS AND METHODS

Chemicals. D-Xylose and triphenylmethyl chloride were purchased from Tokyo Chemical Industries Co., Ltd., Japan. Ethyl n-decanoate, CDCl3 (99.8 atom % D + 0.03% v/v tetramethylsilane (TMS)), and organic solvents used for chemical synthesis were purchased from Kanto Chemical Co. Inc., Japan. N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) was purchased from Regis Chemical Co., USA. (R)-1PE, (S)-1PE, CD3OD (99.8 atom % D + 0.05% v/v TMS), CD3COCD3 (99.9 atom % D + 0.05% v/v TMS), and reagents used for chromatography analysis and chemical synthesis were purchased from Wako Pure Chemical Industries Ltd., Japan. Polyvinylpolypyrrolidone (PVPP), XAD-2, isopropyl-β-D-thiogalactopyranoside (IPTG), HBr/AcOH, β-glucosidase, and Spectrum Plant Total RNA Kit were purchased from Sigma-Aldrich Co. Ltd., USA. βPrimeverosidase was purchased from Amano Enzyme Inc., Japan. ODS-C18 was purchased from Chemco Scientific Co., Ltd., Japan. Sep-PakVac 6 cm3 was purchased from Waters Corp., USA. Coomassie Blue R250, 30% acrylamide/Bis solution, N,N,N′,N′-tetramethylethylenediamine (TMEMD), sodium dodecyl sulfate (SDS), and 2× SYBR Green Universal PCR Mastermix were purchased from Bio-Rad Laboratories, USA. Ni-NTA resin was purchased from Qiagen Inc., USA. Enterokinase was purchased from Novagen, MERCK Eurolab, Germany. Quick RNA Isolation Kit was purchased from Huayueyang Biotechnology Co., Ltd., China. PD-10 desalting column was purchased from GE Healthcare Life Sciences, USA. Plant Materials. The flowers of C. sinensis var. Jinxuan were obtained from tea fields at the South China Agricultural University (Guangzhou, China) between October and November 2012 and 2013. As described in our previous study,12 flower development was divided into three stages: at stage 1 the flower buds are closed, at stage 2 the flower is half open, and at stage 3 the flower is fully open. In addition, the flowers at stage 3 were divided into four parts including petals, filaments, anthers, and other parts. The “other parts” comprise a mixture of carpels, receptacles, sepals, and pedicels. Isolation and Purification of 1PE-Gly from Tea Flowers. Figure S1 (Supporting Information) shows a brief flowchart of the isolation and purification of 1PE-Gly from tea flowers. Eight hundred grams (fresh weight) of the flowers was extracted with 3 L of 80% ethanol overnight at 4 °C and then centrifuged at 17000g and 4 °C for 20 min. The supernatant was filtered, and the filtrate was concentrated to 500 mL by a rotary evaporation. Forty grams of PVPP was added to the concentrated filtrate to remove polyphenols. The mixture was stirred at 4 °C for 60 min and then centrifuged at 17000g and 4 °C for 20 min. Twenty grams PVPP was added to the supernatant, stirred at 4 °C for 30 min, and then centrifuged again at 17000g and 4 °C for 20 min. The resultant supernatant was extracted with 300 mL of CHCl3 three times. The aqueous fraction was evaporated to remove CHCl3 residue and diluted with Milli-Q water to 600 mL. The diluted solution was passed through an XAD-2 column (about 600 mL, i.d. 6 cm × 21 cm) and then eluted with 3 L of Milli-Q water and 3 L of methanol, successively. The methanolic eluate was concentrated to dryness and dissolved in 120 mL of 5% methanol. The 5% methanol-soluble solution was passed through an ODS-C18 open column (60 mL, i.d. 2.4 cm × 13.3 cm) and eluted with 120 mL of 25% methanol, 120 mL of 50% methanol, and 120 mL of 100% methanol, successively. The 50% MeOH/methanolic eluate was concentrated to dryness, dissolved in 1.5 mL of 16% MeCN, and then subjected to the first preparative HPLC (LC pump, Jasco PU-980; detector, 875-UV; solvent mixing module, Jasco LG-1580-02; degasser, Jasco DG-1580-53; column oven, Jasco CO-965; Jasco, Tokyo, Japan) isolation. The first preparative HPLC used a Capcell Pak C18 reversed-phase column (20 mm × 250 mm, 5 μm particle size, Shiseido Co. Ltd., Japan) with 8043

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Figure 1. Schemes of chemical synthesis of (R)/(S)-1PE-Glu (A) and (R)/(S)-1PE-Pri (B). Determination of Stereochemistry of 1PE-Gly by LC-MS and GCMS Analyses. To determine the stereochemistry of 1PE-Gly, LC-MS was employed to analyze the 1PE-Gly in the crude extract of tea flowers (Supporting Information Figure S3) by comparison with the chemically synthesized authentic standards as shown below. The protocol of LC-MS analysis is shown under Quantitative Analysis of 1PE-Gly. Furthermore, the (R)- and (S)-1PE obtained from the enzymatic hydrolysis of the isolated fractions containing 1PE-Gly as described above were analyzed by using a GC-MS QP5000 (Shimadzu) equipped with an InertCap CHIRAMIX (30 m × 0.25 mm × 0.25 μm, GL Sciences, Inc., Torrence, CA, USA). Helium was used as the carrier gas at a flow rate of 1.8 mL/min. The temperatures were 40 °C for 5 min, a ramp of 3 °C/min to 180 °C, and then held at 180 °C for 10 min. MS was performed in full scan mode (mass range m/z 70−200) and SIM mode (m/z 77, 79, 107 and 122).

Determination of Chemical Structures of 1PE-Gly by HighResolution Mass Spectra (HRMS) and NMR. The purified 1PE-Glys were identified by HRMS (JMS-T100LC AccuTOF mass spectrometer, JEOL, Tokyo, Japan) using polypropylene glycol as an internal standard and NMR (JNM-LA 500 FT-NMR, JEOL, Tokyo, Japan) using 1H, 13C, COSY, HSQC, and HMBC with TMS as an internal standard. The MS conditions were as follows: ionization, ESI (positive mode); scan at m/z 50−550. As some 1H NMR signals of the sugar moiety of the purified 1PEGly were overlapped, an acetylation reaction was employed to assign the 1H NMR signal. One milligram of the purified 1PE-Gly was dissolved in 100 μL of dehydrated pyridine and 200 μL of dehydrated acetic anhydride. The solution was stirred for 17 h at room temperature. Afterward, toluene was added and then concentrated to 8044

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min, followed by 35 cycles of 98 °C for 15 s, 54 °C for 15 s, 72 °C for 1 min 30 s, and a final extension at 72 °C for 10 min. The resulting PCR product was purified and subcloned into the pGEM-T vector (Promega) and sequenced to confirm the correctness of ORF. To obtain the mature form of a recombinant protein, the correct ORF was subcloned into pET32a vector (Novagen, Madison, WI, USA) to obtain the expression constructor (pET32a/PRD). After verification by sequencing, the expression constructor was transformed into E. coli Rosetta (Novagen) for inducible His-tagged protein expression. Freshly transformed Rosetta cells harboring pET32a/PRD vector were grown at 37 °C to an OD600 = 0.6. After the addition of 0.05 mM IPTG, the cultures were grown at 20 °C for another 16 h to produce recombinant His-tagged protein. The cells were harvested at 4000g for 10 min and then disrupted by sonication in a 50 mM NaH2PO4 (pH 8.0) buffer containing 300 mM NaCl and 10 mM imidazole. After centrifugation at 10000g for 20 min, the supernatant was collected and purified by using affinity binding on Ni-NTA resin according to the manufacturer’s instruction. The partially purified protein was passed through a PD-10 desalting column for further enzyme activity assay. SDS-PAGE Analysis. The E. coli-expressed proteins were subjected to SDS-PAGE with the use of a separation gel (2.4 mL of 30% acrylamide/bis(acrylamide), 1.8 mL of Tris-HCl (pH 8.8), 0.07 mL of 10% SDS, 0.07 mL of 10% ammonium persulfate, 2.7 mL of ddH2O, 0.003 mL of TMEMD) and a concentration gel (0.34 mL of 30% acrylamide/bis(acrylamide), 0.25 mL of Tris-HCl (pH 6.8), 0.02 mL of 10% SDS, 0.02 mL of 10% ammonium persulfate, 1.4 mL of ddH2O, 0.002 mL of TMEMD). After completion of electrophoresis, the proteins were stained with Coomassie Blue R250 for at least 3 h and then decolored three times. E. coli-Expressed β-Primeverosidase Assay. To identify the E. coli-expressed β-primeverosidase activity, (R)-1PE-Glu, (S)-1PE-Glu, (R)-1PE-Pri, and (S)-1PE-Pri were selected as substrates. The reaction mixture contained 5 μg of substrate, 350 μL of 20 mM sodium citrate buffer (pH 5.0), and 10 μg of partially purified protein. The reactions were incubated at 37 °C for 10 h. After cooling to room temperature, the reaction mixture was extracted with 400 μL of hexane/ethyl acetate (1:1). The upper phase was dried over anhydrous sodium sulfate, and 1 μL of the extract was subjected to GC-MS analysis. Samples were analyzed using a GC-MS QP2010 SE (Shimadzu Corp.) equipped with a SUPELCOWAX 10 column (30 m × 0.25 mm × 0.25 μm, Supelco Inc.). The injector temperature was 240 °C, splitless mode was used with a splitless time of 1 min, and helium was the carrier gas with a velocity 1.0 mL/min. The GC temperatures were 60 °C for 3 min, ramp of 40 °C/min to 180 °C followed by 10 °C/min to 240 °C, and then held 240 °C for 5 min. The mass spectrometer was operated with SIM mode (m/z 77, 79, 107, 122). 1PE was identified by comparing its retention times and mass spectra with those of authentic compound. Transcript Expression Analysis of β-Primeverosidase. Total RNA was isolated immediately after dissection (Fast Plant Total RNA Kit). Ten times-diluted reverse-transcribed cDNA was subjected to qRT-PCR analysis. The qPCR primers for β-primeverosidase were 5′TGCCTCTTCTGCCTACC-3′ (sense) and 5′-TGTTCACTCCTCCGCTAA-3′ (antisense). The qPCR primers for internal standard βactin were 5′-GCCATATTTGATTGGAATGG-3′ and 5′-GGTGCCACAACCTTGATCTT-3′.28 Each qPCR reaction (20 μL) was assembled in a 0.2 mL microtube by adding iTaq Universal SYBR Green Supermix (10 μL) (Bio-Rad, Hercules, CA, USA), 300 nM of each primer, 10 times-diluted cDNA (2 μL), and ddH2O (6 μL). The qRT-PCR was carried out on a Roach LightCycle 480 (Roach Applied Science, Mannheim, Germany) under conditions of one cycle of 95 °C for 60 s, 40 cycles of 95 °C for 15 s, and 60 °C for 30 s. A melt curve was performed at the end of each reaction to verify PCR product specificity. The 2−ΔΔct method was used to calculate the relative expression level.29 β-Actin was used as an internal reference. Change in mRNA level of β-primeverosidase for each treatment was normalized to that of β-actin.

dryness. The acetylated product was dissolved in CD3OD, and analyzed by 1H NMR (500 MHz). Chemical Synthesis of 1PE-Gly. To further confirm the chemical structures of the isolated 1PE-Gly, we chemically synthesized the (R)1PE-Glu, (S)-1PE-Glu, (R)-1PE-Pri, and (S)-1PE-Pri as authentic standards, on the basis of the method described by Ma et al.26 The schemes of chemical synthesis of these 1PE-Glys are shown in Figure 1. The detailed chemical synthesis protocol is described in the Supporting Information. The optical rotation of each compound is shown as (R)-1PE-Glu [α]29D +1.00° (c 1.0, CH3OH), (S)-1PE-Glu [α]29D −18.01° (c 1.1, CH3OH), (R)-1PE-Pri [α]21D −4.24° (c 1.3, CH3OH), and (S)-1PE-Pri [α]21D −4.35° (c 0.2, CH3OH). Figure S4 shows the UV spectra and HR-MS data. Quantitative Analysis of 1PE-Gly. Relative Quantitative Analysis of 1PE-Gly by MSTFA Derivatization and GC-MS Analysis. The crude extract of 1PE-Gly was prepared by using the method described in refs 6 and 12 with a slight modification. Five hundred milligrams (fresh weight, finely powdered) of plant tissues was extracted with 2 mL of cold methanol by vortexing for 2 min followed by ultrasonic extraction in ice-cold water for 10 min. The extracts were mixed with 2 mL of cold chloroform and 0.8 mL of cold water for phase separation, and the resulting upper layer was dried and redissolved in 1 mL of water. The resulting solution was mixed with 30 mg of PVPP, allowed to stand for 90 min, and centrifuged (16400g, 4 °C, 10 min). The resultant supernatant was loaded on an Amberlite XAD-2 column and eluted successively with 3 mL of water, 3 mL of pentane/dichloromethane (2:1), and 5 mL of methanol. The methanol eluate was concentrated in a vacuum, dried under nitrogen gas, derivatized with 70 μL of MSTFA and 70 μL of pyridine at 37 °C for 60 min, and then centrifuged. The MSTFA derivates were then analyzed by a GC-MS QP2010 SE (Shimadzu Corp.) equipped with an HP-5 column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies, Germany). The injector temperature was 240 °C, splitless mode was used with a splitless time of 1 min, and helium was the carrier gas with a velocity 1.0 mL/min. The GC temperatures were 80 °C for 2 min, ramp of 15 °C/min to 300 °C, then held at 300 °C for 10 min. The mass spectrometry was operated with full scan mode and SIM mode (see Table S1 in the Supporting Information). m/z 259 was used as quantifier ion. Quantitative Analysis of 1PE-Gly by LC-MS Analysis. Two grams (fresh weight, finely powdered) of tea flowers at each stage was extracted with 36 mL of methanol containing 5 nmol of p-nitrophenylGlu (pNP-Glu) and 5 nmol of pNP-Pri as internal standards. Nine milliliters of methanol extracts was dried and redissolved in 4 mL of water. The solution was loaded on a Sep-PakVac 6 cm3 (1 g) column and eluted successively with 20 mL of water, 60 mL of 25% methanol (in water), and 20 mL of methanol. The 25% methanol eluate was concentrated in a vacuum and dissolved in 500 μL of water. The resultant solution was analyzed by a LCMS-2010 A system (Shimadzu Corp.) equipped with a UG120 C-18 reversed-phase column (2.0 mm × 150 mm i.d., 5 μm particle size, Shiseido Co. Ltd., Japan) with solvent A (HCOOH.H2O, 0.05:99.95, v/v) and solvent B (MeCN) at a flow rate of 0.2 mL/min at 40 °C. A total of 5 μL of the sample solution was analyzed using a gradient elution. Elution was started from a linear increase of solvent B from 5 to 7% at 60 min and held at 7% of solvent B for 15 min. MS was performed in SIM mode (m/z 329 and 461 for [M + HCOOH]− of 1PE-Glu and 1PE-Pri, respectively, and m/z 346 and 478 for [M + HCOOH]− of pNP-Glu and pNP-Pri, respectively) (Supporting Information Figure S3). Gene Cloning and Recombinant Protein Expression of βPrimeverosidase in Escherichia coli. Finely powdered tea floral tissues (100 mg) were used to isolate RNA, and total RNA was isolated using a Spectrum Plant Total RNA Kit. The first-strand cDNA was synthesized using a Reverse Transcription System (Promega, Madison, WI, USA). The GenBank accession number of βprimeverosidase is AB088027.1. The cDNA coding was amplified by PCR with forward primer 5′-GATATCGCTCAAATCTCCTCCTTCAAC-3′, containing an EcoRV site, and reverse primer 5′-GTCGACCTACTTGAGGAGGAATTTCTT-3′, containing a SalI site.27 The PCR conditions were adjusted as follows: denaturation at 94 °C for 5 8045

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the proposed structures, β-D-glucopyranosides of (R)-1PE and (S)-1PE were chemically synthesized (Figure 1A and Supporting Information). On the basis of the 13C NMR data, the synthetic (R)-1PE and (S)-1PE β-D-glucopyranosides were coincided with those of compounds 1 and 2 (Table S2). The differences between (R)-1PE and (S)-1PE β-D-glucopyranosides were observed in the chemical shifts of C-1, C-2, C-1′, and C-3 (Table S2). On the basis of the above observations, compound 1 is identified as (R)-1PE-Glu and compound 2 as (S)-1PE-Glu. On the basis of the HR-MS data and enzymatic hydrolysis to form (S)-1PE, compound 3 was proposed as a disaccharide of (S)-1PE, which has one additional C5 sugar (Figure 2). The 1H NMR signal of H-1″ is 4.36 with J = 7.6 Hz, suggesting that the sugar moiety with C5 must be a pyranose form not in furanose form (5.10, J = 6.0 Hz) (Supporting Information Table S4). We also need to determine if the disaccharide moiety is primeveroside or vicianoside. The differences between primeveroside and vicianoside are OH orientation at H-4″ (Figure S6A). Due to the differences, the coupling pattern for H-3″ should be different. The signal for H-3″ of primeveroside should have coupling with the same/similar values with H-2″ and H-4″, whereas the signal for H-3″ of vicianoside should have different coupling constants between H-2″ and H-4″ due to the Jax-ax and Jax-eq (Figure S6A). As the H-3″ 1H NMR signals were overlapped, we carried out acylated reaction of compound 3 and then analyzed its 1H NMR. We found that the J H-3″ was a triplet with J = 9.5 Hz (Figure S6B). To further confirm the proposed structures, β-primeverosides of (R)-1PE and (S)-1PE were chemically synthesized (Figure 1B and Supporting Information). The 13C NMR data of the synthetic (S)-1PE-Pri (Table S5) coincided with those of compound 3 (Table S4). On the basis of the above observations, compound 3 is identified as (S)-1PE-Pri. We did not obtain a sufficient purified amount for NMR identification of (R)-1PE-Pri. On the basis of LC-MS data on a column of ODS and the GC-MS equipped with a CHIRAMIX column after enzymatic hydrolysis (Figure S2), (R)-1PE-Pri was tentatively identified in tea flowers. By comparison with the GC-MS chromatogram of the MSTFA-derived product of synthesized (R)-1PE-Pri, tea flowers indeed contained (R)-1PE-Pri (Figure S7D). Moreover, LC-MS analysis by comparison with the chemically synthesized authentic standards also provided evidence that four 1PE-Glys, including (R)/(S)-1PE-Glu and (R)/(S)-1PE-Pri, occurred in the tea flowers at stages 1, 2, and 3 (Figure S3). In addition, the NMR data of the four 1PE-Glys from either tea flowers or chemical synthesis coincided with those from enzymatic syntheses by cultured plant cells that were reported previously.23−25 1PE-Glu Was the Major 1PE-Gly Occurring in Tea Flowers. To further characterize the occurrence of each 1PEGly, we quantified each 1PE-Gly in different floral stages and organs. MSTFA derivatization and GC-MS were employed to analyze the relative content of each 1PE-Gly. Figure S7 in the Supporting Information shows mass spectra and chromatograms of MSTFA-derived products of authentic 1PE-Gly standards and crude 1PE-Gly extract from tea flowers, suggesting the presences of the four 1PE-Glys in tea flowers. Table S1 shows the characteristic ions of MSTFA-derived products of each 1PE-Gly based on GC-MS and GC-TOFMS analyses. Relative quantitation results show that 1PE-Glu had higher molar concentration than 1PE-Pri in each floral stage and organ (Figure 3A,B). On the basis of the calculation of the

RESULTS AND DISCUSSION 1PE-Glys in Tea Flowers Were Determined as (R)/(S)1PE-Glu and 1PE-Pri. 1PE-Gly may be involved in regulating the emission of 1PE from tea flowers.12 To investigate the occurrence of 1PE-Gly in tea flowers, the 1PE-Gly compounds 1, 2, and 3 were isolated (Figure 2). On the basis of HR-MS

Figure 2. HR-MS data and chemical structures of 1PE-monosaccharides (compounds 1 and 2) and 1PE-disaccharide (compound 3) isolated from tea flowers.

data, the isolated compounds 1 and 2 are proposed as diastereomeric isomers of monoglycoside of 1PE (Figure 2). The 13C NMR data show that compounds 1 and 2 have 14 carbon signals (Supporting Information Table S2). 1H NMR showed that both compounds 1 and 2 have anomeric protons with J = 7−8 Hz (Table S3). Moreover, the β-glucosidase could hydrolyze the glycoside to release 1PE (data not shown). These suggest that the glycoside moieties of compounds 1 and 2 have β-connectivity. The 1H chemical shifts of H-1′ significantly differ between (R)- and (S)-1PE glucopyranoside, and that of the (R) type is generally higher than that of the (S) type (Table S3). Moreover, after enzymatic hydrolysis, compounds 1 and 2 were hydrolyzed to release (R)-1PE and (S)-1PE, respectively. These indicate that compound 1 is (R) type and compound 2 is (S) type. The peracetate of compound 1 was obtained, and the structure of its glycosidic part was analyzed on the basis of the coupling patterns of each proton. As shown in Figure S5, from H-1′ to H-5′ all of the protons are trans-diaxial, suggesting that the C6 sugar moiety is β-glucopyranose. Therefore, compounds 1 and 2 were proposed as β-glucopyranose of (R)-1PE and (S)1PE, respectively. On the HMBC, cross peaks between H-1′ /C-1 and H-1/C-1′ revealed their connection between the aglycone and the sugar residue (Figure 2). To further confirm 8046

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Figure 3. Distributions of 1PE-Gly in each floral stages and organ. (A, B) The relative quantitations of (R)-1PE-Glu, (S)-1PE-Glu, (R)-1PE-Pri, and (S)-1PE-Pri were performed using MSTFA derivatization and GC-MS. “Others” is a mixture of carpels, receptacles, sepals, and pedicels. (C−F) The relative ratio (%) of (R) and (S) was calculated on the basis of the peak area of characteristic ion m/z 259 from the MSTFA derivatization and GCMS analysis. (G−I) The quantitations of (R)-1PE-Glu, (S)-1PE-Glu, (R)-1PE-Pri, and (S)-1PE-Pri were performed using LC-MS.

peak area of the characteristic ion m/z 259 (Table S1 and Figure S7), the relative ratio of (R) type of 1PE-Glu was higher than that of (S) type (Figure 3C,D), whereas the relative ratio of the (S) type of 1PE-Pri was higher than that of the (R) type (Figure 3E,F). Considering the possible effects of MSTFA derivatization reaction on the quantitation, we applied LC-MS to directly quantitate the content of each 1PE-Gly (Figure S3). The LC-MS analytical results (Figure 3H,I) show similar trends of ratio of R to S of 1PE-Gly with the GC-MS analytical results (Figure 3C,E), although concentrations of 1PE-Gly analyzed by LC-MS (Figure 3G) were higher than those analyzed by MSTFA derivatization and GC-MS (Figure 3A). In many cases, glycosylated alcoholic volatiles are found in tea leaves as βprimeverosides or glucopyranoside, and the β-primeverosides occur primarily in the glycosidically bound form. 30,31 Glucopyranoside was a major form of 1PE-Gly, although both glycosidic forms of 1PE were identified in tea flowers. Interestingly, the ratio of (R) to (S) differed between the 1PEGlu and the 1PE-Pri. In our previous study, we used exogenous glycoside hydrolases to liberate free 1PE from the crude 1PE glycoside extract of tea flowers12 and found that (R) type was the major hydrolyzed 1PE. In this study, we further

demonstrated that (R)-1PE-Glu is the major 1PE-Gly in tea flowers. 1PE-Gly Hydrolase β-Primeverosidase Transcript Level Was Not Highly Expressed in Floral Tissue. To further clarify the hydrolysis of 1PE-Gly to liberate 1PE, we investigated the 1PE-Gly hydrolase β-primeverosidase. The recombinant β-primeverosidase expressed in E. coli (Figure S8 in the Supporting Information and Figure 4A,B) displayed strong enantioselectivity. Compared with (S)-1PE-Pri, the hydrolysis ability of β-primeverosidase toward (R)-1PE-Pri was significantly higher (Figure 4). On the other hand, neither (S)-1PE-Glu nor (R)-1PE-Glu was found to be hydrolyzed by β-primeverosidase. We attempted to express recombinant βglucosidase in E. coli to verify its function. E. coli itself contained a high amount of its own β-glucosidase, which could hydrolyze 1PE-Glu to liberate 1PE. In contrast to the β-glucosidase occurring in E. coli, the expressed target β-glucosidase was very low. Therefore, it was very hard to distinguish if the expressed target β-glucosidase has a hydrolysis function of 1PE-Gly to form 1PE. β-Primeverosidase transcript level showed a slight but nonsignificant decrease trend during floral development 8047

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Figure 4. SDS-PAGE analysis of recombinant β-primeverosidase (A, B) and GC-MS identifications of formation of 1PE from (R)-1PE-Pri under the action of recombinant β-primeverosidase (C) and relative activities of recombinant β-primeverosidase toward different substrates (D). (A) Arrows indicate target protein. Proteins were resolved by SDS-PAGE on polyacrylamide gel and stained with Coomassie brilliant blue. Lanes: M, protein marker; 1, supernatant of crude extracts from E. coli cells with expression of vector pET32a/PRD; 2, pellet of crude extracts from E. coli cells with expression of vector pET32a/PRD. (B) Arrows indicate target protein. Lanes: M, protein marker; 1, enterokinase digested recombinant βprimeverosidase; 2, recombinant protein purified with Ni-NTA agarose from the supernatant of crude E. coli extracts. (C) 1PE product was identified by GC-MS. Total mass chromatography of characteristic ions of 1PE (m/z 77, 79, 107, and 122) is shown. (D) Activity toward (R)-1PE-Pri was highest and set as 100%. The activity toward other substrate was expressed as percent of maximum activity. N.D., not detected, which indicates that there were no differences between enzymatic reaction and control. The heated enzyme reaction was used as a control to exclude the possible effects of autodegradation of 1PE-Gly.

volatile compounds from the plant. In Rosa damascena flowers, 2-phenylethanol is released from its precursor 2-phenylethyl β37 D-glucoside during or after flower opening. During flower development in R. damascena, β-glucosidase activity increases 5 times,38 and β-glucosidase was suggested to be partly responsible for controlling the diurnal emission of 2-phenylethanol.39 In Gardenia jasmonoides flowers, the formation of flower fragrance compounds was attributed to a glycosidase activity, reaching a maximum level at the flower opening stage.40 In our present study, β-primeverosidase did not show an increase trend during the tea flower development and was relatively lowly expressed in tea flowers, suggesting that 1PEGly may not be easily hydrolyzed to liberate 1PE and be considered as transportable storage compounds. Our previous study showed that tea flowers contained a high amount of endogenous 1PE.12 1PE is used as a fragrance in the food flavor and cosmetics industries because of its mild floral odor and as an intermediate in the pharmaceutical industry. This study provides detailed chemical evidence of occurrences of glycosidically conjugated 1PE in tea flowers for the first time. These results would contribute to our understanding of the formation of 1PE and provide essential information for future potential application of tea flowers in the food flavor and cosmetics industries.

(Figure 5A). β-Primeverosidase was highly expressed in leaves, whereas it was not accumulated in floral tissues (Figure 5B). Furthermore, β-primeverosidase was lowly expressed in anthers, which accumulated the highest amounts of 1PE-Gly and 1PE (Figure 3B and ref 12). β-Primeverosidase has been purified from tea leaves, and its full amino acid sequence and function have been characterized.27,32 β-Glucosidase was only partially characterized in tea leaves.33 However, full the amino acid sequence of β-glucosidase has not been characterized, and its function has not been verified yet. In contrast to β-glucosidase, β-primeverosidase showed more involvement in the hydrolysis of glycosidically bound volatile compounds in tea leaves, because the β-primeverosides occur primarily in the glycosidically bound form.27 The glycosidically bound volatile compounds occur in vacuoles. However, β-primeverosidase was observed to be localized in cell walls and the cavity areas among cells.27 This compartmentation of substrates and enzymes in plant cells leads to the rare available evidence of hydrolysis of glycosidically bound volatile compounds in intact tea leaves.34 In contrast, such enzymatic hydrolysis has mostly been investigated during the manufacturing process of tea leaves, which can have interactions between the enzyme and the substrate.35,36 Investigations in flowers indicate that there is still evidence that the glycoconjugated volatile compounds can be hydrolyzed by endogenous glycosidases to liberate the 8048

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ASSOCIATED CONTENT

* Supporting Information S

Additional experimental details, Figures S1−S8, and Tables S1−S5. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(Z.Y.) Phone: +86-20-38072989. E-mail: [email protected]. Author Contributions ∥

REFERENCES

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Figure 5. Stage-specific (A) and tissue-specific (B) expression profiles of β-primeverosidase. Transcript abundance was calculated on the basis of the difference in cycle threshold (Ct) values between βprimeverosidase and β-actin transcripts by the normalized relative quantitation 2−ΔΔCt method. Data represent the mean value ± standard deviation of three independent experiments performed in triplicate. (B) Leaves were the first to third leaves. The fully open flowers were divided into four parts including petals, filaments, anthers, and other parts. “Others” is a mixture of carpels, receptacles, sepals, and pedicels.

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Article

Ying Zhou and Fang Dong equally contributed to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by “100 Talents Programme of the Chinese Academy of Sciences” (Y321011001 and 201209), the Foundation of Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, the Foundation of Guangdong Food and Drug Vocational College (2013YZ003), and the Japanese Society for the Promotion of Science (P10101). We also thank technical support of Mr. Totsuka for this research.

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ABBREVIATIONS USED HRMS, high-resolution mass spectra; IPTG, isopropyl-β-Dthiogalactopyranoside; MSTFA, N-methyl-N-(trimethylsilyl)trifluoroacetamide; 1PE, 1-phenylethanol; 1PE-Gly, glycosically conjugated 1PE; 1PE-Glu, 1PE β-D-glucopyranoside; 1PE-Pri, 1PE β-primeveroside; pNP-Glu, p-nitrophenyl-β-D-glucopyranoside; pNP-Pri, p-nitrophenyl-β-primeveroside; PVPP, polyvinylpolypyrrolidone; SDS, sodium dodecyl sulfate; TMEMD, N,N,N′,N′-tetramethylethylenediamine; TMS, tetramethylsilane 8049

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