Formation of Volatile Tea Constituent Indole During the Oolong Tea

Jun 5, 2016 - College of Food, South China Agricultural University, Wushan Road, Tianhe District, Guangzhou 510642, China. ⊥ Juxiangyuan Health Food...
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Formation of Volatile Tea Constituent Indole During the Oolong Tea Manufacturing Process Lanting Zeng,†,‡,∇ Ying Zhou,†,∇ Jiadong Gui,†,‡ Xiumin Fu,† Xin Mei,† Yunpeng Zhen,§ Tingxiang Ye,§ Bing Du,∥,⊥ Fang Dong,# Naoharu Watanabe,⊗ and Ziyin Yang*,†,‡ †

Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement & Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Xingke Road 723, Tianhe District, Guangzhou 510650, China ‡ University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China § Waters Technologies (Shanghai) Ltd., No. 1000 Jinhai Road, Shanghai 201203, China ∥ College of Food, South China Agricultural University, Wushan Road, Tianhe District, Guangzhou 510642, China ⊥ Juxiangyuan Health Food (Zhongshan) Co.,Ltd., No. 13, Yandong Second Road, Torch Development Zone, Zhongshan 528400, China # Guangdong Food and Drug Vocational College, Longdongbei Road 321, Tianhe District, Guangzhou 510520, China ⊗ Graduate School of Science and Technology, Shizuoka University, 3-5-1 Johoku, Naka-ku, Hamamatsu 432-8561, Japan S Supporting Information *

ABSTRACT: Indole is a characteristic volatile constituent in oolong tea. Our previous study indicated that indole was mostly accumulated at the turn over stage of oolong tea manufacturing process. However, formation of indole in tea leaves remains unknown. In this study, one tryptophan synthase α-subunit (TSA) and three tryptophan synthase β-subunits (TSBs) from tea leaves were isolated, cloned, sequenced, and functionally characterized. Combination of CsTSA and CsTSB2 recombinant protein produced in Escherichia coli exhibited the ability of transformation from indole-3-glycerol phosphate to indole. CsTSB2 was highly expressed during the turn over process of oolong tea. Continuous mechanical damage, simulating the turn over process, significantly enhanced the expression level of CsTSB2 and amount of indole. These suggested that accumulation of indole in oolong tea was due to the activation of CsTSB2 by continuous wounding stress from the turn over process. Black teas contain much less indole, although wounding stress is also involved in the manufacturing process. Stable isotope labeling indicated that tea leaf cell disruption from the rolling process of black tea did not lead to the conversion of indole, but terminated the synthesis of indole. Our study provided evidence concerning formation of indole in tea leaves for the first time. KEYWORDS: indole, mechanical damage, oolong tea, tea aroma, tryptophan synthase α-subunit, tryptophan synthase β-subunits

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P450s.6−8 Indole is a constituent of herbivore-induced volatiles of many plant species, for instance, peanut, rice, maize, and tea (Camellia sinensis).9−12 It functions as an essential within-plant and plant−plant priming agent that prepares systemic tissues and neighboring plants for incoming attacks. Besides, indole may be incorporated into the biosynthesis of nonvolatile benzoxazinoids, which by themselves can act as defensive inducers in plant.13,14 It is therefore possible that indole-derived metabolites trigger the actual priming response in maize.11 Besides its physiological and ecological functions in plants, indole is a characteristic volatile constituent and potent odorant of oolong tea.15−18 Owing to importance of indole, a wide variety of attempts have been made to probe for the biosynthesis of indole in some plant species, including grass, Zea mays, Arabidopsis thaliana, and wheat.2,19−21 These plant species possess many homolo-

INTRODUCTION Indole, an electron-rich N-aromatic heterocyclic organic compound, is widely distributed and plays an important role in plants. It is a popular component of fragrances, such as jasmine oil and argan essential oil, and the fragrance is one of the key factors for attracting insect pollinators.1 Moreover, several lines of evidence suggest that indole serves as the precursor, core building block, and functional group of many important biochemical molecules and compounds. Tryptophan (Trp), as an essential amino acid for human nutrition, is synthesized from indole.2 Likewise, the phytohormone indole3-acetic acid (IAA) has been proposed to be synthesized from indole through a Trp-independent pathway, which remains to be substantiated from genetic, biochemical, and functional studies.3−5 Indole is also the core of other indolic auxins like indole-3-butyric acid and 4-chloroindole-3-acetic acid (4-CIIAA) etc., which are important in a number of plant developmental processes. In indigoid-producing plants, for example, Chinese woad and woad, indole is present as isatan and indicant.1 The conversion from indole into indigoids is catalyzed by a number of laboratorial-engineering cytochrome © XXXX American Chemical Society

Received: April 18, 2016 Revised: June 2, 2016 Accepted: June 5, 2016

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DOI: 10.1021/acs.jafc.6b01742 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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immediately frozen by liquid nitrogen for further study. Three replicates were processed according to the manufacturing. Simulation of Key Steps During the Manufacturing Processes of Oolong Tea and Black Tea. The tea leaves plucked as described above were continuously shaken and completely crushed to simulate turn over process of oolong tea and rolling process of black tea, respectively. The samples which simulated turn over process of oolong tea were collected after continuous shaking for 0, 4, and 8 h at 25 °C. The middle of leaves collected at 8 h was green, and the margin was red. The plucked tea leaves stored under the same conditions for 0, 4, and 8 h without continuous shaking were used as controls. Simulating key steps of black tea were performed after turn over 1 (T1) and 5 (T5). As treated groups, the tea leaves were completely crushed to make cell disruption and indoorwithered at room temperature for 6 h. Three replicates were processed and all samples were immediately frozen by liquid nitrogen for further study. Extraction and Analysis of Indole in Tea Samples. Analysis of indole used GC-MS analysis method, which was similar as described previously, with a slight modification.17 Five hundred milligrams (fresh weight) of samples (finely powdered) was extracted by 2 mL dichloromethane containing 5 nmol of ethyl decanoate as an internal standard in a shaker at room temperature overnight. The extraction solution was dried over anhydrous sodium sulfate. Then, 1 μL of the extraction was subjected to GC-MS QP2010 SE (Shimadzu Corporation, Japan) equipped with a SUPELCOWAXTM 10 column (30 m × 0.25 mm × 0.25 μm, Supelco Inc., Bellefonte, PA, USA). The injector temperature was 230 °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 oven temperature was 80 °C for 3 min, ramp of 30 °C/min to 240 °C, and then 240 °C for 20 min. The mass spectrometer was operated with full scan mode (mass range m/z 40−200) and the characteristic ion of indole is m/z 117. The relative content of indole in samples was calculated based on comparison with peak area of ethyl decanoate (internal standard). The data represent the mean value ± standard deviation of three in independent experiments performed in triplicate. Extraction and Analysis of Trp in Tea Samples. Analysis of Trp was performed by the method described as the previous report with a slight modification.12 Two hundred milligrams (fresh weight) of samples (finely powdered) was extracted with 0.7 mL cold methanol by vortexing for 2 min followed by ultrasonic extraction in ice-cold water for 20 min. The extracts, after adding 0.5 nmol of ribitol as an internal standard, were mixed with 0.7 mL cold chloroform, and 0.2 mL cold water for phase separation. The resulting upper layer was dried at 45 °C, derivatized with 100 μL of MSTFA at 37 °C for 60 min, and then centrifuged. One microliter of MSTFA derivate was 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 GC oven temperatures were 100 °C for 2 min, ramp of 6 °C/min to 240 and 20 °C/min to 300 °C, and then held at 300 °C for 10 min. The characteristic ion of Trp is m/z 202. The relative content of Trp in samples was calculated based on comparison to peak area of ribitol (internal standard). The data represent the mean value ± standard deviation of three in independent experiments performed in triplicate. Gene Cloning and Recombinant Protein Expressions of CsTSA and CsTSBs in E. coli. Total RNA was obtained

gous indole-3-glycerolphosphate lyases that convert an anthranilic acid (AA)-derivative, indole-3-glycerol phosphate (IGP), to indole. IGP serves as a key branch-point intermediate in Trp-dependent auxin and benzoxazinoid biosynthetic pathways. 3,22 This cleavage of IGP into indole and glyceraldehyde-3-phosphate, catalyzed by benzoxazineless1 (BX1), is the starting step in the biosynthesis of the natural pesticides benzoxazinoids.13 BX1 can also exist as a functionally active monomer, optimized for the production of volatile signal-indole under herbivore attack.14,23 The same reaction (αreaction) is catalyzed by tryptophan synthase α-subunit (TSA) in Trp biosynthesis.21 In contrast to BX1-catalyzed indole, this kind of indole is usually not released but directly converted to Trp (β-reaction) by tryptophan synthase β-subunit (TSB). TSA activity is to a large extent dependent on interaction with TSB.14 However, biosynthesis of indole in tea is still unknown. In our previous study, it was found that indole increased rapidly during manufacturing process of oolong tea, especially at the turn over stage (wounding stress).17 Although wounding stress is also involved in the manufacturing process of black tea, it contains very trace amount of indole.16 Several key questions remain to be answered. (1) How is indole formed in tea leaves? (2) Why does indole accumulate at the turn over stage of oolong tea manufacturing process? (3) Why does wounding stress lead to the difference of indole accumulation between oolong tea and black tea? To answer these questions, the genes involved in formation of indole were isolated, cloned, sequenced, and functionally characterized. Formation mechanism of indole in tea leaves was elucidated in this study.

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MATERIALS AND METHODS Chemicals. Indole was purchased from Wako Pure Chemical Industries Ltd., Japan. Isopropyl-β-D-thiogalactopyranoside (IPTG) was purchased from Sigma-Aldrich Company Ltd., USA. [15N]AA (15N% = 98%) was purchased from Cambridge Isotope Laboratories Inc., Cambridge, MA. 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. Ribose-5-phosphate (R5P) was purchased from macklin, China. Quick RNA isolation kit was purchased from Huayueyang Biotechnology Co., Ltd., China. Ni-NTA resin was purchased from Qiagen Inc., USA. PD-10 desalting column was purchased from GE Healthcare Life Sciences, USA. Manufacturing Processes of Oolong Tea and Black Tea. One bud and two leaves of C. sinensis var. Jinxuan were plucked from Tea Experiment Station at the South China Agricultural University (Guangzhou, China) in October 2015. The manufacturing processes of oolong tea and black tea were carried out according to the previous methods.15,17,24,25 The freshly plucking tea leaves (P) were exposed to sunlight (43 °C and 93500 Lux) for 70 min as solar withering (SW). Afterward, the tea leaves were indoor-withered (IW) at temperature of 30 °C to achieve a relative humidity of 70%. The tea leaves were subsequently turned over by 5 times (T1-T5) every 1.5 h for oolong tea, whereas the tea leaves were rolled for about 30 min to twist and rupture the tissue to express the juice and underwent oxidation for black tea. Then they were both parched in a tea-firing roller machine at 250 °C for 2−3 min to inactivate the enzymatic activity and fix the sample (F). Finally, the tea leaves were rolled at room temperature for 15 min and dried at 105 °C for 1.5 h. The samples of every stage were B

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24 h and redissolved by HCl solution (pH 5.8). Indoleglycerol phosphate synthase (tIGPS), catalyzing conversion of CdRP to IGP, was obtained using E. coli-expressed system. Before E. coliexpressed, BLAST (basic local alignment search tool) search was carried out to reveal the sequence of tIGPS gene, shown in Table S2 (Supporting Information). Then the sequence of tIGPS was compounded by Sangon Biotech company (Shanghai, China). The reaction mixture of IGP synthesis, containing 1900 μL of 100 mM potassium phosphate buffer (PPB, pH 7.5), 50 μL of tIGPS, and 50 μL of CdRP, was incubated at 60 °C for 1 h. The mixture, containing CdRP, was desiccated using vacuum freeze drier for 24 h and redissolved by 1 mL 100 mM PPB (pH 8.0). The reaction mixture containing 50 μL of IGP, 340 μL of 100 mM PPB (pH 8.0), and 10 μL of partially purified E. coli-expressed protein (enzyme) were incubated at 37 °C for 1 h. Seven reaction groups, which used CsTSA, CsTSB1, CsTSB2, CsTSB3, CsTSA combined with CsTSB1, CsTSA combined with CsTSB2, and CsTSA combined with CsTSB3 as enzymes, were conducted. 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 GCMS analysis as analytical method of indole. Transcript Expression Analyses of CsTSA and CsTSBs. Gene transcript expression was determined by quantitative real time PCR (qRT-PCR). The reaction was performed in a 0.2 mL microtube containing iTaq Universal SYBR Green Supermix (10 μL) (Bio-Rad, Hercules, CA, USA), 0.2 μM of each specific primer, 10-fold diluted cDNA (2 μL), and ddH2O (6 μL). The encoding elongation factors (EFs) was used as an internal reference gene.27 The CsEFs, CsTSA, and CsTSBs specific primers of qRT-PCR are shown in Table S3 (Supporting Information). The qRT-PCR was carried out on Roche LightCycle 480 (Roche Applied Science, Mannheim, Germany) under condition 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−△△ct method was used to calculate the relative expression level.28 Changes in mRNA levels of CsTSA and CsTSBs were normalized to that of CsEFs. Administration of [15N] AA into Tea Leaves and Analysis of [15N]Indole, [15N] Oxygenated Indole (OXIndole), and [15N]Trp. Fifty micorliters of solution of [15N]AA (20 mM) was directly injected into each tea leaf blade at the T1 stage by an injector. One part of the tea leaves with [15N]AA were completely crushed to make cell disruption and indoor-withered at room temperature for 6 h. While, other part of the tea leaves with [15N]AA was followed the oolong tea manufacturing and processed to the T5 stage. The amount of [15N]indole and related metabolites was analyzed by UPLCQTOF-MS. One hundred milligrams (dry weight) of samples with [15N]AA (finely powdered) was extracted twice with 2 mL cold 70% methanol by vortexing for 1 min followed by ultrasonic extraction in ice-cold water for 10 min. The combined extracts were diluted with cold 70% methanol to 5 mL and then subjected to UPLC-QTOF-MS analyses. UPLC analysis was conducted using a Waters ACQUITY UPLC-HSS T3 Systems (Waters, Milford, USA) controlled by Masslynx v 4.1. Chromatographic separation was performed with a 2.1 × 100 mmi.d., 1.8 μm UPLC column (Waters, Corp., USA) and kept at a temperature of 40 °C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in

using Quick RNA isolation Kit (Huayueyang Biotechnology) from tea leaves. The cDNA was reversely transcribed from total RNA using PrimeScript RT reagent Kit (Takara) according to the manufacture’s instruction. The cDNA coding was amplified by PCR with primers shown in Table S1 (Supporting Information). The PCR conditions were adjusted as follows: denaturation at 98 °C for 2 min, followed by 35 cycles of 98 °C for 10 s, 69 °C for 15 s, 72 °C for 1 min, and a final extension at 72 °C for 10 min. The resulting PCR product was purified and subcloned into pET32a vector (Novagen, Madison, WI, USA) to obtain the expression constructor. 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 recombinant vector were grown at 37 °C to an OD = 0.6. After the addition of 0.05 mM 600 IPTG, the cultures were grown at 20 °C for another 16 h to produce recombinant Histagged protein. The cells were harvested at 4000 g for 10 min and then disrupted by sonication in a 25 mM Tris-HCl (pH 7.4) buffer. After centrifugation at 12000 g 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, and 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. Western Blotting Analysis. The E. coli-expressed proteins were resolved as above SDS-PAGE analysis and transferred to a polyvinylidene fluoride membrane, which was then probed with an anti-His ×6 antibody (Signalway antibody, Pearland, TX, USA) at a dilution of 1:3000, followed by an antimouse secondary antibody (Signalway antibody, Pearland, TX, USA) at a dilution of 1:10000. The S-tagged proteins were detected with a SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA) according to the manufacturer’s instruction. E. coli-Expressed CsTSA and CsTSBs Assay. The E. coliexpressed CsTSA and CsTSBs were tested for conversion of IGP to indole. The synthesis of IGP was performed according to the reference.26 As precursor of IGP, 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP), was obtained through chemical synthesis method. The steps in detail were as follows: 0.211 mmol of AA (Sigma) were dissolved in 26.4 μL isopropanol and mixed with 0.106 mmol of ribose-5phosphate (R5P) (Macklin) dissolved in 132 μL of water plus 264 μL of isopropanol. The reaction mixture was kept in the dark, at room temperature overnight. Reaction was then cooled (4 °C) for 10 min, after which two layers were formed. The stability of the compound was estimated by UV−vis spectra measured at different times after the solubilization of CdRP in water pH 4.8, observing the maximum absorption peaks of CdRP (350 and 254 nm) as compared with the absorption peaks of AA (maxima at 323 and 240 nm). The mixture, containing CdRP, was desiccated using vacuum freeze drier for C

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Figure 1. Identification and functional characterization of CsTSA and CsTSBs expressed in E. coli. (A) SDS-PAGE analysis of CsTSA and CsTSBs expressed in E. coli. Arrows indicate target proteins. Proteins were resolved by SDS-PAGE on polyacrylamide gel and stained with Coomassie brilliant blue. (B) Western blot analysis of CsTSA and CsTSBs expressed in E. coli. The CsTSA and CsTSBs were the partially purified expressed His-tagged recombinant proteins. (C) GC-MS identification of formation of indole from IGP. Characteristic ion of indole (m/z 117) is shown.

numbers are shown in Table S1 (Supporting Information). The four partly purified recombinant proteins were obtained in an E. coli-expressed manner. Their native molecular masses were estimated by SDS-PAGE analysis, approximate 33.29, 45.38, 51.52, and 55.12 kDa, respectively, indicating that these proteins were monomers in solution (Figure 1A). Figure 1B shows the Western Blot analysis of CsTSA and CsTSBs expressed in E. coli. A series of studies have pointed out that TSA catalyzes indole synthesis only in a TSB-dependent manner.3,21,22,29 Therefore, to investigate whether recombinant CsTSA had tryptophan synthase α-activity, except for four individual purified recombinant proteins, CsTSA was also mixed with CsTSB1, CsTSB2, and CsTSB3, respectively, and these seven groups were tested for conversion of IGP to indole and glyceraldehyde-3-phosphate (α reaction). As Figure 1C showed, only CsTSA/CsTSB2 mixture was able to catalyze the α-reaction, and indole was observed. These results strongly indicated the prerequisite combination of recombinant CsTSA and CsTSB2 converted IGP to indole (α-reaction), and such activating interaction between α- and β-subunit in maize was also well-known.2 Many studies have also proposed that a heterotetramer formation of TSA and TSB (ααββ heterotetramer) in some other plant species, such as Zea mays and Arabidopsis thaliana.2,21 No α-activity of CsTSA/CsTSB1 and CsTSA/CsTSB3 mixtures was detected in the analogous experiments, suggesting that there was no interaction of CsTSB1 and CsTSB3 with CsTSA. CsTSB2 Was Highly Expressed During the Turn Over Process of Oolong Tea. To probe for why indole substantially accumulated during the manufacturing process of oolong tea, the manufacturing process was performed as the former report17 and the changes in content of indole were monitored through GC-MS analysis. Consistent with our previous study,17 indole increased dramatically during manufacturing process of oolong tea, especially at the turn over stage

acetonitrile (B). The linear gradient elution was performed as follows: 0−0.25 min, 3% B; 0.25−8 min, 3−97% B; 8−13 min, 97% B; 13−13.01 min, 97−3% B; 13.01−18 min, 3% B. A flow rate of 0.45 mL/min was employed for elution and the injected sample volume was set at 0.3 μL. Mass spectrometry was recorded using a Xevo G2-XS QTOF (Waters MS Technologies, UK) equipped with an ESI source and controlled by MassLynx v 4.1 software. A full MS scan was performed in the range m/z 50−1200 Da at a resolution mode with scan time 0.2 s. The capillary voltage was set at 3000 V, and the cone voltage was 20 V. The source temperature was 120 °C, and the desolvation temperature was 550 °C. Nitrogen gas was used both for the nebulizer and in desolvation. The desolvation and cone gas flow rates were 1000 and 50 L/h, respectively. The low energy was set at 6 V, and high energy was ramped from 15 to 45 V. The lock mass solution of Leucine Enkephalin was used as the lock mass, which m/z 556.2766 for positive mode. The identification of [15N]labeled metabolite was based on retention time and m/z data, i.e., [15N]indole (tR = 1.95 min, m/z 119.0629 [M + H]+, calcd. for C8H815N1, 119.0627, + 0.2 mmu); [15N]OX-indole 1 (tR = 2.32 min, m/z 135.0612 [M + H]+, calcd. for C8H815N1O1, 135.0576, + 3.6 mmu); [15N]OXindole 2 (tR = 2.9 min, m/z 135.0612 [M + H]+, calcd. for C8H815N1O1, 135.0576, + 3.6 mmu); L-[15N]Trp (tR = 1.95 min, m/z 206.0976 [M + H]+, calcd. for C11H13N115N1O2, 206.0947, + 2.9 mmu). The amount of each metabolite was expressed based on the ion intensity of each compound.

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RESULTS AND DISCUSSION Combination of Recombinant CsTSA and CsTSB2 Converted IGP to Indole. Based on the previous reports,2,13,21 BLAST searches were carried out to reveal putative TSA and TSBs sequences in C. sinensis. Only one TSA (CsTSA) and three TSBs (CsTSB1, CsTSB2, and CsTSB3) genes were highly similar, and the corresponding accession D

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Figure 2. Changes in indole and Trp contents, and CsTSA and CsTSBs expression levels during the manufacturing process of oolong tea. P: freshly plucking tea leaves; SW: solar withering; IW: indoor-withering; T1-T5: turn over 5 times. Data represent the mean value ± standard deviation of three in independent experiments performed in triplicate. Means distinguished with different letters are significantly different from each other (p ≤ 0.05). (A, B) The y-axis unit is peak area ratio of analyte to internal standard per gram of dry weight tea leaves. (C, D, E, F) Transcript abundance was calculated based on the difference in cycle threshold (Ct) values between target gene and internal reference gene transcripts by the normalized relative quantitation 2−△△Ct method.

sometimes exceeds that of Trp, the heterocomplex formation of TSA and TSB prevents the release of the indole intermediate and ensures a basal level of Trp production for protein and auxin biosynthesis.13,22 In addition, indole is also an important component in Japanese green tea (sen-cha), in which enzyme is immediately inactivated after plucking. Occurrence of indole in sen-cha might correlate with plucking time of tea leaf and accumulation during the tea leaf growth. It remains to be determined whether other biosynthetic pathways except the pathway leading from IGP to indole are involved in formation of indole in tea leaves. We also monitored changes in content of indole-derivatived Trp from the plucking process to the fifth turn over process of oolong tea. As shown in the Figure 2B, the amount of Trp as well as increased during the manufacturing process of oolong tea, which was consistent with the previous observation.30 However, the pattern of change in Trp was different from that of indole. The amount of Trp exhibited significant increment during the withering stage, whereas the amount of indole nearly steady. Further studies on enzyme and gene involved in the pathway leading from indole to Trp would help us to

(Figure 2A). This result provides important hints that the expression of some key genes involved in formation of indole may be activated and enhanced and/or the expression of some other key genes involved in conversion of indole may be reduced at the turn over process. We investigated the changes in expression levels of CsTSA, CsTSB1, CsTSB2, and CsTSB3 during the manufacturing process of oolong tea. Surprisingly, the expression levels of CsTSA, CsTSB1, and CsTSB3 almost held steadily (Figure 2C, D, and F), while CsTSB2 gene expression levels significantly increased (Figure 2E) at the turn over stage. These results suggested that the expression of CsTSB2 gene is the constraint factor regulating the formation of indole during the turn over stage. A series of studies have pointed out that TSA catalyze indole synthesis only in a TSBdependent manner.3,21,22,29 Therefore, it can be speculated that an analogous tryptophan synthase ααββ heterotetramer may also exist in C. sinensis, which is in agreement with some other plant species, such as Zea mays and Arabidopsis thaliana.2,21 However, necessary experiments, i.e., enzyme activity test in vitro and vivo and yeast two-hybrid experiment, are needed to confirm the viewpoint. Because the production of indole E

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Figure 3. Changes in indole and Trp contents, and CsTSA and CsTSBs expression levels during continuous mechanical damage. Data represent the mean value ± standard deviation of three in independent experiments performed in triplicate. Significant differences between control and continuous mechanical damage are indicated (* p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001). Solid dot represents the changes in indole and Trp contents, and CsTSA and CsTSBs expression levels during continuous mechanical damage. Hollow dot represents the changes in indole and Trp contents, and CsTSA and CsTSBs expression levels during simple storage process. (A, B) The y-axis unit is peak area ratio of analyte to internal standard per gram of dry weight tea leaves. (C, D, E, F) Transcript abundance was calculated based on the difference in cycle threshold (Ct) values between target gene and internal reference gene transcripts by the normalized relative quantitation 2−△△Ct method.

studies. The amount of Trp increased without continuous mechanical damage (Figure 3B), which was consistent with the changes from the plucking process to the indoor-withering process of oolong tea. It was speculated that the conversion of indole to Trp might be the main factor for the little accumulated indole during withering process of oolong tea. The increasing degree of Trp amount during continuous mechanical damage was greater than the one that was simple stacking (Figure 3B). As shown in Figure 3A and 3E, the continuous mechanical damage enhanced the expression level of CsTSB2, thus contributing to accumulation of indole. However, the expression levels of CsTSA, CsTSB1, and CsTSB3 were not significantly affected by the continuous mechanical damage (Figure 3C, D, and F). Tea Leaf Cell Disruption Terminated the Synthesis of Indole and Did Not Lead to the Conversion of Indole. In general, the contents of total volatiles, aliphatics, aromatics, and terpenoids increased with the fermentation degree, whereas indole was the highest in semifermented tea, i.e., oolong tea.16 Moreover, it is well-known that tea leaves are rolled, leading to cell disruption completely, before fermentation stage during the black tea (complete-fermented tea) manufacturing process.15,25 From these reports, we wondered whether rolling process resulted in the difference, in particular the amount of indole, between oolong tea and black tea. Therefore, to deal with the

understand the different change patterns of indole and Trp during the manufacturing process of oolong tea. Continuous Mechanical Damage Enhanced the Expression Level of CsTSB2 and Amount of Indole. To confirm whether wounding stress from the turn over process was a key factor regulating formation of indole during the oolong tea manufacturing process, we monitored changes of indole and Trp contents, and CsTSA, CsTSB1, CsTSB2, and CsTSB3 genes expression levels during continuous shaking treatment (single wounding stress as a control) which simulated the turning-over step during the oolong tea manufacturing process. The amount of indole was enhanced by continuous mechanical damage (Figure 3A), which was consistent with the changes during the oolong tea manufacturing process. Volatile indole is not emitted from physically wounded rice plants; however, the emission was detected from insect infested.10 Besides, insect attack can also induce the emission of indole in higher concentrations than from nonstressed tea leaves.12,31,32 These literatures show some evidence that simple mechanical damage is not sufficient to induce emission of volatiles while continuous mechanical damage may be sufficient trigger release of specific volatiles, i.e., indole.33 In this study, the amount of indole from plucked tea (simple mechanical damage) was almost unchanged (Figure 3A), thus making a point that was proposed in the above F

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Figure 4. Effect of disruption of tea leaf cell on amounts of indole and the transformation from [15N]AA to [15N]indole, [15N]Trp, [15N]OX-indole 1, and [15N]OX-indole 2. (A) T1: the first turn over, which stage tea leaf cell is not disrupted; CK1: after turn over 1 (T1), which stage tea leaf cell was not disrupted, tea leaves were indoor-withered at room temperature for 6 h; Crush 1: after turn over 1 (T1), parts of tea leaves were completely crushed to make disruption of leaf cell, and indoor-withered at room temperature for 6 h; T5: the 5th turn over, which stage tea leaf cell was not disrupted; CK2: after turn over 5 (T5), which stage tea leaf cell was not disrupted, tea leaves were indoor-withered at room temperature for 6 h; Crush 2: after turn over 5 (T5), parts of tea leaves were completely crushed to make disruption of leaf cell, and indoor-withered at room temperature for 6 h. (B and C) Data represent the mean value ± standard deviation of three in independent experiments performed in triplicate. Means distinguished with different letters are significantly different from each other (p ≤ 0.05). * p ≤ 0.05, ** p ≤ 0.01.

Figure 5. Hypothetic model of different formation of indole between oolong tea process and black tea process. For oolong tea, continuous wounding stress on noncompleted disrupted cell structure of tea leaf activated high expression of CsTSB2, which resulted in accumulation of indole at the turn over stage. For black tea, tea leaf cell disruption from the rolling process did not lead to the conversion of indole, but terminated the synthesis of indole, which explained trace amount of indole in black tea.

doubt, the rolling process was stimulated to make the structure of the leaf cells disrupted after the first and fifth turn over process, and quantitative analysis of indole from these tea leaves

was carried out (Figure 4A). As shown in Figure 4B, there was no significant difference of amount of indole among these three groups, T1, CK1, and Crush1. The amount of indole in tea G

DOI: 10.1021/acs.jafc.6b01742 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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leaves (CK1) which was not processed to the T5 stage according to the oolong tea manufacturing but indoor-withered at room temperature for 6 h was almost unchanged, further confirming that wounding stress from turn over process was a key factor regulating formation of indole. In addition, the nearly invariable amount of indole in tea leaf with cell disruption after the first turn over process suggested that tea leaf cell disruption terminated the synthesis of indole. Stable isotope labeling has demonstrated that AA is one of the precursors of indole in tea leaves.17,30 Besides, the concentration of RNA from a tea leaf whose cell was disrupted was decreased from several hundreds to 20 of ng/μL. To further verify the viewpoint that tea leaf cell disruption terminated the synthesis of indole, we also investigated the formation of indole by feeding of stable isotope-labeled compounds [15N]AA to the tea leaves. It was found that the amount of [15N]indole in tea leaf with cell disruption was much lower than that in tea leaf which was processed to the T5 stage of the oolong tea manufacturing and without cell disruption (Figure 4C). Indole is able to be converted to either Trp or several oxygenated indoles (OXindoles), such as 4-hydroxy- (OX-indole 1), 5-hydroxy- (OXindole 2), and 6-hydroxy-indoles (OX-indole 3) in higher plants.30 P450 enzymes are involved in the oxidation process of indole.34 We therefore also monitored the changes in [15N]OXindole and [15N]Trp to confirm whether tea leaf cell disruption led to the conversion of [15N]indole. The levels of [15N]OXindole and [15N]Trp in tea leaves with cell disruption were much lower than those in tea leaf without cell disruption (Figure 4C). These results to some extent revealed why black tea contained much less indole. We propose a hypothetic model of different formations of indole between oolong tea process and black tea process (Figure 5). In tea leaf, indole was synthesized by CsTSA from IGP (α reaction), and CsTSA activity was to a large extent dependent on interaction with CsTSB2. Therefore, it was accordingly proposed that a tryptophan synthase ααββ heterotetramer also existed in C. sinensis. Continuous wounding stress on noncompleted disrupted cell structure of tea leaf activated high expression of CsTSB2, which resulted in accumulation of indole at the turn over stage of oolong tea manufacturing process. Wounding stress was both involved in manufacturing processes of oolong tea and black tea, however, there was a marked difference of indole amount between these two tea kinds. The reason was that tea leaf cell disruption from the rolling process (wounding stress) of black tea did not lead to the conversion of indole, but terminated the synthesis of indole. This study provided evidence concerning formation of indole in tea leaves for the first time. In addition, it uncovered the truth of accumulation of indole in oolong tea, and shed light on the differences concerning the formation of indole in oolong tea and in black tea.

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Article

AUTHOR INFORMATION

Corresponding Author

*Tel: +86-20-38072989; E-mail: [email protected]. Author Contributions ∇

L.Z. and Y.Z. equally contributed to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the financial support from Guangdong Natural Science Foundation for Distinguished Young Scholar (2016A030306039), and “100 Talents Programme of the Chinese Academy of Sciences” (Y321011001 and 201209).

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ABBREVIATIONS USED EFs, elongation factors; GC-MS, gas chromatography−mass spectrometer; IW, indoor-withered; P, freshly plucking tea leaves; SW, solar withering; T1-T5, turned over by 5 times; TMEMD, N, N, N′, N′-tetramethylethylenediamine; IPTG, isopropyl-β-D-thiogalacto-pyranoside; SDS, sodium dodecyl sulfate; MSTFA, N-methyl-N-(trimethylsilyl)-trifluoroacetamide; Trp, Tryptophan; tIGPS, indoleglycerol phosphate synthase; CdRP, 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate; AA, anthranilic acid; IGP, indole-3-glycerol phosphate; TSA, tryptophan synthase α-subunit; TSB, tryptophan synthase β-subunit

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REFERENCES

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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.6b01742. Table S1: Primers of PCR for cDNA cloning used in this study; Table S2: sequence of indoleglycerol phosphate synthase (tIGPS) used in this study; Table S3: primers of qRT-PCR used in this study (PDF) H

DOI: 10.1021/acs.jafc.6b01742 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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