Biosynthesis of Jasmine Lactone in Tea (Camellia sinensis) Leaves

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Article Cite This: J. Agric. Food Chem. 2018, 66, 3899−3909

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Biosynthesis of Jasmine Lactone in Tea (Camellia sinensis) Leaves and Its Formation in Response to Multiple Stresses Lanting Zeng,†,‡ Ying Zhou,† Xiumin Fu,† Yinyin Liao,† Yunfei Yuan,† Yongxia Jia,† Fang Dong,§ and Ziyin Yang*,†,‡ †

Guangdong Provincial Key Laboratory of Applied Botany & Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, 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 § Guangdong Food and Drug Vocational College, Longdongbei Road 321, Tianhe District, Guangzhou 510520, China S Supporting Information *

ABSTRACT: Jasmine lactone has a potent odor that contributes to the fruity, sweet floral aroma of tea (Camellia sinensis). Our previous study demonstrated that jasmine lactone was mostly accumulated at the turnover stage of the oolong tea manufacturing process. This study investigates the previously unknown mechanism of formation of jasmine lactone in tea leaves exposed to multiple stresses occurring during the growth and manufacturing processes. Both continuous mechanical damage and the dual stress of low temperature and mechanical damage enhanced jasmine lactone accumulation in tea leaves. In addition, only one pathway, via hydroperoxy fatty acids from unsaturated fatty acid, including linoleic acid and α-linolenic acid, under the action of lipoxygenases (LOXs), especially CsLOX1, was significantly affected by these stresses. This is the first evidence of the mechanism of jasmine lactone formation in tea leaves and is a characteristic example of plant volatile formation in response to dual stress. KEYWORDS: aroma, Camellia sinensis, jasmine lactone, tea, volatile

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INTRODUCTION Tea (Camellia sinensis) aroma is one of the main sensory properties that are decisive for the quality of the final product, especially black tea and oolong tea. To date, many key aroma odorants in these two tea kinds have been determined by aroma extract dilution analysis (AEDA) and by the calculation of odor activity values (OAVs).1−4 In black tea, 46 odorants, such as linalool, geraniol, E/Z-2,6-nonadienal, (E)-δ-damascenone, 4hydroxy-2,5-dimethyl-3(2H)-furanone, 2-phenylethanol, (E,E,Z)-2,4,6-nonatrienal, etc., were identified.1,2 In oolong tea, 52 compounds, such as linalool, indole, 3-methylbutanal, 2methylpropanal, (E)-2-heptenal, β-damascenone, dimethyl sulfide, jasmine lactone, etc., were proposed to play important roles in overall aroma of tea.3,4 To improve the quality of tea aroma, it is of significance to have insight into the biosynthesis of these aroma odorants. Formation of many aroma compounds is due to stresses which occur during the growth and manufacturing processes of tea.5,6 The tea growth process (tea growing stage), insects (e.g., tea green leafhoppers), and light conditions (e.g., dark, blue light, and red light) can significantly increase the amount of tea aroma compounds.7−11 During the tea manufacturing processes, mechanical damage and low temperature stress can result in accumulations of some aroma compounds, including jasmine lactone.12−14 Jasmine lactone, which processes a jasmine-like floral and fruity odor, is well-known to be one of the most important compounds to tea aroma.3,14,15 However, stress that induces the biosynthesis of jasmine lactone has not been specified, and its formation in tea leaves has not been investigated. In addition, although many attempts have been made,16,17 the enantiomeric ratio of the © 2018 American Chemical Society

chiral jasmine lactone in tea has also not been determined at present. Volatile lactones are only present in low concentrations in some plants and foods, but they have a high impact on odors due to their low odor detection threshold values.18 Jasmine lactone is a δ-lactone and occurs in a variety of plants, such as Camellia sinensis, Amygdalus persica, Mangifera indica, and flowers of Jasminum, Gardenium, Tuberose, and Mimosa species.19−21 In spite of their considerable biological and biochemical importance, the biosynthetic pathway of many lactones in plants, especially that of jasmine lactone, is still unclear. Isotope labeling experiments in yeast indicated that δjasmine lactone and (Z,Z)-dodeca-6,9-dieno-4-lactone were derived from α-linolenic acid (ALA)21 and that γ-decalactone and γ-dodecalactone were produced from the metabolism of epoxy octadecanoic acid by epoxide hydrolase.22 Moreover, observations in plants provided evidence that epoxy fatty acids are involved in lactone biosynthesis under the action of epoxide hydrolase.23,24 Epoxy fatty acids are derived from ALA under the action of peroxygenase (PGX).25,26 In addition, under the action of 9-lipoxygenase (9-LOX), ALA can be converted to hydroperoxy fatty acids, which are precursors of monohydroxy fatty acids and lactones.27 Taken together, there are two proposed pathways for the formation of lactones19 including (i) one pathway leading from unsaturated fatty acids to lactones via Received: Revised: Accepted: Published: 3899

January 27, 2018 March 28, 2018 March 31, 2018 March 31, 2018 DOI: 10.1021/acs.jafc.8b00515 J. Agric. Food Chem. 2018, 66, 3899−3909

Article

Journal of Agricultural and Food Chemistry

One bud and two leaves plucked in October 2017 were used for a continuous mechanical damage treatment, simulating stresses from the turnover stages of oolong tea manufacture, according to our previous studies.12,13 The picked tea leaves were shaken using a shaking table at 25 °C and collected after continuous shaking for 0, 4, 8, and 12 h. The same tea leaves, without continuous shaking, stored under the same conditions for 0, 4, 8, and 12 h were used as controls. To investigate the effects of dual stress of low temperature and mechanical damage on the formation of jasmine lactone in tea leaves, the leaves (one bud and two leaves, collected in July 2016) were damaged using a needle to cause mechanical damage and then exposed to 25 or 15 °C temperatures for 16 h. There were four different treatments on tea leaves, including tea leaves exposed to 25 °C with no mechanical damage, tea leaves exposed to 25 °C with mechanical damage, tea leaves exposed to 15 °C with no mechanical damage, and tea leaves exposed to 15 °C with mechanical damage. The samples were immediately frozen in liquid nitrogen and stored at −80 °C until further analysis. Analysis of Jasmine Lactone in Tea Samples. Jasmine lactone extraction was carried out from 500 mg of a finely powdered sample (fresh weight) by a solvent extraction method as in our previous study.28 Dichloromethane (2 mL) was used to extract, and 5 nmol of ethyl n-decanoate was added as an internal standard. After an overnight shaking, the extraction solution was collected, dried using anhydrous sodium sulfate, and concentrated to 200 μL under a stream of nitrogen. The extract was then subjected to gas chromatography− mass spectrometry (GC−MS) analysis. The analysis was conducted on a GCMS-QP2010 SE (Shimadzu Corporation, Kyoto, Japan) equipped with a SUPELCOWAX 10 column (30 m × 0.25 mm × 0.25 μm, Supelco Inc., Bellefonte, PA, USA). One microliter samples were injected in a splitless mode, carried by helium at a flow rate of 1.0 mL/min, and held at 230 °C for 1 min. The initial GC oven temperature was 60 °C for 3 min, followed by a 4 °C/min to 240 °C, and then held at 240 °C for 20 min. The GC-MS was operated in a scan mode m/z (mass range, m/z 40−200). The characteristic fragment ions of jasmine lactone are m/z 71 and 99, and the quantitation analysis was based on a calibration curve. A calibration curve was conducted between the concentration and peak area of jasmine lactone authentic standard. Analysis of Enantiomeric Ratio of Jasmine Lactone in Tea Samples. Jasmine lactone extraction was the same as the description in the section “Analysis of Jasmine Lactone in Tea Samples”. The extract was then subjected to GC-MS QP2010 SE equipped with an InertCapTM CHIRAMIX column (30 m × 0.25 mm × 0.25 μm, GL Sciences, Tokyo, Japan). One microliter samples were injected in a splitless mode, carried by helium at a flow rate of 1.0 mL/min, and held at 180 °C for 1 min. The initial GC oven temperature was 60 °C for 3 min, followed by a 2 °C/min to 180 °C, and then held at 180 °C for 20 min. The GC-MS was operated in a scan mode m/z (mass range, m/z 40−200). Apart from the two main characteristic fragment ions (m/z 71 and 99) of jasmine lactone, other fragment ions (m/z 48, 50, and 108) were also detected. Analyses of LA and ALA in Tea Samples. A previously reported method was used to extract LA and ALA.19 A finely powdered sample (500 mg fresh weight) was extracted with 2 mL of 80% methanol, then vortexed, and sonicated for 10 min. After sonication, fatty acids were extracted with 2 mL of hexane. The hexane layer was collected and dried under a stream of nitrogen. The metabolites were then derivatized with 50 μL of MSTFA at 80 °C for 60 min. One microliter of MSTFA derivate was analyzed by a GC-MS QP2010 SE equipped with an HP-5 column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies, California, USA). The GC oven temperature was held at 80 °C for 5 min, then ramped at 5 °C/min to 300 °C, and then held at 300 °C for 15 min. The characteristic fragment ions of the MSTFA derivate of LA are m/z 262, 337, and 352, and the characteristic fragment ions of the MSTFA derivate of ALA are m/z 260, 335, and 350. A full scan mode (mass range, m/z 50−600) was used for qualitative analysis, and a selected ion mode (m/z 260, 262, 335, 337, 350, and 352) was used for quantitative analysis. A calibration curve

hydroperoxy fatty acids and monohydroxy fatty acids under the actions of LOX and PGX and (ii) another pathway leading from unsaturated fatty acids to lactones via epoxy fatty acids and dihydroxy fatty acids under the actions of PGX and epoxide hydrolase. Although many studies have shown that various lactones, including jasmine lactone, are formed from ALA by biodegradation, Haffner et al.21 found that some lactones, such as (R)-decano-5-lactone and (Z)-dodec-6-eno-4-lactone, were derived from linoleic acid (LA) in yeast. The precursor(s) of jasmine lactone is still unclear in many plant species. Therefore, several key questions regarding the occurrence of jasmine lactone in tea leaves remain to be answered: (i) Which is the key stress that induces the biosynthesis of jasmine lactone in tea leaves? (ii) Which biosynthetic pathway is responsible for jasmine lactone formation? (iii) Are LA and ALA precursors for jasmine lactone formation in C. sinensis. The aim of this study was to discover the formation mechanism of jasmine lactone in C. sinensis plants, including stress responses and the biosynthetic pathway during the growth and manufacturing process of tea leaves. The information provided evidence concerning formation of jasmine lactone in tea leaves for the first time.

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MATERIALS AND METHODS

Chemicals. Ethyl n-decanoate was purchased from Wako Pure Chemical Industries Ltd., Japan. Linoleic acid (LA, solution in ethanol, ≥ 98% purity) and α-linolenic acid (ALA, solution in ethanol, ≥98% purity) were purchased from Cayman Chemical Company, USA. [13C18]LA (≥92% purity) and [13C18]ALA (≥93% purity) were purchased from Cambridge Isotope Laboratories Inc., Cambridge, MA. Jasmine lactone (≥97% purity by GC) was purchased from ZEON Corporation, Kyoto, Japan, and kindly provided by T. Hasegawa Flavours & Fragrances (Shanghai) Co., Ltd.. The mass spectra, NMR, and HRMS of jasmine lactone were shown in the Supporting Information (Figure S1). The Quick-RNA isolation kit was purchased from Huayueyang Biotechnology Co., Ltd., Beijing, China. iTaq Universal SYBR Green Supermix was purchased from Bio-Rad Laboratories, CA, USA. N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) was purchased from Regis Technologies Inc., Morton Grove, USA. PrimeScript RT Reagent Kit with gDNA Eraser was purchased from Takara Bio Inc., Kyoto, Japan. Plant Materials and Treatments. Tea leaves were collected from C. sinensis var. Jinxuan plant, a popular tea cultivar in South China, at the Tea Research Institute, Guangdong Academy of Agricultural Sciences (Yingde, China). “One bud and two leaves” is one bud with two leaves on the same branch (bud, the first leaf; two leaves, the second leaf and third leaf), which is generally used for oolong tea manufacturing as a whole in China.3,28 To exactly simulate the oolong tea manufacture, one bud and two leaves were used in the present study. To investigate the effect of insect attack on formation of jasmine lactone, the tea leaves (one bud and two leaves) infested by tea green leafhoppers (Empoasca (Matsumurasca) onukii Matsuda), which appearance showed internode shortening, yellow and curling leaves,7 were obtained in September 2016. The control was the intact tea leaves (one bud and two leaves) without any insect attacks. To investigate the effect of light on formation of jasmine lactone, shading net (5% light transmitted, width: 5.3 m, Xinzhiyuan, Jiangsu, China) was used for the shading treatment. The tea plants were covered by four canopy layers for 14 days in April 2015. The photosynthetic photon flux density (PPFD) was measured by a Lux Meter AS823 (SMART SENSOR, HK, China), and the PPFD under the shading treatment was 0 μmol·m−2·s−1. The tea plants grown in parallel under identical conditions without treatment were used as the control group. After the shading treatment, one bud and two leaves were collected. The samples were immediately frozen in liquid nitrogen and stored at −80 °C until further analysis. 3900

DOI: 10.1021/acs.jafc.8b00515 J. Agric. Food Chem. 2018, 66, 3899−3909

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Journal of Agricultural and Food Chemistry was conducted between the concentration and peak area of the MSTFA derivate of authentic standards. Transcript Expression Analyses of CsLOXs and CsPGXs. Total RNA was obtained using a Quick RNA isolation Kit (Huayueyang Biotechnology Co., Ltd., Beijing, China). The cDNA was reversely transcribed from total RNA using PrimeScript RT Reagent Kit with gDNA Eraser (Takara Bio Inc., Kyoto, Japan) according to the manufacture’s instructions. 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 factor1 (EF1) was used as an internal reference gene. The CsEF1, CsLOXs, and CsPGXs specific primers of qRT-PCR are shown in Table S1 (Supporting Information). The qRTPCR was carried out on Roche LightCycle 480 (Roche Applied Science, Mannheim, Germany) under conditions of one cycle of 95 °C for 5 s, 40 cycles of 95 °C for 5 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. Changes in mRNA levels of CsLOXs and CsPGXs were normalized to that of CsEF1. Supplement of [13C18]LA or [13C18]ALA into Tea Samples and Tentative Identification of [13C10]Jasmine Lactone. One bud and two leaves collected in November 2017 were cultivated in 4 mM [13C18]LA or [13C18]ALA solution (250 μL, total 1 μmol) at 25 °C for 12 h. Four millimoles per liter [13C18]LA or [13C18]ALA solution was made of 20 μmol of [13C18]LA or [13C18]ALA and 10 μL of 1 M NaOH in a 5 mL volumetric flask and filled up with distilled water. Cut-stem feeding was used, and the labeled fatty acids entered the tea leaves via the cut stem driven by the transpiration stream. After complete absorption, the samples were used to conduct continuous wounding stress treatment and dual stress treatment. For the continuous wounding stress treatment, the samples were kept under continuous shaking for 12 h at 25 °C. Samples stored without shaking for 12 h were used as controls. For the dual stress treatment, the samples were exposed to temperatures of 25 or 15 °C for 16 h after a single instance of mechanical damage by a needle. Samples without mechanical damage were used as controls. After the treatments, the samples were immediately frozen with liquid nitrogen and used to tentatively identify [13C10]jasmine lactone. The amount of tentatively identified [13C10]jasmine lactone was analyzed by GC-MS with electron impact ionization as described above with some modifications. Only a 200 mg sample was extracted, and 0.5 nmol of ethyl ndecanoate was used as an internal standard. The characteristic fragment ions of tentatively identified [13C10]jasmine lactone are m/ z 75 and 104. A full scan mode (mass range, m/z 40−200) was used to qualitative analysis, and a selected ion mode (m/z 75 and 104) was used for quantitative analysis. Because of a lack of [13C10]jasmine lactone authentic standard, quantitation analysis was based on a calibration curve acquired from unlabeled jasmine lactone authentic standard as described above. In addition, the [13C10]jasmine lactone was also tentatively identified by a GC-MS with negative ion chemical ionization. The identification was conducted on a GC-MS QP2010 Plus (Shimadzu Corporation, Kyoto, Japan) equipped with a VFWAXms column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies, California, USA). Two microliter samples were injected in a splitless mode, carried by helium at a flow rate of 1.0 mL/min, and held at 250 °C for 1 min. The initial GC oven temperature was 60 °C for 3 min, followed by a 4 °C/min to 240 °C, and then held at 240 °C for 20 min. The temperatures of ion source and transfer line were both kept at 200 °C, with methane as the chemical ionization moderating gas at an ion source pressure of 0.15 MPa. The GC-MS was operated in a scan mode (mass range, m/z 40−200). Gene Cloning and Expression in Escherichia coli (E. coli) of CsLOX1. The cDNA coding was amplified by PCR with primers shown in Table S2 (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, 58 °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 a recombinant vector were grown at 37 °C to an OD600 = 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 4,000 g for 10 min and then disrupted by sonication in a 25 mM Tris-HCl (pH 7.4) buffer. After centrifugation at 12,000 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 of CsLOX1. The E. coli-expressed CsLOX1 was subjected to SDS-PAGE with the use of a separation gel (2.4 mL of 30% acrylamide/bis(acrylamide) (29:1), 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) (29:1), 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, a colloidal staining method was used. The proteins were stained with Coomassie Blue R250 overnight and then decolored three times. Enzyme Assay and Functional Characterization of CsLOX1. Enzyme activity of E. coli-expressed CsLOX1 was determined spectrophotometrically by monitoring the increase in the absorbance at 234 nm due to the transformation of LA or ALA to the respective conjugated hydroperoxydienes. The assay was carried out mainly according to the method proposed by Plagemann et al.29 The details were as follows: in a UV spectrophotometer containing 2.5 μg of partially purified proteins and 2 mL of Tris-HCL buffer (25 mM, pH 7) the reaction was initiated by adding 40 μL of a freshly prepared 2 mM substrate solution, which was made of 20 μL (18 mg) of LA or ALA, 30 μL (33 mg) of Tween 20, and 60 μL of 1 M NaOH in a 2 mL volumetric flask and filled up with distilled water. Absorbance at 234 nm was recorded every 10 s and monitored at least for 5 min using a UV spectrophotometer, which was tempered to 25 °C. Enzyme activity was calculated on the basis of the molar extinction coefficient of the conjugated diene hydroperoxides at 234 nm (ε = 2.5 × 104 M−1 cm−1). Control was carried out using the same amount of partially purified proteins obtained from an empty vector (pET32a). Statistical Analysis. Statistical analysis was performed using SPSS software, version 18.0 (SPSS Inc., Chicago, IL, USA). Two-tailed student’s t test was used to determine the differences between the two groups. One-way ANOVA followed by Duncan’s multiple comparison tests was used to determine the differences among three or more than three groups. A probability level of 5% (p ≤ 0.05) was considered as significant.

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RESULTS AND DISCUSSION Jasmine Lactone Levels Significantly Increased in Tea Leaves Exposed to Continuous Mechanical Damage. Many stresses are involved in the growth and manufacturing processes of tea. We first investigated the effects of environmental factors on jasmine lactone formation during the tea growth process (i.e., leaves on the tea plants). Two factors, including tea green leafhopper attack (biotic factor, insect) and shading treatment (for 14 days) (abiotic factor, light), were selected. These two factors did not affect jasmine lactone levels in tea leaves. The related data are not shown, because the amount of jasmine lactone in the tea leaves after these stress treatments was below the detection limit. We also monitored changes in the amounts of jasmine lactone and its potential precursors, LA and ALA, during the continuous mechanical damage. Details about qualitative analysis of LA and ALA are shown in Figure 1, including the calculation of the characteristic 3901

DOI: 10.1021/acs.jafc.8b00515 J. Agric. Food Chem. 2018, 66, 3899−3909

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Journal of Agricultural and Food Chemistry

Figure 1. Analysis of linoleic acid (LA) and α-linolenic acid (ALA) in tea samples by gas chromatography−mass spectrometry (GC−MS). (A) Mass spectrum of products of LA and ALA derivatized by MSTFA. (B) Calculation of the characteristic fragment ions of products from LA and ALA derivatized by MSTFA. (C) GC−MS chromatography of LA and ALA from tea samples

fragment ions of the products from [13C18]LA and [13C18]ALA derivatized with MSTFA. Continuous mechanical damage significantly enhanced jasmine lactone content, with a decrease in LA and ALA (Figure 2A). Furthermore, expression levels of CsLOXs, especially CsLOX1, were significantly enhanced by the continuous mechanical damage (Figure 2B), consistent with the changes in content of jasmine lactone. We also analyzed enantiomeric distribution of the jasmine lactone in tea. Figure S2 showed that not only the (S)- but also the (R)-jasmine lactone could be detected, and (S)-jasmine lactone was the major one in tea leaves. Moreover, different treatments affect the enantiomeric ratio of jasmine lactone in tea leaves (Figure S2). In our previous study, we found that when tea leaves were exposed to jasmonic acid treatment, emitted jasmine lactone collected by solid-phase microextraction (SPME) was not detected (below detection limit).30 This suggests that jasmine lactone was not readily emitted but stored in tea samples (internal jasmine lactone). In oolong tea, the content of internal jasmine lactone ranges from 230 to 350 μg/g.31 The

detection odor threshold in water of jasmine lactone is 2000 ng/mL.32 It is calculated that the OAV of jasmine lactone is >1, indicating that jasmine lactone has a contribution to the overall tea aroma.2 Moreover, in our previous study, the same materials collected from C. sinensis var. Jinxuan plants were manufactured into oolong tea, and jasmine lactone showed a flavor dilution (FD) factor of 46, which was the second highest in FD factors of potent odorants of the oolong tea product.3 These results showed the important contribution of jasmine lactone to oolong tea aroma. There are two representative types of stress-treated tea leaves used as raw materials for tea manufacture. One is tea leaves infected by tea green leafhoppers, which are used for making a famous oolong tea “Oriental Beauty”.7 The tea green leafhopper attack process endows the oolong tea with characteristic volatile monoterpenes, which contribute to the unique aroma of ripe fruit and honey of the oolong tea.7,11 Another representative of stress-treated raw tea leaves is dark (shading)-treated tea leaves. Shading treatment on tea plants is a common agronomic way of improving tea quality,5 and it can 3902

DOI: 10.1021/acs.jafc.8b00515 J. Agric. Food Chem. 2018, 66, 3899−3909

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Journal of Agricultural and Food Chemistry

Figure 2. Effect of continuous mechanical damage on amount of jasmine lactone, linoleic acid (LA), and α-linolenic acid (ALA) (A) and expression levels of CsLOXs and CsPGXs (B). CK, the tea leaves stored at 25 °C without continuous shaking for 0, 4, 8, or 12 h. Continuous mechanical damage, the tea leaves shaken using a shaking table at 25 °C for 0, 4, 8, or 12 h. Significant differences between control and continuous mechanical damage are indicated (* p ≤ 0.05, and ** p ≤ 0.01). All data are expressed as mean ± SD (n = 3). (A) The characteristic fragment ions of jasmine lactone are m/z 71 and 99, the characteristic fragment ions of the MSTFA derivate of LA are m/z 352, 337, and 262, and the characteristic fragment ions of the MSTFA derivate of ALA are m/z 350, 335, and 260. The contents of jasmine lactone, LA, and ALA were calculated based on corresponding calibration curves. DW, dry weight. (B) 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. The expression level from tea sample collected at 0 h was defined as 1. LOX, lipoxygenase; PGX, peroxygenase.

volatile compounds are derived from different pathways, there may be unknown common factor(s) mediating their formation under continuous mechanical damage. Recently, the tea tree genome has been elucidated36 and may be a valuable resource for elucidating the unknown common factors involved in interaction between tea aromas and stresses in the future. Jasmine Lactone Levels Significantly Increased in Tea Leaves under Dual Stress. Katsuno et al. reported that the amount of jasmine lactone in tea leaves was significantly enhanced after 15 °C treatment for 16 h, compared to 25 °C.14 To further investigate whether there is a synergistic effect of low temperature and mechanical damage on jasmine lactone formation, we compared the jasmine lactone content in tea leaves between single stress treatment (low temperature or mechanical damage) and dual stress treatment. In our preliminary experiments, we found that jasmine lactone accumulated the most abundantly in tea leaves exposed to low temperature for 16 h after mechanical damage, and lower temperatures could enhance increasing degree (Figure S3). Therefore, we selected a treatment time and temperature of 16 h and 15 °C, respectively. Jasmine lactone levels showed no significant changes between the tea leaves exposed to 15 and 25 °C without mechanical damage (Figure 3A). However, when the tea leaves underwent mechanical damage and lowtemperature treatment (15 °C), the amount of jasmine lactone

enhance levels of amino acids and volatile phenylpropanoids/ benzenoids.9,33 Our present study showed that jasmine lactone was not accumulated either in the tea green leafhoppers-treated tea leaves or in the shading-treated tea leaves compared with the control (without treatment). In the present study, the tea leaves without treatments during the tea growth process were used for other stress treatments. The oolong tea manufacturing process involves many stresses, such as plucking-induced wounding, solar withering-induced drought, heat, and UV radiation, indoor withering-induced drought, and turnoverinduced continuous wounding.7,28,34 Therefore, the oolong tea manufacturing process is a good model for studying the formation of tea aromas from the biological viewpoint. Jasmine lactone is highly accumulated at the turnover stage, in which continuous mechanical damage is a key stress.3 Usually, there is five times of turnover treatment, and the interval between each turnover treatment was about 2 h.35 Therefore, the continuous mechanical damage treatment times were set at 4, 8, and 12 h. Our present study provided evidence that the turnover stage significantly enhances jasmine lactone content due to activation of the pathway mainly via hydroperoxy fatty acids under the action of CsLOX1 (Figure 2). Jasmine lactone was not the only volatile compound highly accumulated at the turnover stage due to continuous mechanical damage, and indole and (E)nerolidol showed a similar pattern.3,12,13 Although these three 3903

DOI: 10.1021/acs.jafc.8b00515 J. Agric. Food Chem. 2018, 66, 3899−3909

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Journal of Agricultural and Food Chemistry

Figure 3. Effect of dual stress on amount of jasmine lactone, linoleic acid (LA), and α-linolenic acid (ALA) (A) and expression levels of CsLOXs and CsPGXs (B). −, intact tea leaves; +, tea leaves with mechanical damage. Means distinguished with different letters are significantly different from each other (p ≤ 0.05). All data are expressed as mean ± SD (n = 3). (A) The characteristic fragment ions of jasmine lactone are m/z 71 and 99, the characteristic fragment ions of the MSTFA derivate of LA are m/z 352, 337, and 262, and the characteristic fragment ions of the MSTFA derivate of ALA are m/z 350, 335, and 260. The contents of jasmine lactone, LA, and ALA were calculated based on corresponding calibration curves. DW, dry weight. (B) 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. The expression level from tea samples exposed to 25 °C and nonmechanical damage was defined as 1. LOX, lipoxygenase; PGX, peroxygenase.

combinations of abiotic stresses such as temperature, light, and ozone.40 However, it is still unclear whether responses of plant volatiles under multiple stresses are passive or active behaviors, and whether such responses can positively contribute to plant defense against multiple stresses. Stress-induced plant volatiles may contribute to defense against stresses and can also be quality components of agricultural plants. Therefore, the use of the stress response to improve the aromas of plant-derived foods such as fruits and tea has been attracting increased attention recently. The interaction of multiple stresses on fruit aroma compounds has been examined in strawberries and demonstrated that formation of volatile benzenoids and volatile esters was significantly enhanced by a combination of low temperature and dark conditions than when either stress was applied alone.41 Our previous investigation showed another example of the additive effects of multiple stresses on aromas of plantderived foods. In tea leaves, the combination of low temperature and mechanical damage had a synergistic effect on (E)-nerolidol accumulation, due to the additive effects on the expression level of (E)-nerolidol synthase.13 In the present study, similar to (E)-nerolidol, jasmine lactone was also significantly enhanced by a combination of low temperature and mechanical damage than when either stress was applied alone (Figure 3A). Furthermore, pathways via hydroperoxy fatty acids under the action of CsLOX1 contributed to the formation of jasmine lactone (Figure 3B). This suggests that tea leaves may have unknown transcription factors activated by the

was significantly enhanced (Figure 3A). This suggests that the dual stress had a synergistic effect on jasmine lactone formation. However, the amount of LA and ALA showed inconsistent variation compared with the jasmine lactone content (Figure 3A). We also investigated the effect of the dual stress on the related genes. The results showed that the expression level of CsLOX1 increased in tea leaves exposed to dual stress, whereas the CsPGXs were not activated (Figure 3B). This showed that the pathway via hydroperoxy fatty acids, under the action of CsLOX1, was activated by dual stress, consistent with the result observed with continuous mechanical damage (Figure 2B). Numerous studies have validated that single or independent stress such as insect attack, mechanical damage, temperature, and light can affect the formation and emission of plant volatiles.7,9−13 In recent years, the effect of multiple or cooccurring stress factors on the regulation of plant volatile formation and emission has attracted increasing interest.37 From the viewpoint of plant science, investigations into the relationship between stress and plant volatiles showed that the interaction of multiples stresses has great potential to alter the formation and emission of plant volatiles.38 When two or more stresses co-occur, their effects are sometimes additive, while in other cases the influence of one stress has priority. For example, the additive effects of biotic and abiotic stresses were noted in a study on maize exposed to a combination of high temperature and simulated herbivore attack, which led to higher volatile emissions than the individual stresses.39 Additive effects on plant volatile formation and emission can also be found for 3904

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Figure 4. Qualitative analysis of tentatively identified [13C10]jasmine lactone from [13C18]linoleic acid (LA) or [13C18]α-linolenic acid (ALA) during continuous mechanical damage and dual stress treatment. (A) Treatment of tea samples. One bud and two leaves were cultivated in 4 mM [13C18]LA or [13C18]ALA solution (250 μL, total 1 μmol) at room temperature for 12 h. (B) Calculation of the characteristic fragment ions of tentatively identified [13C10]jasmine lactone. The characteristic fragment ions of jasmine lactone are m/z 71 and 99, and the characteristic fragment ions of tentatively identified [13C10]jasmine lactone are m/z 75 and 104. (C) Qualitative analysis of tentatively identified [13C10]jasmine lactone from [13C18]LA and [13C18]ALA by GC-MS with electron impact ionization. Unlabeled jasmine lactone was used for identification of tentatively identified [13C10]jasmine lactone. (D) Qualitative analysis of tentatively identified [13C10]jasmine lactone from [13C18]LA and [13C18]ALA by GC-MS with negative ion chemical ionization. Unlabeled jasmine lactone was used for identification of tentatively identified [13C10]jasmine lactone. The qualitative ion of jasmine lactone is m/z 169 corresponding to [M + H]+, and the qualitative ion of tentatively identified [13C10]jasmine lactone is m/ z 179. Continuous mechanical damage or dual stress treatment resulted in high accumulation of unlabeled jasmine lactone, which retention time is overlapped with that of tentatively identified [13C10]jasmine lactone. However, the m/z 179 was not detected in the control sample (tea leaf without treatments of labeled fatty acids), and it was detected in samples treated with [13C18]LA or [13C18]ALA.

temperature should be introduced into the tea manufacturing process to improve the floral aroma quality of tea. CsLOX1 Is Involved in the Biosynthesis of Jasmine Lactone Derived from LA and ALA in Tea Leaves. To confirm whether LA and/or ALA are the precursor(s) of jasmine lactone, we fed a stable isotope-labeled compound, either [ 13 C 18 ]LA or [ 13 C 18 ]ALA, to tea samples and investigated the formation of tentatively identified [13C10]jasmine lactone under continuous mechanical damage and the dual stress of low temperature and mechanical damage (Figure 4A). Based on the calculation of the characteristic fragment ions of tentatively identified [13C10]jasmine lactone (Figure

dual stress that regulate genes involved in multiple biosynthetic pathways of tea aromas. In the biology of tea, mechanical damage stress is activated once the tea leaves are picked, thus there are combinations of other stresses and mechanical damage during the tea manufacturing process. Therefore, further studies on the discovery of key transcription factors from the multiple stresses, on top of mechanical damage, will advance our knowledge of the relationship of stresses and tea aromas. In addition, (E)-nerolidol and jasmine lactone contribute considerably to the floral aromas of tea quality. Both our previous study13 and present study indicate that low 3905

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Figure 5. Quantitative analysis of tentatively identified [13C10]jasmine lactone from [13C18]linoleic acid (LA) or [13C18]α-linolenic acid (ALA) during continuous mechanical damage (A) and dual stress treatment (B). Content of tentatively identified [13C10]jasmine lactone was calculated based on a calibration curve acquired from unlabeled jasmine lactone. DW, dry weight. All data are expressed as mean ± SD (n = 3). (A) CK, the tea leaves stored at 25 °C without continuous shaking for 12 h. Continuous mechanical damage, the tea leaves shaken using a shaking table at 25 °C for 12 h. For continuous mechanical damage, significant differences between control and continuous mechanical damage are indicated (* p ≤ 0.05, and ** p ≤ 0.01). (B) −, intact tea leaves; +, tea leaves with mechanical damage. For dual stress treatment, means distinguished with different letters are significantly different from each other (p ≤ 0.05).

Figure 6. Identification and functional characterization of CsLOX1 recombinant protein expressed in Escherichia coli (E. coli). LOX, lipoxygenase; M, marker. (A) SDS-PAGE analysis of E. coli-expressed CsLOX1. Arrows indicate target proteins. (B) The E. coli-expressed CsLOX1 was assayed from the increase in absorbance at 234 nm due to the transformation of linoleic acid (LA) or α-linolenic acid (ALA) to the respective conjugated hydroperoxydienes.

can be sequentially desaturated to ALA in plants.45 Therefore, it is conceivable of the presented study that LA is desaturated to ALA prior to conversion to jasmine lactone. However, in the present work, we were not able to determine whether LA was desaturated to ALA prior to conversion to jasmine lactone in tea leaves. Therefore, further studies are needed to confirm the speculation. Continuous mechanical damage can activate CsLOXs and result in high accumulation of jasmine lactone in tea leaves (Figure 2). However, only the expression level of CsLOX1 showed a positive correlation to jasmine lactone levels under the dual stress of low temperature and mechanical damage (Figure 3). The results show that CsLOX1 may be the key gene that regulates jasmine lactone formation under these stresses. Therefore, to confirm our hypothesis, we obtained the CsLOX1 recombinant protein through E. coli expression (Figure 6A) and used partly purified proteins to catalyze LA and ALA. The results showed that E. coli-expressed CsLOX1 had enzyme activity and could transform LA and ALA to the respective conjugated hydroperoxydienes (Figure 6B). Nevertheless, some other CsLOXs, such as CsLOX2 and CsLOX4 that were also activated during the continuous mechanical damage, did not catalyze the ALA (data not shown). The CsLOX1 activity was determined spectrophotometrically by monitoring the increase in the absorbance at 234 nm; however, the downstream products could not be detected and identified. Therefore, the current results only indicate that CsLOX1 can catalyze LA and ALA, but further studies are needed to show that CsLOX1 can convert these unsaturated fatty acids into jasmine lactone. Deshpande et al.19 showed that Mi9LOX had a higher affinity

4B), we made a qualitative analysis of tentatively identified [13C10]jasmine lactone from [13C18]LA and [13C18]ALA by GCMS with electron impact ionization (Figure 4C) and negative ion chemical ionization (Figure 4D). In the present study, [13C10]jasmine lactone biodegraded from [13C18]LA or [13C18]ALA was detected and tentatively identified (Figures 4C and 4D), indicating that LA and ALA are both precursors of jasmine lactone. In addition, the accumulation of tentatively identified [13C10]jasmine lactone, either from [13C18]LA or [13C18]ALA, was enhanced by both continuous mechanical damage and dual stress (Figure 5). Tea leaves put under stress, especially continuous mechanical damage, may induce more catalytic conversion of unsaturated fatty acids into jasmine lactone, as decreased LA and ALA levels were observed during continuous mechanical damage (Figure 2A). Many attempts have been made to demonstrate that many lactones, including jasmine lactone, are mainly synthesized from ALA.19,21,24 However, there are some studies showing that LA is also a precursor of lactones.19,21 In the present study, labeled jasmine lactone was produced in tea leaves when fed with labeled unsaturated fatty acids, [13C18]LA and [13C18]ALA, respectively. Based on the calculation between feeding amounts of [13C18]LA and [13C18]ALA and product of tentatively identified [13C10]jasmine lactone, turnover rates from fatty acid into jasmine lactone were very low (nearly 0.11−0.05%), because there are many steps involved in the transformation. Besides, many other compounds, such as (Z)-3-hexenol, (E)-3-hexenyl acetate, jasmonic acid, methyl jasmonate, etc., are also converted from fatty acids.42−44 LA and ALA are not necessarily independent precursors of jasmine lactone, as LA 3906

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Journal of Agricultural and Food Chemistry of toward ALA than LA, similar to that of olive 9LOX,46 suggesting possible tuning of this enzyme to the in vivo availability of the substrate, as more ALA accumulated during the ripening of mango fruit.47 However, in our study, no significant difference in the affinity of CsLOX1 toward ALA and LA was observed. In addition, levels of both LA and ALA were decreased under continuous mechanical damage (Figure 2A). Therefore, more studies are needed to provide in vivo evidence of CsLOX1 affinity toward ALA and LA in tea leaves. The present study investigated the biosynthetic pathways of jasmine lactone, a characteristic aroma compound of oolong tea, in tea leaves. Since additive effects on (E)-nerolidol under the dual stress of low temperature and mechanical damage have been observed,13 our present study provided another example that jasmine lactone also exhibited such a phenomenon (Figure 7). Furthermore, only one pathway via hydroperoxy fatty acids

ORCID

Ziyin Yang: 0000-0003-3112-3479 Author Contributions

L.Z. and Y.Z. are co-first authors and contributed equally to this work. Funding

This work was supported by grants from the Guangdong Natural Science Foundation for Distinguished Young Scholars (2016A030306039), the Youth Innovation Promotion Association of Chinese Academy of Sciences, the Pearl River Science and Technology New Star Fund of Guangzhou, the Guangdong Innovation Team of Modern Agricultural Industry Technology System (2017LM1143), and the Guangdong Special Support Plan for Training High-Level Talents (2016TQ03N617). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank T. Hasegawa Flavours & Fragrances (Shanghai) Co., Ltd. for kindly providing the jasmine lactone authentic standard.

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ABBREVIATIONS USED ALA, α-linolenic acid; EF1, encoding elongation factor 1; GCMS, gas chromatography−mass spectrometer; IPTG, isopropylβ-D-thiogalactopyranoside; LA, linoleic acid; LOX, lipoxygenase; MSTFA, N-methyl-N-(trimethylsilyl)trifluoroacetamide; PGX, peroxygenase; PPFD, photosynthetic photon flux density; SDS, sodium dodecyl sulfate; SPME, solid-phase microextraction; TMEMD, N,N,N′,N′-tetramethylethylenediamine

Figure 7. Formation of jasmine lactone in tea (Camellia sinensis) leaves exposed to multiple stress during the tea manufacturing process. LA, linoleic acid; ALA, α-linolenic acid; LOX 1, lipoxygenase 1; PGX, peroxygenase.

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(1) Joshi, R.; Gulati, A. Fractionation and identification of minor and aroma-active constituents in Kangra orthodox black tea. Food Chem. 2015, 167, 290−298. (2) Schuh, C.; Schieberle, P. Characterization of the key aroma compounds in the beverage prepared from Darjeeling black tea: quantitative differences between tea leaves and infusion. J. Agric. Food Chem. 2006, 54 (3), 916−924. (3) Gui, J. D.; Fu, X. M.; Zhou, Y.; Katsuno, T.; Mei, X.; Deng, R. F.; Xu, X. L.; Zhang, L. Y.; Dong, F.; Watanabe, N.; Yang, Z. Y. Does enzymatic hydrolysis of glycosidically bound volatile compounds really contribute to the formation of volatile compounds during the oolong tea manufacturing process? J. Agric. Food Chem. 2015, 63 (31), 6905− 6914. (4) Zhu, J. C.; Chen, F.; Wang, L. Y.; Niu, Y. W.; Yu, D.; Shu, C.; Chen, H. X.; Wang, H. L.; Xiao, Z. B. Comparison of aroma-active volatiles in oolong tea infusions using GC−olfactometry, GC−FPD, and GC−MS. J. Agric. Food Chem. 2015, 63 (34), 7499−7510. (5) Yang, Z. Y.; Baldermann, S.; Watanabe, N. Recent studies of the volatile compounds in tea. Food Res. Int. 2013, 53 (2), 585−599. (6) Ho, C. T.; Zheng, X.; Li, S. Tea aroma formation. Food Sci. Hum. Wellness 2015, 4 (1), 9−27. (7) Cho, J. Y.; Mizutani, M.; Shimizu, B.; Kinoshita, T.; Ogura, M.; Tokoro, K.; Lin, M. L.; Sakata, K. Chemical profiling and gene expression profiling during the manufacturing process of Taiwan oolong tea ″Oriental Beauty″. Biosci., Biotechnol., Biochem. 2007, 71 (6), 1476−1486. (8) Dong, F.; Yang, Z. Y.; Baldermann, S.; Sato, Y.; Asai, T.; Watanabe, N. Herbivore-induced volatiles from tea (Camellia sinensis) plants and their involvement in intraplant communication and changes in endogenous nonvolatile metabolites. J. Agric. Food Chem. 2011, 59 (24), 13131−13135. (9) Yang, Z. Y.; Kobayashi, E.; Katsuno, T.; Asanuma, T.; Fujimori, T.; Ishikawa, T.; Tomomura, M.; Mochizuki, K.; Watase, T.;

under the action of CsLOX1 was significantly affected by the dual stresses, although both jasmine lactone formation pathways may occur in tea leaves (Figure 7). Moreover, the results confirmed that two unsaturated fatty acids, LA and ALA, were both precursors of jasmine lactone. This study provided the first evidence concerning jasmine lactone formation in tea leaves and identified jasmine lactone accumulation in oolong tea. The results obtained from this study will contribute to draw a unified mechanism of formations of characteristic aromas in response to multiple stresses from the tea manufacturing process.

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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.8b00515. Table S1, primers of qRT-PCR used in this study; Table S2, primers of PCR for cDNA cloning used in this study; Figure S1, identification of jasmine lactone authentic standard; Figure S2, determination of the enantiomeric ratio of jasmine lactone in Camellia sinenesis; Figure S3, effect of treatment time and temperature after mechanical damage caused by needle on jasmine lactone formation (PDF)

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REFERENCES

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Corresponding Author

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benthamiana for biotechnological application. BMC Biotechnol. 2011, 11, 30. (28) Zeng, L. T.; Zhou, Y.; Fu, X. M.; Mei, X.; Cheng, S. H.; Gui, J. D.; Dong, F.; Tang, J. C.; Ma, S. Z.; Yang, Z. Y. Does oolong tea (Camellia sinensis) made from a combination of leaf and stem smell more aromatic than leaf-only tea? Contribution of the stem to oolong tea aroma. Food Chem. 2017, 237, 488−498. (29) Plagemann, I.; Zelena, K.; Arendt, P.; Ringel, P. D.; Krings, U.; Berger, R. G. LOXPsa1, the first recombinant lipoxygenase from a basidiomycete fungus. J. Mol. Catal. B: Enzym. 2013, 87, 99−104. (30) Zeng, L. T.; Liao, Y. Y.; Li, J. L.; Zhou, Y.; Tang, J. C.; Dong, F.; Yang, Z. Y. α-Farnesene and ocimene induce metabolite changes by volatile signaling in neighboring tea (Camellia sinensis) plants. Plant Sci. 2017, 264, 29−36. (31) Wang, D. M.; Kubota, K.; Kobayashi, A.; Juan, I. M. Analysis of glycosidically bound aroma precursors in tea leaves. 3. Change in the glycoside content of tea leaves during the oolong tea manufacturing process. J. Agric. Food Chem. 2001, 49 (11), 5391−5396. (32) Engel, K. H.; Flath, R. A.; Buttery, R. G.; Mon, T. R.; Ramming, D. W.; Teranishi, R. Investigation of volatile vonstituents in nectarines. 1. analytical and sensory characterization of aroma components in some nectarine cultivars. J. Agric. Food Chem. 1988, 36 (3), 549−553. (33) Chen, Y. Y.; Fu, X. M.; Mei, X.; Zhou, Y.; Cheng, S. H.; Zeng, L. T.; Dong, F.; Yang, Z. Y. Proteolysis of chloroplast proteins is responsible for accumulation of amino acids in dark-treated tea (Camellia sinensis) leaves. J. Proteomics 2017, 157, 10−17. (34) Baldermann, S.; Yang, Z. Y.; Katsuno, T.; Tu, V. A.; Mase, N.; Nakamura, Y.; Watanabe, N. Discrimination of green, oolong, and black Teas by GC-MS analysis of characteristic volatile flavor compounds. Am. J. Anal. Chem. 2014, 5 (9), 620−632. (35) Kobayashi, A.; Tachiyama, K.; Kawakami, M.; Yamanishi, T.; Juan, I. M.; Chiu, W. T. F. Effects of solar-withering and turn over treatment during indoor-withering on the formation of pouchong tea aroma. Agric. Biol. Chem. 1985, 49, 1655−1660. (36) Xia, E. H.; Zhang, H. B.; Sheng, J.; Li, K.; Zhang, Q. J.; Kim, C.; Zhang, Y.; Liu, Y.; Zhu, T.; Li, W.; Huang, H.; Tong, Y.; Nan, H.; Shi, C.; Shi, C.; Jiang, J. J.; Mao, S. Y.; Jiao, J. Y.; Zhang, D.; Zhao, Y.; Zhao, Y. J.; Zhang, L. P.; Liu, Y. L.; Liu, B. Y.; Yu, Y.; Shao, S. F.; Ni, D. J.; Eichler, E. E.; Gao, L. Z. The tea tree genome provides insights into tea flavor and independent evolution of caffeine biosynthesis. Mol. Plant 2017, 10 (6), 866−877. (37) Holopainen, J. K.; Gershenzon, J. Multiple stress factors and the emission of plant VOCs. Trends Plant Sci. 2010, 15 (3), 176−184. (38) Dong, F.; Fu, X.; Watanabe, N.; Su, X.; Yang, Z. Recent advances in the emission and functions of plant vegetative volatiles. Molecules 2016, 21 (2), 124. (39) Gouinguene, S. P.; Turlings, T. C. The effects of abiotic factors on induced volatile emissions in corn plants. Plant Physiol. 2002, 129 (3), 1296−1307. (40) Vickers, C. E.; Gershenzon, J.; Lerdau, M. T.; Loreto, F. A unified mechanism of action for volatile isoprenoids in plant abiotic stress. Nat. Chem. Biol. 2009, 5, 283−291. (41) Fu, X. M.; Cheng, S. H.; Zhang, Y. Q.; Du, B.; Feng, C.; Zhou, Y.; Mei, X.; Jiang, Y. M.; Duan, X. W.; Yang, Z. Y. Differential responses of four biosynthetic pathways of aroma compounds in postharvest strawberry (Fragaria × ananassa Duch.) under interaction of light and temperature. Food Chem. 2017, 221 (15), 356−364. (42) Koch, T.; Bandemer, K.; Boland, W. Biosynthesis of cisjasmone: a pathway for the inactivation and the disposal of the plant stress hormone jasmionic acid to the gas phase. Helv. Chim. Acta 1997, 80, 838−850. (43) Hatanaka, A.; Kajiwara, T.; Sekiya, J. Biosynthetic pathway for C6-aldehydes formation from linolenic acid in green leaves. Chem. Phys. Lipids 1987, 44, 341−361. (44) Vick, B. A.; Zimmerman, D. C. Biosynthesis of jasmonic acid by several plant species. Plant Physiol. 1984, 75, 458−461. (45) Chen, R.; Matsui, K.; Ogawa, M.; Oe, M.; Ochiai, M.; Kawashima, H.; Sakuradani, E.; Shimizu, S.; Ishimoto, M.; Hayashi, M.; Murooka, Y.; Tanaka, Y. Expression of Δ6, Δ5 desaturase and

Nakamura, Y.; Watanabe, N. Characterisation of volatile and nonvolatile metabolites in etiolated leaves of tea (Camellia sinensis) plants in the dark. Food Chem. 2012, 135 (4), 2268−2276. (10) Fu, X. M.; Chen, Y. Y.; Mei, X.; Katsuno, T.; Kobayashi, E.; Dong, F.; Watanabe, N.; Yang, Z. Y. Regulation of formation of volatile compounds of tea (Camellia sinensis) leaves by single light wavelength. Sci. Rep. 2015, 5, 16858. (11) Mei, X.; Liu, X. Y.; Zhou, Y.; Wang, X. Q.; Zeng, L. T.; Fu, X. M.; Li, J. L.; Tang, J. C.; Dong, F.; Yang, Z. Y. Formation and emission of linalool in tea (Camellia sinensis) leaves infested by tea green leafhopper (Empoasca (Matsumurasca) onukii Matsuda. Food Chem. 2017, 237, 356−363. (12) Zeng, L. T.; Zhou, Y.; Gui, J. D.; Fu, X. M.; Mei, X.; Zhen, Y. P.; Ye, T. X.; Du, B.; Dong, F.; Watanabe, N.; Yang, Z. Y. Formation of volatile tea constituent indole during the oolong tea manufacturing process. J. Agric. Food Chem. 2016, 64 (24), 5011−5019. (13) Zhou, Y.; Zeng, L. T.; Liu, X. Y.; Gui, J. D.; Mei, X.; Fu, X. M.; Dong, F.; Tang, J. C.; Zhang, L. Y.; Yang, Z. Y. Formation of (E)nerolidol in tea (Camellia sinensis) leaves exposed to multiple stresses during tea manufacturing. Food Chem. 2017, 231, 78−86. (14) Katsuno, T.; Kasuga, H.; Kusano, Y.; Yaguchi, Y.; Tomomura, M.; Cui, J. L.; Yang, Z. Y.; Baldermann, S.; Nakamura, Y.; Ohnishi, T.; Mase, N.; Watanabe, N. Characterisation of odorant compounds and their biochemical formation in green tea with a low temperature storage process. Food Chem. 2014, 148, 388−395. (15) Yamanishi, T.; Kosuge, M.; Tokitomo, Y.; Maeda, R. Flavor constituents of pouchong tea and a comparison of the aroma pattern with jasmine tea. Agric. Biol. Chem. 1980, 44, 2139−2142. (16) Kobayashi, A.; Kubota, K. Chirality in tea aroma compounds. In Olfaction and Taste XI; Kurihara, K., Suzuki, N., Ogawa, H., Eds.; Springer-Verlag: Tokyo, 1994; pp 260−261, DOI: 10.1007/978-4-43168355-1_5. (17) Werkhoff, P.; Brennecke, S.; Bretschneider, W.; Guntert, M.; Hopp, R.; Surburg, H. Chirospecific analysis in essential oil, fragrance and flavor research. Z. Lebensm.-Unters. Forsch. 1993, 196, 307−328. (18) Kulkarni, R. S.; Chidley, H. G.; Pujari, K. H.; Giri, A. P.; Gupta, V. S. Geographic variation in the flavour volatiles of Alphonso mango. Food Chem. 2012, 130 (1), 58−66. (19) Deshpande, A. B.; Chidley, H. G.; Oak, P. S.; Pujari, K. H.; Giri, A. P.; Gupta, V. S. Isolation and characterization of 9-lipoxygenase and epoxide hydrolase 2 genes: Insight into lactone biosynthesis in mango fruit (Mangifera indica L.). Phytochemistry 2017, 138, 65−75. (20) Demole, E. P. The Fragrance of Jasmine. In Fragrance Chemistry, the Science of the Sense of Smell; Theimer, E. T., Ed.; Academic Press: New York, 1982; pp 349−396. (21) Haffner, T.; Nordsieck, A.; Tressl, R. Biosynthesis of δ-jasmin lactone (=(Z)-dec-7-eno-5-lactone) and (Z, Z)-dodeca-6, 9-dieno-4lactone in the yeast Sporobolomyces odorus. Helv. Chim. Acta 1996, 79, 2088−2099. (22) Haffner, T.; Tressl, R. Stereospecific metabolism of isomeric epoxyoctadecanoic acids in the lactone-producing yeast Sporidiobolus salmonicolor. Lipids 1998, 33, 47−58. (23) Schottler, M.; Boland, W. Biosynthesis of dodecano-4-lactone in ripening fruits: crucial role of an epoxide-hydrolase in enantioselective generation of aroma components of the nectarine (Prunus persica var Nucipersica) and the strawberry (Fragaria ananassa). Helv. Chim. Acta 1996, 79, 1488−1496. (24) Vecchietti, A.; Lazzari, B.; Ortugno, C.; Bianchi, F.; Malinverni, R.; Caprera, A.; Mignani, I.; Pozzi, C. Comparative analysis of expressed sequence tags from tissues in ripening stages of peach (Prunus persica L. Batsch). Tree Genet. Genomes 2009, 5 (3), 377−391. (25) Meesapyodsuk, D.; Qiu, X. A peroxygenase pathway involved in the biosynthesis of epoxy fatty acids in oat. Plant Physiol. 2011, 157 (1), 454−463. (26) Fuchs, C.; Schwab, W. Epoxidation, hydroxylation and aromatization is catalyzed by a peroxygenase from Solanum lycopersicum. J. Mol. Catal. B: Enzym. 2013, 96, 52−60. (27) Huang, F. C.; Schwab, W. Cloning and characterization of a 9lipoxygenase gene induced by pathogen attack from Nicotiana 3908

DOI: 10.1021/acs.jafc.8b00515 J. Agric. Food Chem. 2018, 66, 3899−3909

Article

Journal of Agricultural and Food Chemistry GLELO elongase genes from Mortierella alpina for production of arachidonic acid in soybean [Glycine max (L.) Merrill] seeds. Plant Sci. 2006, 170, 399−406. (46) Padilla, M. N.; Hernandez, M. L.; Sanz, C.; Martinez-Rivas, J. M. Molecular cloning, functional characterization and transcriptional regulation of a 9-lipoxygenase gene from olive. Phytochemistry 2012, 74, 58−68. (47) Deshpande, A. B.; Chidley, H. G.; Oak, P. S.; Pujari, K. H.; Giri, A. P.; Gupta, V. S. Data on changes in the fatty acid composition during fruit development and ripening of three mango cultivars (Alphonso, Pairi and Kent) varying in lactone content. Data Brief 2016, 9, 480−491.

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