Increasing Temperature Changes Flux into Multiple Biosynthetic Pathways for 2-Phenylethanol in Model Systems of Tea (Camellia sinensis) and Other Plants

Article abstract of DOI:10.1021/acs.jafc.9b03749

2-Phenylethanol (2PE) is a representative aromatic aroma compound in tea (Camellia sinensis) leaves. However, its formation in tea remains unexplored. In our study, feeding experiments of [²H₈]L-phenylalanine (Phe), [²H

Full text of DOI:10.1021/acs.jafc.9b03749

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Chemistry and Biology of Aroma and Taste
Increasing temperature changes the flux into the multiple
biosynthetic pathways for 2-phenylethanol in model
systems of tea (Camellia sinensis) and other plants
Lanting Zeng, Haibo Tan, Yinyin Liao, Guotai Jian, Ming
Kang, Fang Dong, Naoharu Watanabe, and Ziyin Yang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03749 • Publication Date (Web): 16 Aug 2019
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Title: Increasing temperature changes the flux into the multiple biosynthetic pathways for
2-phenylethanol in model systems of tea (Camellia sinensis) and other plants
Authors: Lanting Zeng a, b, #, Haibo Tan a, b, #, Yinyin Liao a, c, Guotai Jian a, c, Ming Kang a,
*
Fang Dong d, Naoharu Watanabe e, Ziyin Yang a, b, c,
Affiliation:
a 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, No. 723 Xingke Road, Tianhe District,
Guangzhou 510650, China
b Center of Economic Botany, Core Botanical Gardens, Chinese Academy of Sciences, No.
723 Xingke Road, Tianhe District, Guangzhou, 510650, China
c University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China
d Guangdong Food and Drug Vocational College, No. 321 Longdongbei Road, Tianhe
District, Guangzhou 510520, China
e Graduate School of Science and Technology, Shizuoka University, No. 3-5-1 Johoku,
Naka-ku, Hamamatsu 432-8561, Japan
# Co-first authors. * Corresponding author. Ziyin Yang, Tel: +86-20-38072989, Email
address: zyyang@scbg.ac.cn.
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ABSTRACT
2-Phenylethanol (2PE) is a representative aromatic aroma compound in tea (Camellia sinensis)
leaves. However, its formation in tea remains unexplored. In our study, feeding experiments of
[2H8]L-phenylalanine (Phe), [2H5]phenylpyruvic acid (PPA), or (E/Z)-phenylacetaldoxime
(PAOx) showed that three biosynthesis pathways for 2PE derived from L-Phe occurred in tea
leaves, namely, pathway I (via phenylacetaldehyde (PAld)), pathway II (via PPA and PAld), and
pathway III (via (E/Z)-PAOx and PAld). Furthermore, increasing temperature resulted in
increased flux into the pathway for 2PE from L-Phe via PPA and PAld. In addition, tomato fruits
and petunia flowers also contained the 2PE biosynthetic pathway from L-Phe via PPA and PAld,
and increasing temperatures led to increased flux into this pathway, suggesting that such a
phenomenon might be common among most plants containing 2PE. This represents a
characteristic example of changes in flux into the biosynthesis pathways of volatile compounds
in plants in response to stresses.
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KEYWORDS: tea; Camellia sinensis; aroma; 2-phenylethanol; phenylpyruvic acid; volatile
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INTRODUCTION
Tea (Camellia sinensis) aroma compounds determine tea aroma quality.1–3 Tea aroma
compound formation and regulation are hot topics in tea-related research.2–4 Since the isolation
and identification of some glycosidically bound aroma compounds from tea leaves have been
achieved, the transformations between glycosidically bound aroma compounds and free aroma
compounds have become of great interest.2,5 In recent years, with developments in tea plant
biology, aroma compound formation from de novo enzymatic reactions in live tea leaves has
attracted increasing research attention.4 Biochemical pathways, enzymes, or genes regulating the
formation of several tea characteristic aroma compounds, for example indole, jasmine lactone,
(S)-linalool, (E)-nerolidol, -ocimene, and (Z)-3-hexenal have been studied and elucidated.6–13
Furthermore, tea aroma compound formation under stresses during the preharvest and
postharvest tea stages have become of increasing interest, with the specific relationships between
stresses and typical aromas considered key to the effective and safe improvement of tea aroma
quality.4 For example, biotic stresses (such as attack by tea green leafhoppers) can enhance
volatile terpene compounds, such as linalool and diendiol,4,10,14 while abiotic stresses (such as
continuous wounding, and dual stress of wounding and low temperature) can lead to the high
accumulation of, for example, jasmine lactone, (E)-nerolidol, and indole.7,9,12 However, most
studies have concentrated on how stresses affect the contents of volatile compounds in plants,
while little is known about their effect on the flux of plant volatile compounds into biosynthesis
pathways. In contrast to volatile terpenes and volatile fatty acid derivatives, there is few research
on the biosynthesis of aromatic aroma compounds, volatile phenylpropanoids/benzenoids
(VPBs), and their formation in response to stresses, in tea leaves.
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In most tea cultivars, fresh tea leaves contain a relatively high percentage of VPBs, such as
methyl salicylate, benzyl alcohol, benzaldehyde, phenylacetaldehyde (PAld), and
2-phenylethanol (2PE).2,15 Among these VPBs, 2PE is a rose-like aromatic alcohol and
representative aroma compound found in tea leaves.2 Presently, in tea leaves, the formation of
VPBs such as 2PE refers to studies on other plants, such as petunia flowers, rose flowers, and
tomatoes.16–21 Based on these reports, several pathways for 2PE synthesis have been identified in
plants, as follows: (i) Derivation from L-phenylalanine (Phe) via PAld;17,18 (ii) derivation from
L-Phe via phenylpyruvic acid (PPA) and PAld;21–23 and (iii) derivation from L-Phe via
(E/Z)-phenylacetaldoxime (PAOx) and PAld, which was recently discovered in Plumeria.24
However, the 2PE synthesis pathway(s) in tea leaves are unknown. In this study, we aimed to
investigate how many pathways synthesize 2PE in tea leaves and, if more than one 2PE synthesis
pathway was present, whether the flux into the different pathways was affected by stresses.
Accordingly, [2H8]L-Phe (stable isotope-labeled) was used to confirm the occurrences of 2PE
biochemical pathway(s) in tea leaves. We chemically synthesized [2H5]PPA (stable
isotope-labeled) and (E/Z)-PAOx for tracing in a model system of tea leaves to confirm
alternative pathways for 2PE synthesis. Furthermore, the effect of increasing temperature on flux
into the different 2PE pathways in tea leaves was investigated. Finally, other plants, including
tomato fruits and petunia flowers, were also investigated to determine whether the identified 2PE
biochemical pathways and change in flux in response to increasing temperature were specific to
tea leaves or common in most plants. This study aimed to elucidate 2PE formation in tea leaves,
including biochemical pathways and their flux changes in response to increasing temperature
stress. This is a characteristic example of changing flux into the biosynthesis pathways of
volatile compounds in plants in response to stresses.
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MATERIALS AND METHODS
Chemical reagents
Ethyl n-decanoate (purity, ≥99%) and sodium phenylpyruvate (purity, ≥98%) were obtained
from Aladdin Industrial Co., Ltd (Shanghai, China). PAld (purity, ≥90%) was obtained from
Sigma-Aldrich Company, Ltd. (Louis, MO, USA). [2H8]L-Phe (purity, ≥98%) and
[2H6]benzaldehyde (≥ 98%) were obtained from Cambridge Isotope Laboratories Inc.
(Cambridge, MA, USA). 4-Phenylimidazole (purity, ≥98%) was purchased from Aikon
Biomedical Research and Development Co., Ltd (Jiangsu, China). 2PE (≥ 98%) was obtained
from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). [2H5]PPA (purity, ≥95%) and
(E/Z)-PAOx (purity, ≥95%) were synthesized in our laboratory.
Plant materials and growth conditions
C. sinensis cv. ‘Jinxuan’ are widely cultivated in South China and suitable for making oolong
tea. These tea plants were grown at the Tea Research Institute (Yingde Tea Experimental Station,
Yingde, China), Guangdong Academy of Agricultural Sciences. Tea branches with one bud and
three leaves from nearly twenty-year-old C. sinensis plants were used in this study.
Solanum lycopersicum cv. ‘Micro Tom’ and Petunia × hybrida cv. ‘Mitchell Diplod’ were
planted in a greenhouse with controlled conditions (relative humidity, 70±2%; temperature, 25±2
°C; dark/light photoperiod, 8 h/16 h). Developing petunia flowers at about stages 6–7 based on
the previous standard,25 and tomato fruits (immature and mature) were used in this study.
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Chemical synthesis of [2H5]PPA
[2H5]PPA was synthesized using the method of Liu et al.,26 as shown in Figure 1A. The first
step was the synthesis of [2H6](Z)-2-methyl-4-(phenylmethylene)oxazol-5(4H)-one (3). A
solution of sodium acetate (10 mmol, 820 mg) dissolved in 4 mL acetic anhydride,
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[2H6]benzaldehyde (1, 2 mmol, 224 mg) and N-acylglycine (2, 2.4 mmol, 280 mg) were added in
one portion. Then, the mixture was heated at 120 °C for 7 h under a nitrogen atmosphere and
analyzed by thin-layer chromatography (TLC). When most of starting materials had disappeared,
the reaction mixture cooled to room temperature. The resulting solution was stored in a
refrigerator at 0 °C overnight to acquire a yellowish solid. After further filtration and washing
with cold water, the resulting yellow solid was collected and subjected to drying under vacuum
to obtain crude product [2H6](Z)-2-methyl-4-(phenylmethylene)oxazol-5(4H)-one (3, 310 mg).
The second step was the synthesis of [2H5](Z)-2-acetamido-3-(phenyl)acrylic acid (4). Crude
product [2H6](Z)-2-methyl-4-(phenylmethylene)oxazol-5(4H)-one (3, 310 mg) was redissolved in
an acetone/H2O mixture (5:7, 12 mL). Then, the solution was heated to reflux and kept at that
time for 7 h under a nitrogen atmosphere. After most of starting material was consumed, the
solvent was dried by evaporation to acquire crude product
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[2H5](Z)-2-acetamido-3-(phenyl)acrylic acid (4, 367 mg) as a residual solid. The third step was
the synthesis of [2H5]PPA (5). Crude product [2H5](Z)-2-acetamido-3-(phenyl)acrylic acid (4,
367 mg) from the previous step was directly added to 4 mL HCl aqueous solution (1 mM). Then,
the mixture was heated to dissolve the starting material (solid) and analyzed by TLC. After most
of the starting material had been consumed and the reaction mixture cooled to room temperature,
the mixture was purified using 10 mL ethyl acetate for 4 times, and the organic layers were
collected and combined. It was dried with anhydrous Na2SO4, and filtrated with buchner funnel,
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and the solvent was removed to obtain an oil (slight yellow) under reduced pressure via rotary
evaporation. This crude product was further re-crystallized from hexane to obtain pure product (5,
0.64 mmol, 108 mg, 32% yield).
[2H5]PPA (5) was white solid with a purity of ≥95%. The purity of [2H5]PPA was
1
determined by its H nuclear magnetic resonance (NMR) spectra (Figure S1A). Detailed NMR
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data for 5 was as follows: H NMR (500 MHz, CD3OD) H 6.51 (s, 1H); C NMR (125 MHz,
CD3OD) C 110.1, 127.1, 127.3, 127.5, 128.7, 128.9, 129.1, 134.8, 134.8, 140.8, 140.9, 167.0,
172.0. HRESIMS m/z 503.2436 [M–H] (calcd. for C31H35O6, 503.2439).
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Chemical synthesis of (E/Z)-PAOx
The synthesis of (E/Z)-PAOx was conducted using a literature method,27 as shown in Figure
1B. Sodium carbonate (0.6 mmol, 63 mg) and hydroxylamine hydrochloride (1.2 mmol, 83 mg)
were dissolved in a mixed solvent (4 mL, methanol/H2O, 1:1) and stirred for 10 min with a
magnetic stir bar. PAld (6, 1 mmol, 120 mg) was then added in one portion at 0 °C, and the
mixture was further subjected to 24 h-stirring under a nitrogen atmosphere (room temperature),
and analyzed by TLC. After most of starting materials were consumed, the reaction mixture was
extracted with ethyl acetate (5 mL  5). The organic layers were combined, dried with anhydrous
Na2SO4, and filtrated with buchner funnel, and the solvent was removed to obtain a slight yellow
solid under reduced pressure via rotary evaporation. Purification of the yellow slight solid was
used flash column chromatography (silica gel, hexane/ethyl acetate, 5:1), and desired product 7
(120 mg, 89% yield) was obtained as a pair of cis and trans isomers with a ratio of about 0.3:1.
(E/Z)-PAOx (7) was a yellow solid with purity of ≥95%. The purity of (E/Z)-PAOx was
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determined by its H NMR spectra (Figure S1B). Detailed NMR data for 7 was as follows: H
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NMR (500 MHz, CDCl3) H 3.57 (d, J = 6.30, 0.6H), 3.78 (d, J = 5.33, 2H), 6.93 (t, J = 5.33,
1H), 7.20–7.40 (m, 6.5H), 7.57 (t, J = 6.30, 0.3H); HRESIMS m/z 503.2436 [M–H] (calcd. for
C31H35O6, 503.2439).
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Feeding experiments using [2H8]L-Phe in different plants and under different temperature
treatments
Tea branches from C. sinensis cv. ‘Jinxuan’ plants plucked in October 2016 were used to
conduct the feeding experiment with [2H8]L-Phe. The tea branches were cultivated in [H8]L-Phe
solution (12 mM) (Figure S2A)28 and the tea leaves were then kept in an incubator under
controlled conditions (humidity, 70%; dark/light photoperiod, 8 h/16 h) at 15, 25, or 35 °C. The
suitable growth temperature of tea plant is 20–25 °C. Therefore, the lower, medium, and higher
temperature were set at 15, 25, and 35 °C, respectively. After treating for 3 d and 7 d, the
samples were stored at 80 °C until further analysis after freezing with liquid N2. Three samples
were processed in parallel as replicates.
Fruits of S. lycopersicum cv. Micro-Tom plucked in January 2017 were used to conduct a
feeding experiment with [2H8]L-Phe. Injecting the solution into tomato fruit was difficult using
an injector, especially for mature fruit. Therefore, some part of the fruit was removed, and the
solution was added, as shown in Figure S2B. However, the wounded tomato fruit could not be
kept for long periods, especially at high temperatures. Therefore, the fruit was first subjected to
different temperature treatments for 2 d, and then fed with [2H8]L-Phe and cultivated at the
corresponding temperature for another 1 d. First, the fruit was kept in an incubator under
controlled conditions (humidity, 70%; dark/light photoperiod, 8 h/16 h) at 10, 25, or 35 °C. The
suitable growth temperature of tomato is 13–28 °C. Therefore, the lower, medium, and higher
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temperature were set at 10, 25, and 35 °C, respectively. In contrast to tea plant, tomato is more
resistant to low temperature. Therefore, the lower temperature in tomato treatment was set at 10
°C. After treating for 2 d, every fruit was fed with [2H8]L-Phe solution (300 μL, 12 mM) and
continually treated in the corresponding incubator for another 1 d.29 After treatment, the samples
were stored at 80 °C until further analysis after freezing with liquid N2. Three samples were
processed in parallel as replicates for immature fruit, and two samples were processed in parallel
as replicates for mature fruit.
Flowers of P. hybrida cv. ‘Mitchell Diplod’ plucked in May 2017 were used to conduct the
feeding experiment with [2H8]L-Phe. Each flower was cultivated in [2H8]L-Phe solution (12 mM)
(Figure S2C)28,29 and then kept in an incubator under controlled conditions (humidity, 70%;
dark/light photoperiod, 8 h/16 h) at 25 °C for 1 d. The samples were then divided into three parts.
The first was kept in the incubator at 25 °C, the second was transferred to the incubator at 10 °C,
and the third was transferred to the incubator at 35 °C. The suitable growth temperature of
tomato is 13–24 °C. Therefore, the lower, medium, and higher temperature were set at 10, 25,
and 35 °C, respectively. In contrast to tea plant, petunia is more resistant to low temperature.
Therefore, the lower temperature in petunia treatment was set at 10 °C. After treating for a
further 1 d, headspace solid-phase microextraction was applied to collect the aroma compounds
emitted from the flowers. After collection, the samples were stored at 80 °C until further
analysis after freezing with liquid N2. Three samples were processed in parallel as replicates.
Feeding experiments using PPA, [2H5]PPA, (E/Z)-PAOx, and 4-phenylimidazole combined
with [2H8]L-Phe in tea leaves
Tea branches from C. sinensis cv. ‘Jinxuan’ plants plucked in April 2019 were used to conduct
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the feeding experiment with PPA. Branches with one bud and three leaves were cultivated in two
different solutions (Figure S2A): (i) H2O (Control 1) and (ii) 6 mM PPA (PPA Treatment). In
PPA Treatment, each branch (nearly 700800 mg) absorbed 1.5 mL PPA solution via
transpiration, and was then cultivated in H2O. As PPA is water insoluble, commercial sodium
phenylpyruvate (water soluble) was used to prepare the PPA solution.
Tea branches from C. sinensis cv. ‘Jinxuan’ plants plucked in May 2019 were used to conduct
the feeding experiment with [2H5]PPA. Branches with one bud and three leaves were cultivated
in two different solutions (Figure S2A): (i) 0.1 mM phosphate buffer (pH 8) (Control 2) and (ii)
6 mM [2H5]PPA resolved in sodium bicarbonate buffer (0.1 mM, pH 8) ([2H5]PPA Treatment).
In [2H5]PPA Treatment, each branch (nearly 700800 mg) absorbed 1.5 mL [2H5]PPA solution
via transpiration, and was then cultivated in phosphate buffer (0.1 mM, pH 8). As PPA is water
insoluble, a slightly alkaline phosphate buffer (pH 8) was used to dissolve [2H5]PPA.
Tea branches from C. sinensis cv. ‘Jinxuan’ plants plucked in May 2019 were used to conduct
the feeding experiment with (E/Z)-PAOx. The branches with one bud and three leaves were
cultivated in two different solutions (Figure S2A): (i) H2O containing 1.5% dimethyl sulfoxide
(DMSO) (Control 3) and (ii) 6 mM (E/Z)-PAOx dissolved in H2O containing 1.5% DMSO
(PAOx Treatment). In PAOx Treatment, each branch (nearly 700800 mg) absorbed 1.5 mL
PAOx solution via transpiration, and was then cultivated in H2O containing 1.5% DMSO. Based
on the phenotype of tea leaves after PAOx treatment (data not shown in the study), it could be
speculated that the concentration of 2PE after PAOx treatment was tolerable for tea branches.
Tea branches from C. sinensis cv. ‘Jinxuan’ plants plucked in May 2019 were used to conduct
the feeding experiment with [2H8]L-Phe or 4-phenylimidazole and [2H8]L-Phe. Branches with
one bud and three leaves were cultivated in two different solutions (Figure S2A): (i) 12 mM
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[2H8]L-Phe resolved in H2O containing 0.5% ethanol and (ii) 12 mM [2H8]L-Phe and 1 mM
4-phenylimidazole resolved in H2O containing 0.5% ethanol. 4-Phenylimidazole is an inhibitor
of CYP450 enzymes.24
All samples were placed in an incubator under controlled conditions (humidity, 70%;
dark/light photoperiod, 8 h/16 h) at 25 °C for 2 d. After treatment, the samples were stored at
80 °C until further analysis after freezing with liquid N2. Four samples were processed in
parallel as replicates.
Determination of 2PE in samples
Organic reagent extraction was used to extract endogenous 2PE from tissues based on a
previous method with some modifications.12 All samples were ground into powder with liquid N2
before extraction. Dichloromethane (1.8 mL) was selected to extract aroma compounds from tea
leaves or tomato fruit (500 mg, fresh weight), and ethyl n-decanoate (internal standard, 5 nmol)
was added to the mixture. Dichloromethane (1.5 mL) was selected to extract aroma compounds
from petunia flowers (100 mg, fresh weight), and ethyl n-decanoate (internal standard, 3 nmol)
was added to the mixture. A horizontal shaker kept at room temperature was used to conduct
oscillating extraction of samples overnight. After oscillating extraction, the extract was gathered,
dried with anhydrous Na2SO4, and concentrated to 100 L using gaseous N2. An aliquot of the
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extract (1 L) was then separated and analyzed by gas chromatography–mass spectrometer
(GC–MS).
The collection of 2PE emitted from petunia flowers was achieved using solid-phase
microextraction (SPME) fibers (2 cm, 50/30 m DVB/CarboxenTM/PDMS Stable FlexTM,
Supelco Inc., Bellefonte, PA, USA), with a method similar to that published elsewhere.30
A
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petunia flower was kept in a sealed container (1 L) and the emitted aroma compounds were
collected at 25±2 °C using a SPME fiber for 1 h. The aroma compounds absorbed in SPME fiber
were separated and analyzed by GC–MS QP2010 SE.
The injection port of GC was kept at 230 °C for 1 min, all injections were conducted in
splitless mode, and a velocity of 1 mL/min helium was used. For the separation of aroma
compounds, a SUPELCOWAX 10 column of 30 m  0.25 mm  0.25 μm (Supelco Inc.,
Bellefonte, PA, USA) was selected. Initial temperature of GC oven was 60 °C after maintaining
for 3 min, which was then up to 240 °C (at 4 °C/min) and held at 240 °C for 20 min. A full-scan
mode with a mass range of m/z 40–200 was used for mass spectrometry. Both non-labeled or
labeled 2PE were identified and quantitatively analyzed based on the non-labeled 2PE authentic
standard.
Expression level analyses of the related genes
The gene expression level was measured using quantitative real time PCR (qRT-PCR)
according to the previous study.30 The internal reference gene was encoding elongation factor1
(EF1).31 The specific primers of the related genes used for qRT-PCR analysis are listed in Table
S1. Detailed steps are provided in supplementary materials.
Analysis of glycosidically conjugated 2-phenylethanol (2PE-Gly) in tea samples
Extraction and analysis of 2PE-Gly in tea samples were referred to the previous studies with
some modifications.30,32 Change in the content of non-labeled and labeled 2PE-Gly was
monitored by GC–MS combined with enzymatic hydrolysis. Detailed steps are provided in
supplementary materials. The relative content of 2PE-Gly was based on the content of the 2PE
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from enzymatic hydrolysis of 2PE-Gly.
Statistical analysis
One-way analysis of variance (ANOVA) or student’s t-test were used to determine statistical
significance performing on SPSS software version 18 (SPSS Inc., Chicago, IL, USA).
Differences among three groups were evaluated using ANOVA, followed by Duncan’s multiple
comparison test. Differences of p ≤ 0.05 were defined as significant. Difference between two
groups was evaluated using a two-tailed Student’s t-test.
RESULTS AND DISCUSSION
Identification of 2PE biosynthesis pathways in tea leaves
To determine the biochemical pathway(s) for the synthesis of 2PE in tea leaves, we first
treated the tea leaves with [2H8]L-Phe. Two types of deuterium-labeled 2PE products, namely,
[2H7]2PE and [2H8]2PE, were detected, suggesting that there was more than one pathway for the
synthesis of 2PE in tea leaves (Figure 2). To confirm the occurrence of proposed pathway II
(Figure 2A) in the tea leaves, we first chemically synthesized [2H5]PPA, which was not
commercially available (Figure 1A). After supplementation of the tea leaves with [2H5]PPA,
[2H5]2PE was detected (Figure 2B). Furthermore, after supplementation of the tea leaves with
non-labeled and labeled PPA, the 2PE and [2H5]2PE contents were both increased (Figures 3A
and 3B). These results indicated that pathway II (Figure 2A) occurred in tea leaves. To confirm
the occurrence of the proposed pathway III (Figure 2A) in tea leaves, we also chemically
synthesized (E/Z)-PAOx, which are not commercially available (Figure 1B). After
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supplementing the tea leaves with (E/Z)-PAOx, the 2PE content was dramatically increased
(Figure 3C). Furthermore, we supplied 4-phenylimidazole, an inhibitor of CYP450 enzymes
potentially related to the pathway derived from L-Phe to PAOx, to combine with [2H8]L-Phe in
the tea leaves. The ratio of [2H8]2PE to [2H7]2PE decreased (Figure 3D), suggesting that the flux
into [2H8]2PE decreased in tea leaves when fed with 4-phenylimidazole.
VPBs are ubiquitous in plant species and rank the second most ubiquitous among all volatile
classifications.33 In most cases, volatile terpenes and volatile fatty acid derivatives are proposed
to be mainly used for defense against herbivores, while VPBs seem to be primarily used for
pollinator attraction.34 In addition to ecological functions, VPBs have abundant value for
economic applications, such as in improving flavor and memory, sedation, and food storage.33,35
Current knowledge regarding VPB biosynthesis in plants has mainly been obtained from
researches on flowers from Petunia, which is a model plant species for studying VPBs because
metabolic background of Petunia is comparatively simple.16 Furthermore, some ornamental
flowers, such as rose flowers, contain abundant VPBs.18,19,22,23 Most VPBs belong to the
shikimate pathway and are generally derived from L-Phe. The most-studied first step is catalytic
conversion into trans-cinnamic acid (CA) from L-Phe by L-phenylalanine ammonia lyase
(PAL).33 Some VPBs are synthesized via non--oxidation, a coenzyme A (CoA) independent
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pathway, or a -oxidation CoA-dependent pathway.16 The -oxidation pathway involves CA
activation through cinnamoyl-CoA ester formation, the formation of
3-hydroxy-3-phenylpropionyl-CoA from CoA ester hydration, and the formation of
3-oxo-3-phenylpropionyl-CoA by hydroxyl group oxidation.16 In general, studies on the
biosynthesis of VPBs have mostly focused on the last biosynthetic steps of VPBs, such as
functional characterization of the involved enzymes and genes. Recently, several studies on VPB
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upstream pathways have led to important discoveries. Firstly,
3-deoxy-D-arabino-heptulosonate-7-phosphate synthase has been proposed to be a critical
enzyme controlling flux via the shikimate pathway based on an expression study on a
3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (bacterial feedback-insensitive) involved
in the shikimate pathway in Arabidopsis.36 Secondly Chorismate mutase 1 was reported to be
related to the formation of VPBs derived from L-Phe in Petunia flower using an RNAi
suppression system.37 Thirdly, the synthesis of L-Phe via arogenate from prephenate under
catalysis by prephenate aminotransferase and arogenate dehydratase is a dominant pathway in
Petunia flowers.38,39 Furthermore, similar to some microorganisms, plants can synthesize L-Phe
from PPA.40 Finally, CoA ligases have been found to play important roles in benzenoid
biosynthesis in Petunia flowers. A close positive correlation was detected between the
expression of petunia cinnamate:CoA ligase (Ph-CNL) and emission of some VPBs, such as
methylbenzoate, phenylethylbenzoate, and benzylbenzoate. In addition, during -oxidative
pathway, formation of cinnamoyl-CoA under the action of peroxidosomal-located Ph-CNL was
the key procedure, and was closely involved in benzoic acid formation from L-Phe.41 However,
direct investigations of VPB biosyntheses in tea plants have been limited.
Among VPBs in plants, 2PE has been widely studied. The biosynthesis of other VPBs, such as
PAld and 2PE, competes with PAL for L-Phe utilization and is not derived from CA. Previous
studies on other plants, such as tomato and rose flowers, found that 2PE was derived from L-Phe
via another VPB compound, PAld, under the action of two enzymes, namely, aromatic
phenylacetaldehyde synthase (PAAS)/amino acid decarboxylase (AADC), and
phenylacetaldehyde reductase (PAR).17,18,42 In tea plants, PAAS/AADC has yet to be
functionally characterized, but a PAR that can catalyze PAld to 2PE has been identified.29
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Although the pathway for 2PE derived from L-Phe via PAld in tea plants is generally accepted,
further in vivo evidence is required (Figure 2). In recent years, another 2PE biosynthesis pathway
has been identified in rose flowers and melon, starting from L-Phe via PPA and PAld under
catalysis by aromatic amino acid aminotransferase (AAAT), phenylpyruvic acid decarboxylase
(PPDC), and PAR.22,23,43 AAAT1 in tea leaves showed L-Phe transaminase functions and convert
L-Phe into PPA using in vitro and in vivo systems.44 Furthermore, based on evidence obtained
from the present study, the 2PE biosynthesis from L-Phe via PPA and PAld was proposed to
occur in tea plants (Figure 2). In 2019, a third pathway for 2PE formation was discovered in
Plumeria, derived from L-Phe via (E/Z)-PAOx and PAld. Furthermore, a cytochrome P450
enzyme PrCYP79D73 was isolated, identified, and functionally characterized to contribute to
2PE production.24 In the present study, based on the feeding experiments of (E/Z)-PAOx and
inhibitor 4-phenylimidazole combined with [2H8]L-Phe (Figures 3C and 3D), the third pathway
from L-Phe via PAOx and PAld was also confirmed in a model system of tea plants (Figure 2A).
Obtaining direct evidence of secondary metabolite synthesis in tea in vivo is difficult because,
to date, it is unavailable using a well-established genetic transformation system. Therefore,
obtaining direct evidence of metabolic networks in plants lacking transformation systems based
on the trace using stable isotope-labeled precursors in a model system is available and
effective.45 In previous studies, investigations into the pathways of secondary metabolites in tea
have been successfully performed using the technique based on stable isotope labeling.7,12,41,46 In
our study, using this method, multiple pathways of 2PE formation in tea leaves were confirmed,
including the pathways from L-Phe via PAld, via PPA and PAld, and via (E/Z)-PAOx and PAld
(Figure 2A).
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Effect of increasing temperature on flux into 2PE pathway from L-Phe via PPA and PAld
To study the effect of increasing temperature on flux into the pathways for 2PE synthesis, we
supplied [2H8]L-Phe to the tea leaves at different temperatures, namely 15, 25, or 35 °C, for 3 d
and 7 d, and then analyzed the ratio of [2H8]2PE to [2H7]2PE. The results showed that the ratio of
[2H8]2PE to [2H7]2PE decreased with increasing temperature (Figure 4). [2H7]2PE was derived
from [2H8]L-Phe via [2H7]PPA and [2H7]PAld (Figure 2A, pathway II), which suggested that
increasing the temperature resulted in increased flux into the pathway of 2PE from L-Phe via
PPA and PAld in tea leaves. To further determine whether this phenomenon specifically
occurred in tea leaves or was commonly present in most plants, investigations into other plants,
including tomato fruit and petunia flowers, have been performed. Non-labeled 2PE, [2H8]2PE,
and [2H7]2PE were also measured, but the content of these compounds did not exhibit a changing
tendency in these plants that was consistent with tea plants (Figures 4, 5, and S3). In our previous
experiments, jasmonic acid, as a marker of wounding stress, in tea leaves that were cultivated in
water remains at low level (nearly 20 ng per 1 g tea leaves), suggesting that cut branches may not
be exposed to stress for several days. In addition, the comparison among different temperatures
was at the same treatment time. Although 2PE contents in tea leaves were slightly higher after 7
d at 25 and 35 °C (Figure 4A), there were significant differences in 2PE content among the
different temperature treatments. From the results (Figures 4A, 5A, 5D, S3A and S3D), it was
found that the contents of 2PE in different plant species showed different change patterns under
the increasing temperatures. Increasing temperatures resulted in an increase in 2PE in tea leaves
(Figure 4A), while it led to a decrease in 2PE in petunia flowers (Figures 5D and S3D).
Furthermore, 2PE showed no significant change in tomato fruit under increasing temperatures
(Figures 5A and S3A). However, the ratio of [2H8]2PE to [2H7]2PE, either in mature or immature
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fruit, and either endogenous 2PE or emitted 2PE, decreased with increasing temperature in these
samples (Figures 5C, 5F, S3C, and S3F), suggesting that such a phenomenon might be common
in most plants containing 2PE, regardless of different plants and tissues.
The expression level of genes, including CsAADC, CsAAAT, CsCYP79D73, CsPPDC, and
CsPAR (being involved in 2PE synthesis derived from L-Phe) in tea leaves under increasing
temperature were also investigated. The expression levels of CsAAAT1 and CsPPDC (being
involved in pathway II) increased, while other two genes, CsAADC and CsCYP79D73 (being
involved in pathway I and III) were not up-regulated under increasing temperature treatment for
7 d (Figure 6). Furthermore, expression level of CsPARs were also enhanced by higher
temperature (Figure 6), which may contribute to increase in 2PE in tea leaves under increasing
temperature. Change in the content of non-labeled and labeled 2PE-Gly was monitored by
GC–MS combined with enzymatic hydrolysis. Relative contents of 2PE-Gly and [2H7]2PE-Gly
also increased under the increasing temperature, while the ratio of [2H8]2PE-Gly to
[2H7]2PE-Gly decreased (Figure 7), which were consistent with the changing tendencies of
non-labeled and labeled 2PE (Figure 4).
2PE is abundant in tea products, such as green tea, oolong tea and black tea. Many studies
have confirmed that 2PE is identified as an important aroma contributor to these tea
products.47–49 On the other hand, 2PE is a taxonomically widespread floral volatile compound
and has been reported to be involved in plant-insect interactions.50 Hence, the increased 2PE may
also play a role in ecological function in tea plants. The suitable growth temperature varies with
plant species (details were shown in materials and methods). In contrast to tea plant, tomato and
petunia are more resistant to low temperature. Therefore, 15 °C was set as lower temperature
treatment for tea leaves, and 10 °C was set as lower temperature treatment for tomato fruits and
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petunia flowers. In addition, the medium and higher temperature were all set at 25 and 35 °C,
respectively. Apart from the temperature, the treatment time was another factor affecting the
different pathways into 2PE. In the present study, the ratio of [2H8]2PE to [2H7]2PE showed
significant difference after different temperature treatment for 2-3 d. In tea leaves, there was the
same changing tendency under increasing temperature for a longer treatment time (7 d) (Figure
4C). However, for tomato fruits and cut petunia flowers, the treatment time could not be too long,
especially at higher temperature. Further studies are need to investigate the change in the
preharvest tomato fruits and petunia flowers. In the present study, the different pathways always
existed in three plant species, including tea, tomato and petunia, regardless of temperature. In tea
leaves, increasing temperature resulted in an increase in [2H7]2PE, thus inducing a decrease in
the ratio of [2H8]2PE to [2H7]2PE. In tomato fruits and petunia flowers, increasing temperature
resulted in a decrease in [2H8]2PE, thus inducing a decrease in the ratio of [2H8]2PE to [2H7]2PE.
Increasing temperature affected the content of [2H7]2PE in tea leaves, and content of [2H8]2PE in
tomato fruit and petunia flowers. It did not terminate the pathway into [2H8]2PE. Therefore, there
was only slight change in ratio of [2H8]2PE to [2H7]2PE under increasing temperature. Usually,
plants have the ability to adapt to their environment, so a certain temperature difference is
needed to make a difference in plant species. Ten degrees of temperature difference in these
plant species may be too low to induce the significant difference (Figures 4C, 5C, 5F, S3C and
S3F), and a greater temperature differences, such as twenty degrees or even twenty-five degrees
were needed.
Under abiotic and biotic stresses, plants have evolutionarily formed a series of diverse
protective mechanisms. For example, to adapt to environmental conditions, plants generally
produce specialized metabolites, such as volatile metabolites.51 In most cases, volatile fatty acid
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derivatives and terpenes are closely related to several types of biotic interactions, while VPBs are
mostly reported to be involved in abiotic interactions.20,51,52 Temperature is one of mostly studied
abiotic stresses. In the last several decades, the average global temperature has increased
significantly, with the rate of increase accelerating. It has been shown that, in the last 1400 years,
the average temperature of the northern hemisphere during the period from 1983 to 2012 might
be the warmest period (30 years).53 Furthermore, in the last four decades, springtime has
advanced at the rate of 2.5 d/decade, while heat wave events and warmer nights have occurred
more frequently.53,54 Global climate change has attracted the attention of researchers regarding
interactions between temperature and plant volatile compounds.54 Many studies have
investigated the effects of increasing temperature on VPB formation in Petunia flowers, but the
changing tendency varied in previous studies. In P. axillaris flowers, augmenting the
environmental temperature to 30 °C for one week could enhance the emission of floral VPBs.55
Nonetheless, a high-temperature growth condition with long-term (one month, 28 °C) negatively
influenced the floral VPBs of P. hybrida.54 In the cut flowers of P. hybrida, a high-temperature
treatment with a shorter time, namely, at 28 °C for 10−12 h, reduced most VPB emissions,
whatever the light conditions are.20 The reduction in VPB emissions from petunia flowers under
a high temperature treatment with long-term at 28 °C for one month was due to the
down-regulation of genes related to the shikimate pathway, and up-regulation of the negative
regulation factor of floral production.54 Increasing the temperature short-term can also affect
VPB emissions under regulation factors and structural genes.20 In response to temperature
changes in different seasons, 2PE formation via two different biosynthetic pathways changed in
rose flowers.23 In addition to the effects of seasons, the evaluated temperature can also activate
the pathway into 2PE from L-Phe via PPA in postharvest cut rose flowers.56 Most studies of
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interactions between temperature and VPBs are reported for plant floral parts, while little is
known about the vegetative parts. Furthermore, plants produce some specialized metabolites
through more than one pathway. Currently, most studies have concentrated on the effects of
stresses on plant volatile metabolite contents, while little is known about the effects of stresses
on flux into their biosynthesis pathways. In tea plants, 2PE was not significantly affected by
insect attacks and light wavelengths, but was significantly enhanced under dark conditions
(shading treatment) owing to the increase in its precursor, L-Phe.6,15,57 In the present study,
increasing the temperature resulted in increased flux into the pathway of 2PE from L-Phe via
PPA and PAld in tea leaves (pathway II, Figures 2A and 4). An increase in expression level of
CsAAAT1 and CsPPDC might be a reason to account for the phenomenon (Figure 6).
Furthermore, this phenomenon might be common in most plants containing 2PE, regardless of
different species and tissues (Figures 5 and S3). It would be interesting to know whether this
plant response had some special biology functions for plants in response to high-temperature
stress. Furthermore, in fresh tea leaves, the PAld content is generally much lower than the 2PE
content, which led to PAld in tea plants receiving little attention. As the biological functions of
both PAld and 2PE in tea plants are presently unknown, it would be interesting to investigate
their roles in tea plants exposed to abiotic stresses.
In the present study, we chemically synthesized precursors, including [2H5]PPA and
(E/Z)-PAOx. [2H8]L-Phe and the as-synthesized precursors were applied to tea leaves to obtain
evidence of 2PE biosynthesis in a model system and its response to increasing high-temperature
stress. Three pathways toward the synthesis of 2PE in tea leaves were elucidated, namely, the
pathways from L-Phe via PAld, via PPA and PAld, and via (E/Z)-PAOx and PAld. Furthermore,
flux into the pathway of 2PE from L-Phe via PPA and PAld was increased by increasing the high
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temperature stress, which might be common in most plants containing 2PE, regardless of
different species and tissues (Figure 8). The results obtained from this study not only provided
evidence of 2PE formation in a model system of tea leaves, which is not a model crop, but also
gave a characteristic example of changes in flux into the biosynthesis pathways of volatile
compounds in plants in response to stresses. The information will provide critical guidance for
improving the aroma quality of tea leaves and other agricultural and food products.
ASSOCIATED CONTENT
Supporting information
Table S1 Primer Used for Quantitative Real Time PCR (qRT-PCR) in the Study.
Figure S1 Identification of [2H5]Phenylpyruvic Acid (A) and (E/Z)-Phenylacetaldoxime (B)
Authentic Standard Based on Nuclear Magnetic Resonance Analysis.
Figure S2 Feeding Methods of Tea Leaves (A), Tomato Fruit (B), and Petunia Flowers (C).
Figure S3 Effect of Increasing Temperature on Endogenous 2-Phenylethanol (2PE) in Mature
Tomato Fruit and Emitted 2PE in Petunia Flowers.
AUTHOR INFORMATION
Corresponding Author
*(Z.Y.) Phone: +86-20-38072989. E-mail: zyyang@scbg.ac.cn.
Author Contributions
# Lanting Zeng and Haibo Tan equally contributed to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
This study was financially supported from the National Key Research and Development
Program of China (2018YFD1000601), the Strategic Leading Science & Technology Programme
(XDA13020604), the Guangdong Natural Science Foundation for Distinguished Young Scholar
(2016A030306039), and the Guangdong Province Higher Vocational Colleges & Schools Pearl
River Scholar (50117G25002).
ABBREVIATIONS
FW, fresh weight; PAld, phenylacetaldehyde; (E/Z)-PAOx, (E/Z)-phenylacetaldoxime; 2PE,
2-phenylethanol; 2PE-Gly, glycosidically conjugated 2-phenylethanol; L-Phe, L-phenylalanine;
PPA, phenylpyruvic acid; VPBs, volatile phenylpropanoids/ benzenoids.
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