Alternative Pathway to the Formation of trans-Cinnamic Acid Derived from l -Phenylalanine in Tea (Camellia sinensis) Plants and Other Plants

Article abstract of DOI:10.1021/acs.jafc.9b07467

trans-Cinnamic acid (CA) is a precursor of many phenylpropanoid compounds, including catechins and aroma compounds, in tea (Camellia sinensis) leaves and is derived from l-phenylalanine (l-Phe) deamination. We have discovered an alternative CA formation pathway from l-Phe via phenylpyruvic acid (PPA) and phenyllactic acid (PAA) in tea leaves through stable isotope-labeled precursor tracing and enzyme reaction evidence. Both PPA reductase genes (CsPPARs) involved in the PPA-to-PAA pathway were isolated from tea leaves and functionally characterized in vitro and in vivo. CsPPAR1 and CsPPAR2 transformed PPA into PAA and were both localized in the leaf cell cytoplasm. Rosa hybrida flowers (economic crop flower), Lycopersicon esculentum Mill. fruits (economic crop fruit), and Arabidopsis thaliana leaves (leaf model plant) also contained this alternative CA formation pathway, suggesting that it occurred in most plants, regardless of different tissues and species. These results improve our understanding of CA biosynthesis in tea plants and other plants.

Full text of DOI:10.1021/acs.jafc.9b07467

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Agricultural and Environmental Chemistry
An alternative pathway to the formation of trans-cinnamic acid derived
from L-phenylalanine in tea (Camellia sinensis) plants and other plants
Lanting Zeng, Xiaoqin Wang, Haibo Tan, Yinyin Liao, Ping Xu, Ming Kang, Fang Dong, and Ziyin Yang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b07467 • Publication Date (Web): 20 Feb 2020
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Page 1 of 38
Journal of Agricultural and Food Chemistry
Title: An alternative pathway to the formation of trans-cinnamic acid derived from
L-phenylalanine in tea (Camellia sinensis) plants and other plants
Authors: Lanting Zeng a, b, Xiaoqin Wang a, Haibo Tan a, Yinyin Liao a, c, Ping Xu d, Ming
*
Kang a, Fang Dong 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 Department of Tea Science, Zhejiang University, No. 388 Yuhangtang Road, Hangzhou
310058, China
e Guangdong Food and Drug Vocational College, No. 321 Longdongbei Road, Tianhe
District, Guangzhou 510520, China
* Corresponding author. Ziyin Yang, Tel: +86-20-38072989, Email address:
zyyang@scbg.ac.cn.
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ABSTRACT
trans-Cinnamic acid (CA) is a precursor of many phenylpropanoid compounds, including
catechins and aroma compounds, in tea (Camellia sinensis) leaves, and is derived from
L-phenylalanine (L-Phe) deamination. We have discovered an alternative CA formation pathway
from L-Phe via phenylpyruvic acid (PPA) and phenyllactic acid (PAA) in tea leaves through
stable isotope-labeled precursor tracing and enzyme reaction evidence. Both PPA reductase
genes (CsPPARs) involved in the PPA-to-PAA pathway were isolated from tea leaves and
functionally characterized in vitro and in vivo. CsPPAR1 and CsPPAR2 transformed PPA into
PAA and were both localized in the leaf cell cytoplasm. Rosa hybrida flowers (economic crop
flower), Lycopersicon esculentum Mill fruits (economic crop fruit), and Arabidopsis thaliana
leaves (leaf model plant) also contained this alternative CA formation pathway, suggesting that it
occurred in most plants, regardless of different tissues and species. These results improve our
understanding of CA biosynthesis in tea plants and other plants.
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KEYWORDS: Tea; Camellia sinensis; trans-Cinnamic acid; L-Phenylalanine; Phenylpyruvic
acid; Phenyllactic acid; Phenylpyruvic acid reductase
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INTRODUCTION
Tea is an aqueous infusion of processed leaves from the plant Camellia sinensis (L.) O.
Kuntze, which is the second most popular beverage globally next to water owing to its
characteristic functions and quality. Many specialized secondary metabolites derived from
L-phenylalanine (L-Phe) contribute to tea function and quality. Catechins, accounting for
12%–24% of dried tea leaves by weight, are considered natural antioxidants with dietary
importance for human health.1,2 Catechins are also responsible for the taste and color of tea,
which affect tea quality.3 Volatile phenylpropanoids/benzenoids (VPBs) are another class of
L-Phe-derived metabolites and the second most ubiquitous volatile class in plants.4 In tea, VPBs
can impact tea quality through contributions to tea aroma and odor.3 Therefore, the metabolism
of L-Phe plays an important role in tea quality and function, and deserves further investigation.
Recently, catechins and some characteristic VPBs have been directly investigated in tea leaves,
containing the biosynthetic pathways involved in their production, enzymes that catalyze these
pathways, genes that encode these enzymes, and the responses to abiotic and biotic stresses on
the formation of these metabolites in tea leaves.5–10 Among these studies, most have focused on
the final biosynthetic steps of catechins and VPBs, such as functional characterization of the
related genes and enzymes. However, upstream pathway information on tea is very limited, with
most reports referring to other plants.
Recently, much research has focused on the upstream pathway of L-Phe-derived metabolites in
various plants, resulting in useful and insightful discoveries, including determination of the role
of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase in the shikimate pathway in
Arabidopsis,11 the participation of Chorismate mutase 1 in VPB formations in Petunia flowers,12
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the synthesis of L-Phe from prephenate in Petunia flowers and from phenylpyruvic acid (PPA) in
plants,13–15 and the role of CoA ligases in benzenoid biosynthesis in Petunia flowers.16
Furthermore, some studies have investigated the synthesis of trans-cinnamic acid (CA) derived
from L-Phe owing to its important roles. In plants, although CA is involved in plant metabolism
and physiology, its biosynthesis remains unclear. The only pathway to CA formation in plants
that is generally accepted is the deamination of L-Phe under the action of phenylalanine
ammonia-lyase (PAL).17 In some microorganisms, such as Propionibacterium freudenreichii,
L-Phe can be transaminated to PPA, which is then further transformed into phenyllactic acid
(PAA).18 Related pathways have also been confirmed in some plant species.6,19 Previous review
has proposed that there are two candidate pathways of CA formation by the dehydration of
α-hydroxy acid or removing the element of ammonia from α-amino acid.20 In addition, it remains
to distinguish between these two possible routes in the previous tracer study.20 This led us to
consider whether an alternative pathway for CA formation from L-Phe via PPA and PAA occurs
in plants, such as tea.
This study aimed to investigate the occurrence of an alternative pathway to CA in tea leaves.
Accordingly, L-[2H8]Phe (stable isotope-labeled precursor) was applied to determine the
occurrence of CA biochemical pathway(s) in tea leaves. The chemical synthesis of [2H6]CA
(stable isotope-labeled compound) was used as an authentic standard for the L-[2H8]Phe-derived
CA product. Furthermore, two phenylpyruvic acid reductase genes (CsPPARs) involved in the
pathway from PPA to PAA were also functionally identified. We also chemically synthesized
[13C6]PAA for tracing in tea leaves to verify the pathway from PAA to CA. Finally, other plants,
including Rosa hybrida flowers (economic crop flower), Lycopersicon esculentum Mill fruits
(economic crop fruit), and Arabidopsis thaliana leaves (leaf model plant), were also investigated
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to confirm whether the identified alternative pathway to CA was specific to tea leaves or
ubiquitous in most plants. This study aimed to discover an alternative pathway for CA formation
from L-Phe in plants, and enhance our understanding of L-Phe metabolism and CA biosynthesis.
Chemical reagents
CA was obtained from Wako Pure Chemical Industries Ltd., Japan. L-[2H8]Phe, L-[13C6]Phe,
[2H6]aldehyde and [2H7]CA were obtained from Cambridge Isotope Laboratories Inc.,
Cambridge, MA, USA. N-Methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) was obtained
from Regis Chemical Co., Morton Grove, Illinois, USA. [2H6]CA (purity, ≥95%) and [13C6]PAA
(purity, ≥95%) were synthesized in our lab.
Plant materials and growth conditions
In this study, the tea sample was obtained from cultivar ‘Jinxuan’ grown in Yingde town,
Guangdong province, China. Tea cultivar ‘Jinxuan’ is mainly grown in southern China. R.
hybrida ‘Tineke’ flower and L. esculentum Mill fruit were purchased from the market. A.
thaliana was grown under set conditions (22±2 °C, 70±2% relative humidity, photoperiod of 8 h
dark/16 h light).
Plants treatment with L-[2H8]Phe
Plant treatments with L-[2H8]Phe were performed as reported in a previous study.21,22 C.
sinensis ‘Jinxuan’ branches with one bud and three leaves collected in March 2019 were
cultivated in two different solutions (Figure S1A), namely, H2O and 12 mM L-[2H8]Phe. In the
previous study,21 the times for feeding L-[2H8]Phe into ‘Jinxuan’ branches were set on 3 and 7 d,
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and the labelled L-Phe-derived metabolites could be detected on 3 d. Therefore, in the study, the
feeding time was only set on 3 d for C. sinensis. R. hybrida ‘Tineke’ flowers were purchased
from the market in March 2019. Petals were separated from R. hybrida ‘Tineke’ flower, and the
bases were immersed in two different solutions (Figure S1B), namely, H2O and 12 mM
L-[2H8]Phe. Former study has confirmed that labelled L-Phe-derived metabolites could be
detected on 1.5 d in R. hybrida ‘Tineke’ flower when fed with L-[2H8]Phe.22 To obtain more
labelled L-Phe-derived metabolites, the feeding time was set on 2 d for R. hybrida ‘Tineke’. L.
esculentum Mill fruit was purchased on the market in March 2019. The fruit of L. esculentum
Mill was treated with two different solutions (Figure S1C), namely, H2O and 12 mM L-[2H8]Phe.
Every fruit was treated with solutions (300 L) every 12 h (total of 14.4 mol for L-[2H8]Phe).
Former study has confirmed that labelled L-Phe-derived metabolites could be detected on 1 d
when fed with L-[2H8]Phe in L. esculentum Mill fruit.21 After 1 d-feeding, the tomato fruit was in
a good state and could be kept for longer periods. In addition, to obtain more labelled
L-Phe-derived metabolites, the feeding time was set on 2 d for L. esculentum Mill. A. thaliana
(4–5 weeks old) cultivated in the laboratory was used in the stable isotope tracing experiment. A.
thaliana leaves were treated with two different solutions (Figure S1D), namely, H2O and 12 mM
L-[2H8]Phe. Former study has confirmed that labelled Phe-derived metabolites could be detected
on 18 h when fed with L-[2H8]Phe in A. thaliana leaves.23 After 18 h-feeding, the A. thaliana
plants were in a good state and could be kept for longer period. In addition, to obtain more
labelled L-Phe-derived metabolites, the feeding time was set on 1 d for A. thaliana. Feeding time
of 3 days was too long for cut R. hybrida ‘Tineke’ flower, postharvest L. esculentum Mill fruit
and soil-free A. thaliana leaves. Therefore, the sample treatments were conducted under a set
conditions of 70% humidity, 25 °C, and a photoperiod of 8 h dark/16 h light for several days (3
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days for C. sinensis, 2 days for R. hybrida ‘Tineke’ and L. esculentum Mill, and 1 day for A.
thaliana). L-[2H8]Phe entered the tea leaves and R. hybrida ‘Tineke’ flower via the cut stem, and
entered A. thaliana leaves via root driven by the transpiration stream.
Chemical synthesis of [2H6]CA
The synthesis of [2H6]CA was performed according to a previously reported method.24
[2H6]Aldehyde (560 mg, 5.0 mmol) and malonic acid (520 mg, 5.0 mmol) were added to
pyridine solution (6 mL) followed by a catalytic amount of morpholine (44 mg, 44 L, 0.5
mmol). The reaction was conducted by slowly heating to 110 °C and maintaining this
temperature for 6 h. Thin-layer chromatography (TLC) was used to monitor the reaction. The
reaction was quenched into water (30 mL) when cooled to room temperature. After filtration, the
precipitated solid was washed with water (5 mL) two times. The product was then dried by
suction filtration using a Buchner funnel for 15–60 min. The resulting solid product was dried in
an oven at 50–60 °C overnight. The yield of [2H6]CA was 85% (654 mg).
As-obtained [2H6]CA was a white solid with more than 95% purity. The purity of [2H6]CA
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was determined based on its NMR spectra (Figure S2A). H NMR (500 MHz, CD3OD,
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ppm) H 6.49 (s, 1H); 13C NMR (125 MHz, CD3COCD3, ppm) C 117.2, 127.8, 128.0, 128.1,
128.3, 128.5, 128.7, 130.1, 130.3, 130.4, 133.8, 146.6, 146.7, 146.9, 172.5; HRESIMS (m/z)
calcd. for C31H35O6 [M–H] 503.2439, found 503.2436.
Determination of labeled CA
Identification and analysis of [2H7]CA and [2H6]CA were using gas chromatography mass
spectrometer (GC–MS) combined with MSTFA derivatization according to the previous study
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with some modification.25 Finely powdered samples (500 mg for C. sinensis leaves and A.
thaliana leaves, fresh weight) were extracted in cold methanol (4 mL) by ultrasonic extraction
for 20 min. The extract was purified in chloroform (4 mL) and water (1.6 mL). The labelled
metabolites of L. esculentum Mill Fruit and R. hybrida ‘Tineke’ flower (2 g, fresh weight) were
extracted using 8 mL of cold methanol, and partially purified using 8 mL of cold chloroform and
3.2 mL of cold water with the above extraction method. After the extraction and preliminary
purification, all samples were subjected to centrifugation (10,000 g, 4 °C, 10 min), and the
supernatants (4 mL for C. sinensis leaves and A. thaliana leaves, and 9 mL for L. esculentum
Mill Fruit and R. hybrida ‘Tineke’ flower) were collected. The supernatants (methanol) were
dried under gaseous N2, and the resulting solution (only water) was diluted to 2 mL using water.
The extract in water was added with 48 μL hydrochloric acid (0.25 M) and extracted twice with
ethyl acetate (2 mL). The ethyl acetate phase was collected and dried under gaseous N2. The dry
power was derivatized with MSTFA (70 μL) at 37 °C for 60 min and then centrifuged. The
MSTFA derivates (1 μL) were then subjected to gas chromatography mass spectrometer
(GC–MS) analysis carried on a GC–MS QP2010 SE (Shimadzu Corporation, Kyoto, Japan)
equipped with GCMS Solution software (Ver. 2.72. Samples were injected into the injection port
of GC under 230 °C temperature condition for 1 min in splitless mode. Labelled metabolites
were separated on an HP-5 column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies,
California, USA). As the carrier gas, helium was in a velocity of 1 mL/min. The temperature of
GC oven was initially set at 100 °C for 5 min, ramped up to 300 °C at a rate of 10 °C/min, and
then kept at 300 °C for 15 min. Mass spectrometry was operated in a full scan mode (mass range,
m/z 40−350), and a selected ion mode (m/z 138, 212 and 227 for [2H7]CA; m/z 137, 211 and 226
for [2H6]CA).
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Chemical synthesis of [13C6]PAA
[13C6]PAA was prepared according to a previously reported method.26 L-[13C6]Phe (171 mg,
1.0 mmol) was dissolved using water (3.0 mL), and NaNO2 (412 mg, 6.0 mmol) was added in
one portion, followed by the addition of concentrated H2SO4 (105 L) at 0 °C. The mixture was
then stirred under a N2 atmosphere at room temperature for 12 h. The reaction solution was
extracted using EtOAc (5 mL) five times. The obtained EtOAc phases were combined, dried by
anhydrous Na2SO4, and filtered through a Buchner funnel. Solvent was removed from the
resulting solution by rotary evaporation to give the product as a slightly yellow solid. The
product was further washed with hexane to give pure product [13C6]PAA in 78% yield (129 mg,
0.78 mmol).
As-obtained [13C6]PAA was a white solid with more than 95% purity. The purity of
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[13C6]PAA was determined based on its NMR spectra (Figure S2B). H NMR (500 MHz,
CD3OD, ppm) H 2.93 (m, 1H), 3.14 (m, 1H), 4.41 (m, 1H), 7.05–7.47 (m, 5H); HRESIMS (m/z)
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calcd. for C31H35O6 [M–H] 503.2439, found 503.2436.
Recombinant expression of CsPPARs and their enzyme assay
Recombinant expression of CsPPARs was performed according to previous studies.6,22 PCR
was used to obtain the full length open reading frames (ORFs) of CsPPAR1 and CsPPAR2. The
primers are shown in Table S1. Details of CsPPARs recombinant expression are provided in the
supporting information. After recombinant expression, the Escherichia coli-expressed proteins
were subjected to SDS-PAGE and western-blot analysis (the related description also provided in
supporting information). The identified recombinant proteins were used in the enzyme assay and
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substrate selectivity analysis according to a previously reported method.19 The reaction mixture
(500 L) contained 2 mM PAA or p-hydroxyphenylpyruvic acid (p-HPPA) as substrate, 2 mM
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NADPH in potassium phosphate (pH 8.0, 50 mM), and semi-purified protein (25 g). Empty
vector (pET32a) enzyme was used as a negative control. As PPA and p-HPPA were almost
water-insoluble, methanol was added to achieve dissolution, resulting in a reaction mixture
containing 1% methanol. The temperature and time of reaction was 30 °C and 2 h, respectively.
The reaction solution (500 L) was mixed using 0.5 M hydrochloric acid (20 L). The reaction
mixture was then extracted twice with ethyl acetate (1 mL). The extract was dried by blowing
with nitrogen, and the residue was redissolved in acetonitrile (200 L). After filtering through a
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0.22-m membrane, the catalytic products (100-fold diluted for CsPPAR2) were analyzed by
UPLC–QTOF MS (ACQUITY UPLC I-Class/Xevo G2-XS QTOF, Waters Corp., Milford, MA,
USA). Compound identification was conducted using the retention time (tR) and mass data (PAA,
tR = 3.41 min, m/z 165.0552 [M–H]; 4-hydroxyphenyllactic acid, tR = 2.02 min, m/z 181.0501
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[M–H]). Details of UPLC–QTOF MS analysis are provided in our previous study and the
supporting information.6
Identification of function of CsPPAR in Nicotiana benthamiana overexpression lines
Analysis of CsPPAR activity in N. benthamiana overexpression lines was conducted
according to previous studies.6,22 The full-length ORFs were subcloned into pCAMBIA3300
vector
to
obtain
expression
constructs
(pCAMBIA3300-CsPPAR1
and
pCAMBIA3300-CsPPAR2) using the primers listed in Table S1. Details of the vector
constructions and transformation of recombinant vector into N. benthamiana leaves are provided
in the supporting information. After 5 days of infiltration, the tobacco leaves were plucked and
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each leaf was placed in a 1.5-mL tube containing 3 mM sodium phenylpyruvate dissolved in
distilled water (1 mL), and water alone as control. All samples were subjected to an 8 h dark/16 h
light photoperiod at 25 °C in an incubator. After 24 h, 500 L of the phenylpyruvate solution
was absorbed by the tobacco leaf in each treatment, and the leaves were harvested and stored at
80 °C for metabolite analysis. As the PAA content in N. benthamiana overexpression lines was
below the detection limit of UPLC−QTOF MS, GC–MS analysis combined with MSTFA
derivatization was used to investigate changes in the PAA content in N. benthamiana leaves after
transient overexpression of CsPPARs, as shown in the supporting information.
Subcellular localization of CsPPARs in transient transgenic leaves of N. benthamiama
Leaves of CsPPARs-overexpressed N. benthamiama were cut into pieces (nearly 1-2 cm2), and
observed under confocal laser microscope (Zeiss LSM 510 META, Carl Zeiss, Jena, Germany).
For GFP fluorescence observation, the images of confocal microscope were taken with a 40
water objective under 488 nm excitation wavelength.
Subcellular localization of CsPPARs in protoplasts of Arabidopsis leaves
To investigate the subcellular localization of CsPPARs, the full-length ORFs were subcloned
into pSAT6-EYFP-N1 vector to obtain expression constructs (pSAT6-CsPPAR1 and
pSAT6-CsPPAR2) using the primers listed in Table S1. The protoplasts from Arabidopsis leaves
were prepared according to a previous report.27,28 Nearly 3–4 weeks old A. thaliana plants were
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applied to protoplast isolation. The lower epidermal surface cell layer was peeled away, and the
peeled leaves were placed on a Petri dish containing the enzyme solution (total volume, 10 mL;
comprising 20 mM 2-(N-morpholino)ethanesulfonic acid (MES, pH 5.7), 0.3% macerozyme R10,
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20 mM KCl, 1.5% cellulase R10, 0.4 M mannitol, and 10 mM CaCl2). After allowing to stand
for 1 h, protoplasts were released into the solution. According to a previously reported method,28
the constructed expression vectors were introduced into protoplast cells from Arabidopsis. The
protoplasts were centrifuged (100 g, 2 min) and then washed twice with cold W5 solution (2
mM MES (pH 5.7) comprising 154 mM NaCl, 5 mM KCl and 125 mM CaCl2). Finally, the
protoplasts were resuspended in an MMG solution (4 mM MES (pH 5.7) comprising 0.4 M
mannitol and 15 mM MgCl2). pSAT6-CsPPARs plasmid DNA (10 L, 10–20 g of plasmid
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DNA) and polyethylene glycol 4000/Ca (PEG/Ca) solution (110 L) were added to the
protoplasts (100 L, equivalent to 2104 protoplasts). The mixture was incubated at room
temperature for 15 min and then diluted with W5 solution (400–440 L). The diluted solution
was centrifuged at 100 g for 2 min. The resulting pelleted protoplasts were re-suspended in W5
solution (100 L) and incubated at 23 °C for 12–16 h. A Zeiss LSM 510 META confocal laser
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microscope was used to observe the protoplasts using a 40 water objective in multitrack
channel mode. The excitation wavelengths and emission filters were 488 nm/band-pass 505–530
nm for yellow fluorescent protein (YFP), and 488 nm/band-pass 650–710 nm for chloroplast
autofluorescence. LSM 510 version 4.2 software (Zeiss) was used to perform image processing.
Feeding experiment using [13C6]PAA in tea leaves and determination of [13C6]CA
To confirm the role of PAA in CA synthesis in tea leaves, the tea leaves were supplemented
with [13C6]PAA. C. sinensis ‘Jinxuan’ branches collected in May 2019 were cultivated in two
different solutions (Figure S1A), namely, H2O (control) and 6 mM [13C6]PAA ([13C6]PAA
treatment). The samples were treated under set conditions of 70% humidity, an 8 h dark/16 h
light photoperiod, and 25 °C for 2 days. The method used for [13C6]CA determination was the
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same as that used for [2H6]CA identification.
Statistical analysis
One-way analysis of variance (ANOVA) was applied to determine the differences among
three groups and a two-tailed Student’s t-test was used to determine the differences between two
groups.
RESULTS AND DISCUSSION
There are two pathways leading from L-Phe to CA in tea leaves
The deamination of L-Phe under action of PAL (Figure 1, pathway I) is widely considered the
only pathway to CA formation in plants. To investigate whether an alternative pathway for CA
formation from L-Phe occurs in plants (Figure 1, pathway II), tea leaves, were supplemented
with L-[2H8]Phe.1,29 First, we obtained authentic standards of [2H6]CA (Figure 2A) through
chemical synthesis and [2H7]CA commercially. Using these authentic standards, both [2H6]CA
and [2H7]CA were identified in tea leaves supplemented with L-[2H8]Phe (Figures 2B and 2C).
This suggested that an alternative pathway for CA formation from L-Phe occurred in tea leaves.
Relative contribution of two pathways leading to CA have been calculated by [2H6] and [2H7]
concentrations based on the peak areas of their MSTFA-derivates, and the ratio of [2H6]-pathway
to [2H7]-pathway is nearly 1.32. Previous studies have indicated that another biosynthetic
pathway for CA from L-Phe exists, proceeding via PPA and PAA.18,20 The present study showed
that this alternative pathway to CA from L-Phe (Figure 1, pathway II) indeed occurred not only
in microorganisms, but also in some plants, such as tea leaves. Stable isotope-labeled precursor
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L-[2H8]Phe was used to trace the pathway in tea leaves and investigate CA products with
different numbers and positions of 2H, to propose the potential multiple pathways (Figure 1).
Using L-[2H8]Phe tracing in plants, multiple pathways from L-Phe to its downstream volatile
products, such as 2-phenylethanol, have been proposed in tea leaves, rose flowers, petunia
flowers, and tomato fruits.21,22,30–32 Three biosynthetic pathways have been discovered to
2-phenylethanol from L-Phe in plants.21 Furthermore, using L-[2H8]Phe tracing in flowers of tea
plants, two potential pathways from L-Phe to its downstream volatile products, such as
acetophenone and 1-phenylethanol, have been proposed.25 Furthermore, based on L-[2H8]Phe
tracing investigations in plants, the formation of acetophenone from L-Phe in plants other than
flowers of tea plants was small, resulting in little accumulation of 1-phenylethanol in these other
plants.23,33 In this study, using L-[2H8]Phe tracing in tea leaves, an alternative pathway for CA
formation from L-Phe was identified (Figure 2). As a successful transgenic system has not be
established for tea plants, stable isotope-labeled precursor L-[2H8]Phe tracing in tea plants might
be a suitable approach to discovering unknown L-Phe-derived metabolic pathways through in
vivo evidence.
CsPPAR1 can convert PPA to PAA, which is a precursor of CA in tea leaves
According to the position of 2H substituents in [2H6]CA, we proposed that [2H7]PPA and
[2H7]PAA were the key intermediate metabolites in the pathway from L-[2H8]Phe to [2H6]CA
(Figure 1, pathway II). The pathway to PPA from L-Phe has been validated and the involved
enzyme, AAAT, has been functionally identified in tea leaves and other plants, such as rose
flowers and melon.6,22,30,34 Therefore, we further investigated the pathway from PPA to PAA.
Two enzymes (PPA reductases, PPARs) were functionally identified in tea leaves. A comparison
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between homologous sequences of PPAR in tea leaves and a reported AbPPAR (which has been
validated to be involved in the pathway from PPA to PAA) from Atropa belladonna was
performed.19 By searching against the Transcriptome Shotgun Assembly (TSA) database,
CsPPAR1 and CsPPAR2 were found, showing 52.48% and 43.15% similarity with AbPPAR,
respectively (Figure S3). The CsPPAR1 and CsPPAR2 recombinant proteins produced in E. coli
(Figures 3A and 3B) were able to transform PPA into PAA in vitro, while CsPPAR2 preferred
p-HPPA as substrate compared with PPA (Figure 3C). Functional identification in N.
benthamiana plants showed that CsPPAR1 and CsPPAR2 both converted PPA into PAA in
plants (Figure 4). CsPPAR1 and CsPPAR2 were located in the cytoplasm using N. benthamiana
leaf system (Figure 5A). In addition, the subcellular location of CsPPARs was used to confirm
once again based on co-localization of maker protein in the A. thaliana leaf protoplast system,
and the result was the same with the one obtained using N. benthamiana leaf system (Figure 5B).
To further confirm the involvement of PAA in CA formation, [13C6]PAA authentic standard was
chemically synthesized (Figure 6A) and supplied to tea leaves. More [13C6]CA was detected in
[13C6]PAA treatment leaves compared with the control (Figure 6B), suggesting that PAA was
converted into CA in tea leaves. Overall, these results indicated that an alternative pathway to
CA derived from L-Phe via PPA and PAA occurred in tea leaves.
The pathway from L-Phe to PPA has been confirmed in tea leaves, melon, rose flowers,
petunia flowers, and tomato fruits when studying 2-phenylethanol pathways in these plants, as
the pathway from L-Phe to PPA is the alternative upstream pathway of
2-phenylethanol.21,22,30–32,34 Furthermore, the pathway involving enzyme AAAT was identified
and functionally characterized in melon, rose flowers, and tea leaves.6,22,30,34 In contrast to the
pathway from L-Phe to PPA, very little information is available for the pathway from PPA to
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PAA. We chemically synthesized [2H7]PPA, supplied it to the tea leaves, and attempted to detect
the formation of product [2H7]PAA in tea leaves. As PPA is unstable, the synthesized [2H7]PPA
compound contained [2H7]PAA. Furthermore, [2H7]PPA readily autotransformed into [2H7]PAA,
even in sodium bicarbonate buffer (pH 8). Therefore, [2H7]PPA tracing in tea leaves failed to
elucidate the pathway from PPA to PAA in tea leaves. Until now, the PPARs involved in the
pathway from PPA to PAA have been characterized in Wickerhamia fluorescens TK1,
Lactobacillus sp. CGMCC9967 and Atropa belladonna.19,35,36 In Atropa belladonna, AbPPAR is
the key upstream enzyme determining the biosynthesis of tropane alkaloids that are active
compounds, because suppression of AbPPAR disrupted tropane alkaloid biosynthesis.19
Biochemically, PPAR may catalyze the similar type of reaction to hydroxyphenylpyruvate
reductases (HPPR). Because PPAR has a hydroxyl acid dehydrogenase domain, which also
exists in formate/glycerate dehydrogenase like family.19 HPPR belongs to this family and is able
to catalyze p-HPPA into p-hydroxyphenyllactic acid (p-HPLA). In substrate selectivity analysis,
p-HPPA was used as a substrate to determine the function of E. coli-expressed AbPPAR in
vitro,19 which was also carried out in the functional characterization of CsPPARs. In the present
study, CsPPARs from tea leaves were also shown to catalyze the reduction of PPA to PAA both
in vitro (E. coli system) and in vivo (N. benthamiana overexpression system) (Figures 3 and 4).
These results suggested that PPAR was present not only in microorganisms, but also in some
plants. As a successful transgenic system has yet to be established for tea plants, overexpression
or RNAi of CsPPARs in tea leaves cannot be performed at present. In future, direct in vivo
evidence of the involvement of PPAR in CA biosynthesis in tea plants needs to be obtained.
Compared with the pathway from PPA to PAA, almost no confirmed information on the pathway
from PPA to CA is available in plants. In the present study, [13C6]PAA was investigated to
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determine whether it could be transformed into [13C6]CA in tea leaves. Similar to PPA, PAA was
unstable and the [13C6]PAA standard contained [13C6]CA (Figure 6B). However, after
supplementing [13C6]PAA into tea leaves, the [13C6]CA content significantly increased compared
with that automatically derived from the [13C6]PAA standard (Figure 6B). This suggested that
PAA was transformed into CA in tea leaves. In-depth studies on the enzyme(s) participating in
the pathway are needed to obtain further evidence of the pathway from PAA to CA in tea leaves.
The research on the key enzymes involved in the pathway from PAA to CA is of significance.
However, there are two huge challenges in investigating the unknown key enzymes involved in
this pathway. At present, there is no available reference information on this pathway in other
organisms, even microorganism. Therefore, it was impossible to obtain the key genes by blasting
analysis. A classical purification approach is needed to identify a new enzyme. In this approach,
the unknown enzyme is obtained using biochemical chromatography, and the sequence
information of desired protein is applied to isolate the gene from the cDNA of plant.37 However,
the method is time-consuming and of uncertainty, and the desired enzyme may finally not be
obtained. In addition, there is no stable genetic transformation system in tea plants, so it is
impossible to confirm the real function of new enzymes in vivo. The related research is of
interest and significance, and deserves to be carried out in the future study.
An alternative pathway leading from L-Phe to CA also occurs in other plants
To further determine whether the alternative pathway to CA was present specifically in tea
leaves or ubiquitous among most plants, other plants, including R. hybrida flowers (economic
crop flower), L. esculentum Mill fruits (economic crop fruit), and A. thaliana leaves (leaf model
plant), were investigated. Similar to tea leaves, these selected plants contained [2H6]CA and
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[2H7]CA when supplied with L-[2H8]Phe (Figure 7), suggesting that the alternative pathway to
CA might occur in most plants, regardless of different tissues and species.
Phenylpropanoid compounds occur widely in the vegetable kingdom and have various
functions. Structural compositions, such as suberin, lignin, and other cell-wall-associated
phenolics, serve as physical barriers to provide plant protection against invasion from herbivores
and microbes. Phenylpropanoid/benzenoid volatiles and pigments (flavonoid and anthocyanin)
contribute to the aroma and color of fruits and flowers, and make sure pollination and seed
dispersal. Some defense chemicals, including condensed tannins, lignans, flavonoids,
isoflavonoids, and some simple phenolic compounds, possess antifeedant and antimicrobial
functions. Flavonoids and other phenolics act as UV protectants by absorbing UV light that can
damage DNA. Some other substances, such as isoflavonoids and salicylic acid (a ubiquitous
plant hormone), play important roles in signaling.13,38–41 The deamination of L-Phe to give CA
under the action of PAL is the first step in the phenylpropanoid metabolic pathway and acts as a
critical regulating point between primary and secondary metabolism.42 Therefore, CA serves as a
precursor of many plant-derived metabolites, and has key functions in regulating growth,
development, reproduction, and environmental responses of plants.42
In this study, we have provided the first chemical and biochemical evidence of CA formation
from L-Phe via PPA and PAA in tea leaves, which is leaves of economic crop, but also
confirmed that the alternative pathway was ubiquitous in most plants regardless of tissues,
including leaves of model crop, fruit of economic crop, and flower of economic crop (Figure 8).
Furthermore, our findings provide in-depth information on the biosynthetic pathway of CA and
its regulation in plant species to help guide the rational engineering of biosynthetic pathways to
CA and many phenolic compounds. Further detailed genetic analyses of the enzymes/genes
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participating in the pathway from PAA to CA will address the biological significance of the
alternative pathway to CA formation in plants.
ASSOCIATED CONTENT
Supporting information
Table S1 Primers Used in the Study.
Figure S1 Feeding Methods of Tea Leaves (A), Rose Flowers (B), Tomato Fruit (C), and
Arabidopsis Leaf (D).
Figure S2 Identification of [2H6]trans-Cinnamic Acid (A) and [13C6]Phenyllactic Acid (B)
Authentic Standard Based on NMR Analysis.
Figure S3 Amino Acid Sequence Alignment of Phenylpyruvic Acid Reductase.
AUTHOR INFORMATION
Corresponding Author
*(Z.Y.) Phone: +86-20-38072989. E-mail: zyyang@scbg.ac.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This study was financially supported from the Basic Frontier Science Research Program of
Chinese Academy of Sciences (ZDBS-LY-SM032), the National Key Research and
Development Program of China (2018YFD1000601), the Strategic Leading Science &
Technology Programme (XDA13020604), the Guangdong Natural Science Foundation for
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Distinguished Young Scholar (2016A030306039), and the Guangdong Province Higher
Vocational Colleges & Schools Pearl River Scholar (50117G25002).
ABBREVIATIONS
CA, trans-cinnamic acid; PAA, phenyllactic acid; L-Phe, L-phenylalanine; PPA,
phenylpyruvic acid; PPAR, phenylpyruvic acid reductase.
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Figure Caption
Figure 1 Proposed biosynthetic pathways into trans-cinnamic acid (CA) with isotope.
PAA, phenyllactic acid; L-Phe, L-phenylalanine; PPA, phenylpyruvic acid.
Figure 2 Determination of labeled trans-cinnamic acid (CA) in tea leaves fed with
L-[2H8]phenylalanine (Phe).
(A) Schematic presentation of synthesis of [2H6]CA. (B) Identification of [2H7]CA and
[2H6]CA standards. (C) GC–MS chromatography of [2H7]CA and [2H6]CA in tea leaves fed with
L-[2H8]Phe.
Figure 3 Functional characterization of phenylpyruvic acid reductases (CsPPARs) in vitro.
(A) SDS-PAGE analysis of CsPPARs expressed in Escherichia coli. (B) Western blot analysis
of CsPPARs expressed in E. coli. (C) Enzymatic activity analysis and substrate selectively of
CsPPARs expressed in E. coli. N.D., not detected. Data are expressed as mean ± standard
deviation (SD) (n = 3). Means with different letters are significantly different from each other (p
≤ 0.05).
Figure 4 Functional characterization of phenylpyruvic acid reductases (CsPPARs) in vivo.
(A) Enzymatic activity analysis of CsPPARs transiently overexpressed in Nicotiana
benthamiana leaves. Vector, empty vector GV3101 overexpressed in N. benthamiana; CsPPAR1,
vector GV3101/CsPPAR1 overexpressed in N. benthamiana; CsPPAR2, vector
GV3101/CsPPAR2 overexpressed in N. benthamiana. FW, fresh weight. Data are expressed as
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mean ± standard deviation (SD) (n = 6). Means with different letters are significantly different
from each other (p ≤ 0.05). (B) Characteristic ions of product from phenyllactic acid (PAA)
derivatized by MSTFA. (C) GC–MS chromatography of PAA in N. benthamiana leaves after
CsPPARs overexpression.
Figure 5 Subcellular localization analyses of phenylpyruvic acid reductases (CsPPARs).
(A) Subcellular localization analyses of CsPPARs-GFP. The CsPPARs-GFP fusion proteins
were transiently expressed in Nicotiana benthamiana leaves. Green fluorescence indicated
CsPPARs-GFP fusion protein. Red showed the chloroplast auto-fluorescence, respectively.
Merged panel shows overlay of gren and red fluorescence images. (B) Subcellular localization
analyses of CsPPARs-YFP. The CsPPARs-YFP fusion proteins were transiently expressed in
Arabidopsis protoplasts. Yellow fluorescence indicated CsPPARs-YFP fusion protein. Red
showed the chloroplast auto-fluorescence, respectively. Merged panel shows overlay of yellow
and red fluorescence images.
Figure 6 Identification of the biosynthetic pathway into trans-cinnamic acid (CA) from
phenyllactic acid (PAA) in tea leaves.
(A) Schematic presentation of synthesis of [13C6]PAA. L-Phe, L-phenylalanine. (B) Change in
content of [13C6]CA in tea leaves fed with [13C6]PAA. Control, sum of [13C6]CA content from tea
leaves and [13C6]PAA standard; [13C6]PAA Treatment, [13C6]CA content from tea leaves. Data
are expressed as mean ± standard deviation (SD) (n = 4). **p ≤ 0.01, comparison between
control and treatment. FW, fresh weight.
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Journal of Agricultural and Food Chemistry
Figure 7 GC–MS chromatography of [2H7]CA and [2H6]CA in plants fed with
L-[2H8]phenylalanine (Phe).
R. hybrida, Rosa hybrida; L. esculentum Mill, Lycopersivon esculentum Mill; A. thaliana,
Arabidopsis thaliana.
Figure 8 An alternative pathway for the formation of trans-cinnamic acid (CA) derived from
L-phenylalanine (Phe) in plants.
AAAT, aromatic amino acid aminotransferase; PAA, phenyllactic acid; PAL, phenylalanine
ammonia-lyase; PPA, phenylpyruvic acid; PPAR, phenylpyruvic acid reductases.
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