Studies on the Biochemical Formation Pathway of the Amino Acid l -Theanine in Tea (Camellia sinensis) and Other Plants

Article abstract of DOI:10.1021/acs.jafc.7b02437

Tea (Camellia sinensis) is the most widely consumed beverage aside from water. The flavor of tea is conferred by certain metabolites, especially l-theanine, in C. sinensis. To determine why more l-theanine accumulates in C. sinensis than in other plants, we compare l-theanine contents between C. sinensis and other plant species (Camellia nitidissima, Camellia japonica, Zea mays, Arabidopsis thaliana, and Solanum lycopersicum) and use a stable isotope labeling approach to elucidate its biosynthetic route. We quantify relevant intermediates and metabolites by mass spectrometry. l-Glutamic acid, a precursor of l-theanine, is present in most plants, while ethylamine, another precursor of l-theanine, specifically accumulates in Camellia species, especially C. sinensis. Most plants contain the enzyme/gene catalyzing the conversion of ethylamine and l-glutamic acid to l-theanine. After supplementation with [²H₅]ethylamine, all the plants produce [²H₅]l-theanine, which suggests that ethylamine availability is the reason for the difference in l-theanine accumulation between C. sinensis and other plants.

Full text of DOI:10.1021/acs.jafc.7b02437

Article
pubs.acs.org/JAFC
Studies on the Biochemical Formation Pathway of the Amino Acid
L‑Theanine in Tea (Camellia sinensis) and Other Plants
Sihua Cheng,†,‡,⊥ Xiumin Fu,†,⊥ Xiaoqin Wang,†,‡ Yinyin Liao,† Lanting Zeng,†,‡ Fang Dong,§
and Ziyin Yang*,†,‡
†Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement & Guangdong Provincial Key
Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Xingke Road 723, Tianhe District,
Guangzhou 510650, China
‡University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China
§Guangdong Food and Drug Vocational College, Longdongbei Road 321, Tianhe District, Guangzhou 510520, China
S
* Supporting Information
ABSTRACT: Tea (Camellia sinensis) is the most widely consumed beverage aside from water. The flavor of tea is conferred by
certain metabolites, especially L-theanine, in C. sinensis. To determine why more L-theanine accumulates in C. sinensis than in
other plants, we compare L-theanine contents between C. sinensis and other plant species (Camellia nitidissima, Camellia japonica,
Zea mays, Arabidopsis thaliana, and Solanum lycopersicum) and use a stable isotope labeling approach to elucidate its biosynthetic
route. We quantify relevant intermediates and metabolites by mass spectrometry. L-Glutamic acid, a precursor of L-theanine, is
present in most plants, while ethylamine, another precursor of L-theanine, specifically accumulates in Camellia species, especially
C. sinensis. Most plants contain the enzyme/gene catalyzing the conversion of ethylamine and L-glutamic acid to L-theanine.
After supplementation with [2H5]ethylamine, all the plants produce [2H5]L-theanine, which suggests that ethylamine availability
is the reason for the difference in L-theanine accumulation between C. sinensis and other plants.
KEYWORDS: alanine, amino acid, Camellia sinensis, ethylamine, stable isotope labeling, tea, theanine
via the conversion of toxic ammonia into the nontoxic amino
acid form.14−16 As L-theanine accounts for more than 50% of
total amino acids of C. sinensis plants, it fixes a similar amount
of nitrogen as do Glu and Gln combined.17
INTRODUCTION
■
Next to water, tea (Camellia sinensis) is the most popular
beverage worldwide. Numerous studies have validated the
health benefits of tea, and consumers appreciate the unique
taste and aroma.1 In contrast to other plants, C. sinensis pro-
duces specialized metabolites that determine the functions and
quality attributes of tea. It is an interesting and important
scientific question to investigate why these specialized
metabolites accumulate in C. sinensis, but barely accumulate
in other plant species. Theanine (γ-glutamylethylamine) is one
of the specialized metabolites present in C. sinensis. Like other
natural amino acids, theanine occurs in its L-enantiomer form in
nature. L-Theanine is a unique amino acid in C. sinensis leaves
that has a special umami flavor, which confers the special
taste of green tea.2,3 L-Theanine has many biological effects on
humans, including promoting relaxation, concentration, and the
ability to learn. It also has neuroprotective effects, coopera-
tive effects with antitumor agents, protects against vascular
diseases, counteracts the effects of caffeine, and has antiobesity
effects.4−11 Besides its contribution to the function and quality
of tea, L-theanine plays important physiological roles in plants.
Amino acids are not only the basic units for protein synthesis,
but also serve as an energy source and as precursors for
the biosynthesis of many bioactive molecules.12 Among them,
L-glutamic acid (Glu) and L-glutamine (Gln) are two crucial
amino acids that contribute to the nitrogen cycle. In general,
plants assimilate nitrogen via the synthesis of Glu and Gln.13
L-Theanine is a Gln analog and shares many of its properties.
Similar to Gln, L-theanine is involved in ammonia detoxification
Since L-theanine has important functions in humans and
plants, its distribution and biosynthesis in plants have been
topics of interest. A wide variety of Camellia species contain
L-theanine, and it is especially abundant in C. sinensis, which is
used to produce tea.18 A few Ericales plants and the edible
mushroom Xerocomus badius also contain L-theanine.14,19 In
C. sinensis plants, L-theanine is biosynthesized by L-theanine
synthetase (TS, EC 6.3.1.6) from Glu and ethylamine.20
As L-theanine is very similar to Gln, TS is highly homologous to
Gln synthetase (GS, EC 6.3.1.2). A previous study reported
that CsTS1 shows 99% homology to CsGS1.3, and CsTS2 shows
97% to CsGS1.1.21 Wu et al.22 identified 20 CsGS genes,
including several unconfirmed CsTSs. Studies on bacteria,
C. sinensis, and pea seeds have shown that GS can convert Glu
and free ammonia to Gln, and also transfer Glu to L-theanine in
the presence of ethylamine.20,23−26 These findings suggest that
GS and TS are very similar not only in their DNA sequences,
but also in their enzymatic activities. As GS/TS are present in
many plants, it is still unknown why L-theanine accumulates
only in C. sinensis, and barely or not at all in other plant species.
Received: May 26, 2017
Revised: July 27, 2017
Accepted: July 29, 2017
© XXXX American Chemical Society
A
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operated using a mobile phase consisting of lithium citrate pH 2.9, pH
4.2, pH 8.0, and using UV−vis detection at 570 and 440 nm. The flow-
rate of the mobile phase was 0.45 mL/min, and the flow-rate of the
derivatizating reagent was 0.25 mL/min. The column temperature was
set at 38 °C, and the post column reaction equipment was kept at
130 °C temperature. The temperature of the autosampler was kept at
5 °C, and the injection volume was 50 μL for both standard and
samples. The areas under the peaks corresponding to the amounts of
amino acids were compared to the authentic standards of amino acids.
Analyses of Gln by Gas Chromatography−Mass Spectrom-
etry (GC−MS). The MSTFA derivatization and GC−MS were
employed to analyze Gln.29 The crude extract of amino acids as
described above was dissolved in 100 μL of pyridine and derivatized
with 50 μL of MSTFA at 37 °C for 60 min, cooled, and then centri-
fuged. The MSTFA derivates were then analyzed by a GC−MS
QP2010 SE (Shimadzu Corporation, Kyoto, Japan). The injector
temperature was 240 °C, splitless mode was used with a splitless time
of 1 min, and helium was the carrier gas with a velocity of 1.0 mL/min.
An HP-5 column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies,
California, USA) was used with an initial temperature of 100 °C for
2 min, a ramp of 5 °C/min to 300 °C, and then a hold at 300 °C for
10 min. The MS was operated with selective ion monitoring mode
(m/z 156 and 245 for Gln-MSTFA derivate).
Analysis of Ethylamine by GC−MS. The extraction protocol of
internal ethylamine in fresh samples was described by Tsushida and
Takeo.30 Fresh samples were steamed for 60 s and immediately frozen
at −20 °C. Then the frozen samples were lyophilized and pulverized.
Powdered samples (100 mg) were put into a 10 mL volumetric flask
and diluted with 5 mL of distilled water. The flask was warmed at
80 °C for 5 min and allowed to stand for 2 h at room temperature.
Then the extract was filtered and centrifugated at 3500 × g for 5 min.
The analytical method of ethylamine was described by Almeida
et al.31 with a modification.
An aliquot of sample (1 mL) was transferred to a 4 mL silanized
screw-capped glass vial containing 1 mL of toluene, and the mixture
was adjusted to alkaline using 1 mL of 0.5 M phosphate buffer (pH
12.0), and 25 μL of IBCF and a deuterated internal standard (IS)
[2H5]ethylamine:HCl (2 mg mL−1) were added. The vial was shaken
for 10 min at 250 rpm and centrifugated at 3500 × g for 5 min. The
resultant toluene (upper) layer (200 μL) was transferred to another
vial, and 200 μL of alkaline methanol, which prepared by dissolving
KOH in methanol until saturation and followed by filtration through a
0.45 μm filter, was added. The tube was shaken for 5 min, then 600 μL
of 5 M NaOH was added, and the mixture was shaken for another
5 min. After the mixture was centrifugated, the toluene layer was used
for analyzing ethylamine. The ICBF derivatizated products were
analyzed by the GC−MS. GC separation was performed on an HP-5
column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies,
California, USA). The injector temperature was 280 °C, splitless mode
was used with a splitless time of 1 min, and helium was the carrier gas
with a velocity of 1.0 mL/min. The oven temperature program was as
follows: 40 °C held for 3.0 min, ramped to 140 °C at 40 °C/min, then
ramped to 280 °C at 80 °C/min, and held for 10 min. The MS was
operated with selective ion monitoring mode (m/z 90 and 72 for
ICBF-ethylamine derivate).
To answer this question, we determined the contents of
L-theanine and its related metabolites (ethylamine, Glu, and
L-alanine) in two model plant species (Arabidopsis thaliana and
Solanum lycopersicum), the economic plant Zea mays, and two
Camellia species (Camellia nitidissima and Camellia japonica),
and compared them with those in C. sinensis. Then stable
isotope-labeled ethylamine was supplied to each species to
investigate the pathway leading to the specific accumulation
of L-theanine. The results of this study help to explain why
C. sinensis accumulates L-theanine, which determines the quality
of tea products, and will be useful for breeding and screening
high-L-theanine lines of C. sinensis.
MATERIALS AND METHODS
■
Chemicals. Amino acid standard solutions for the amino acid
analyzer were purchased from SYKAM GmbH, Eresing, Germany.
Amino acid authentic standards for mass analysis were purchased
from Chengdu Mansite Pharmacetical Co., Ltd., Chengdu, China.
N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) was pur-
chased from Regis Technoloogies Inc., Morton Grove, USA.
Ethylamine:HCl was purchased from Shanghai Aladdin Bio-Chem
Technology Co., Ltd., Shanghai, China. [2H5]Ethylamine:HCl
(2H5% = 98%) was purchased from Cambridge Isotope Laboratories
Inc., MA, USA. Isobutyl chloroformate (IBCF) was purchased from
Shanghai Macklin Biochemical Co., Ltd., Shanghai, China. Isopropyl-
β-D-thiogalacto-pyranoside (IPTG) was purchased from Sigma-Aldrich
Company Ltd., USA. Coomassie Blue R250, 30% acrylamide/bis solu-
tion, N,N,N′,N′-tetramethylethylenediamine (TMEMD), and sodium
dodecyl sulfate (SDS) were purchased from Bio-Rad Laboratories,
California, USA. Ni-NTA resin was purchased from Qiagen Inc.,
Hilden, Germany. PD-10 desalting column was purchased from GE
Healthcare Life Sciences, Chicago, USA.
Plant Materials and Soils. The roots and leaves of A. thaliana,
S. lycopersicum cv. Micro-Tom, Z. mays var. rugosa Bomaf., C. nitidissima,
C. japonica, and C. sinensis var. Jinxuan were used for the present study.
The seeds of A. thaliana were scattered on the wet filter paper under
4 °C for 3 days. Afterward, these A. thaliana seeds were dispersed 4−6
per pot. The seedlings of A. thaliana were transferred for one seedling
per pot after budding. The seeds of S. lycopersicum and Z. mays were
sown one per pot. The seeds of A. thaliana, S. lycopersicum, and
Z. mays were grown in a growth chamber controlled at 25 °C humidity
80%, under 16-h light and 8-h dark cycles and were watered one time
per 3 days before budding and one time per week after budding.
The plants of C. nitidissima, C. japonica, and C. sinensis were grown in
fields. The soils for cultivating A. thaliana, S. lycopersicum, and Z. mays
were purchased from Treffex AS, Parnumaa, Estonia (producer) on
̈
behalf of Jiffy Products International BV, Moerdijk, Netherlands. The
soils for cultivating each plant species were also collected for analyses.
All the samples were collected in October, 2016.
Analysis of L-Alanine, L-Theanine, and Glu by an Amino Acid
Analyzer. The extraction protocol and analysis method of free amino
acids were described by the previous studies.27−29 Plant tissues
(200 mg fresh weight, finely powdered) were extracted with 0.7 mL of
cold methanol (100%) by vortexing for 2 min followed by ultrasonic
extraction in ice cold water for 20 min. The extracts were mixed with
0.7 mL of chloroform and 0.2 mL of cold water for phase separation.
The resulting upper layer was dried and used as the crude extract of
amino acids. For analysis of all the free amino acids, the dried crude
extract was dissolved in 1 mL of 5% sulfosalicylic acid and stood for
1 h. After being centrifuged at 12 000 × g for 10 min, the supernatant
was filtered through a 0.45 μm membrane and subjected to an amino
acid analyzer. A Sykam S433D Physiological Li C4 system (SYKAM
GmbH, Eresing, Germany) equipped with quaternary pump, column
oven, refrigerated auto sampler, and UV−vis detector was used.
The Physiological Li C4 system was coupled with S4300 postcolumn
derivatization system. The experiments were performed on a high
efficiency sodium cation-exchange Pickering Laboratories column
(4.0 × 150 mm2). The Sykam S433D Physiological Li C4 system was
Supplement of [2H5]Ethylamine to Different Plants and
Identification of [2H5]L-Theanine by Ultra-Performance Liquid
Chromatography−Quadrupole Time-of-Flight Mass Spectrom-
etry (UPLC−QTOF-MS). The roots and shoots obtained from plant
materials mentioned above were placed in tubes with 2 mL of distilled
water containing [2H5]ethylamine (2 mg) and blank as controls.
The tubes were incubated at 25 °C for 24 h. Then samples were
harvested, frozen in liquid N2 immediately, and extracted by the
protocol mentioned above. O-Phthalaldehyde (OPA, 25 μL)
derivation buffer, which added 50 μL of OPA alcohol solution and
25 μL of β-mercaptoethanol into 450 μL of 3% boric acid buffer
(pH 10.5), was added into 300 μL of sample solution and stood at
25 °C for 10 min. Afterward, 25 μL of 0.1 M acetic acid was added
to end the reaction, and then 200 μL of distilled water was added.
After centrifugation, the resultant supernatant was filtered through a
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0.45 μm membrane and subjected to an UPLC−QTOF-MS (Acquity
UPLC I-Class/Xevo G2-XS QTOF, Waters Corporation, MA, USA).
Each sample (5 μL) was injected onto a Waters ACQUITY UPLC C18
column (1.7 μm, 2.1 mm × 100 mm, Waters Corporation, MA, USA).
Solvent A was water with 0.1% (v/v) formic acid. Solvent B was
acetonitrile with 0.1% (v/v) formic acid. The flow rate was set at
0.3 mL/min with a gradient elution, which started at 76% of solvent A
and kept for 8 min, then decreased linearly to 10% in 0.1 min and kept
for 4 min, and raised up to 76% in 0.1 min and kept for 4 min.
The column was maintained at 40 °C, and the samples were main-
tained at a temperature of 4 °C prior to injection. The electrospray
ionization used a capillary voltage of 3.0 kV for positive mode; cone
voltage 50 V; source temperature 100 °C; desolvation temperature
350 °C; cone gas flow 50 L/h; and desolvation gas flow 650 L/h.
The scan range was m/z 50−1000 Da and the mass spectrometer
resolution was 30 000, which enabled mass accuracy within 10 mDa.
The Leu-enkephalin (m/z 556.2771) was used as the lock mass solu-
tion for accurate mass calibration during long analytical sequences to
counteract the potential effect of calibration drift during the long
analytical run time. The characteristic m/z values ([M + H]+) of OPA
derivatizated products of [2H5]L-theanine and L-theanine were 356.1692
and 351.1379, respectively.
Phylogenetic Analyses of CsTSs and GS/TS from Other
Plants. A similarity search for all the unique sequences was carried
on National Center for Biotechnology Information (NCBI) database.
Sequences were aligned using the ClustalX program. The phylogenetic
tree was calculated using neighbor-joining algorithms and generated by
MEGA 5 software. Bootstrap analysis of the NJ tree was performed
using 1000 replicates
AtGSs Recombinant Expression and Function Identification.
AtGS1 was amplified using primers (Table S1) with an EcoRI site
(bold) in the forward primers and a SalI site (bold) in the reverse
primers; AtGS2 was amplified using primers (Table S1) with an
EcoRI site (bold) in the forward primers and a XhoI site (bold) in the
reverse primers. The above amplified cDNAs were subcloned into
the corresponding sites of digested pET32a plasmid and renamed
pET32a-AtGS1 and pET32a-AtGS2, respectively. After verification
by sequencing, the expression construct was transformed into E. coli
Rosetta for recombinant protein expression. A single colony from
each transformed E. coli Rosetta cell line was incubated overnight at
37 °C in 1 mL of LB containing ampicillin, chloramphenicol,
and kanamycin at a concentration of 100 μg/mL, 34 μg/mL, and
15 μg/mL, respectively. Inocula were then individually added to
200 mL of LB, containing 100 μg/mL ampicillin, 34 μg/mL chlor-
amphenicol, and 15 μg/mL kanamycin, and grown at 37 °C to an
OD600 at 0.6. After addition of 0.1 mM IPTG, the cultures were
grown at 20 °C for another 16 h to produce recombinant protein.
Cells were harvested at 10 000 × g for 10 min, then disrupted by
sonication for 20 min in a 50 mM NaH2PO4 (pH 8.0) buffer
containing 300 mM NaCl and 10 mM imidazole. After centrifuga-
tion at 10 000 × g for 20 min, the supernatant was collected and
purified by using affinity binding on Ni Sepharose 6 Fast Flow
(GE Healthcare Life Sciences, Chicago, USA) according to the
manufacturer’s instruction. The purified protein was passed through
a PD-10 (GE Healthcare Life Sciences, Chicago, USA) desalting
column. The protein samples were diluted 1:2 with SDS loading
buffer and denatured for 10 min at 100 °C. SDS-PAGE was per-
formed using 10% (w/v) polyacrylamide gels.
Reaction solution (total volume 800 μL) for L-theanine synthesis
activity contained 25 μmol of Glu, 10 μmol of ethylamine:HCl,
25 μmol of β-mercaptothanol, 25 μmol of MgCl2, 100 μmol of
Tris-HCl (pH 7.5), 3 μmol of K3PO4, 25 μmol of KOH, 10 μmol
of ATP, and 100 μL of AtGSs recombinant protein supernatant
(50 μg).20,32 The reaction mixture was incubated at 30 °C for 2 h,
terminated by heating at 96 °C for 2 min, and filtrated through a
PD-10 (GE Healthcare Life Sciences, Chicago, USA) desalting column.
The reaction solution for Gln synthesis activity contained the same
compounds but ethylamine replaced with ammonium. After drying, the
L-theanine and Gln products were detected by MSTFA derivatization
and GC−MS analysis as described above.
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RESULTS AND DISCUSSION
■
Ethylamine Availability Explains the Difference in
L-Theanine Accumulation between C. sinensis and Other
Plants. First, we quantified L-theanine in roots and leaves of
C. sinensis C. nitidissima, C. japonica, Z. mays, A. thaliana, and
S. lycopersicum. L-Theanine specifically accumulated in Camellia
plants, especially C. sinensis, and also occurred in Z. mays with
tract amount. Furthermore, in Camellia plants, the L-theanine
content was higher in the roots than in the leaves (Table 1).
Because Glu and ethylamine are the precursors of L-theanine in
C. sinensis, we also quantified these compounds. All of the
tested species contained Glu, but only Z. mays and Camellia
species, especially C. sinensis, accumulated ethylamine (Table 1).
In the plant species containing ethylamine, the ethylamine
content was higher in the roots than in the leaves (Table 1).
Deng et al. investigated the L-theanine content in the leaves
of 27 species or varieties of Theaceae plants. They found that
21 species or varieties contained L-theanine, but at much lower
levels than those in C. sinensis. Ethylamine and Glu, the
substrates of TS, were found to be distributed almost uniformly
among all tested parts of the seedlings.33 In our study, we
quantified L-theanine and its related metabolites in plant
species that are not in the Theaceae and found that all of
the plants containing L-theanine also contained ethylamine.
Since L-theanine is a unique amino acid in C. sinensis, it was paid
more attention in Theaceae plants and less in others. Both
L-theanine and ethylamine were detected in Z. mays (Table 1),
which indicated that Z. mays could also transform ethylamine to
L-theanine. To investigate whether ethylamine is the restricting
factor for the differential accumulation of L-theanine between
C. sinensis and other plants, we conducted stable isotope-labeling
experiments (Figure 1A). After [2H5]ethylamine was supplied to
the roots and leaves of C. sinensis, C. nitidissima, C. japonica,
Z. mays, A. thaliana, and S. lycopersicum, [2H5]L-theanine
was detected in all plants, even including A. thaliana and
S. lycopersicum, which did not contain endogenous ethylamine
(Figure 1B,C). Tracer experiments in plants with isotope-labeled
precursors have been used to discover biochemical pathways of
secondary metabolites in several studies.34−36 In fact, L-theanine
was found to be derived from ethylamine in C. sinensis by
using 14C-labeled ethylamine.34 A previous study showed that
L-theanine is produced from ethylamine in several different tissues
of C. sinensis (cotyledons, shoots, and roots).34 In the present
study, isotope-labeled ethylamine was converted into labeled
L-theanine in C. nitidissima, C. japonica, Z. mays, A. thaliana, and
S. lycopersicum. This finding indicates that the enzymes/genes
involved in the pathway leading from ethylamine and Glu to
L-theanine are present in a wide variety of plants.
Figure 1. Supplement of [2H5]ethylamine to different plants and
identification of [2H5]L-UPLC−QTOF-MS. CK, H2O treatment.
Treatment (T), [2H5]ethylamine treatment. (A) Leaf and root of the
selected plants were treated with [2H5]ethylamine and then analyzed by
UPLC−QTOF-MS. 1, C. sinensis; 2, C. japonica; 3, C. nitidissima;
4, Arabidopsis thaliana; 5, Zea mays; 6, Solanum lycopersicum.
(B) UPLC−QTOF-MS analyses of authentic standard of theanine
and [2H5]theanine in C. sinensis (sample 1), Zea mays (sample 5), and
Solanum lycopersicum (sample 6) treated with [2H5]ethylamine.
The characteristic m/z values ([M + H]+) of OPA derivatizated
products of [2H5]theanine and theanine were 356.1692 and 351.1379,
respectively. (C) UPLC−QTOF-MS peak areas of [2H5]theanine in
leaves and roots of the selected plants treated with [2H5]ethylamine.
N.D., not detected. Data are expressed as mean SD (n = 3).
labile and contained ATP-hydrolyzing enzymes. Therefore,
a creatine phosphate and creatine phosphokinase system
was required to generate ATP.37 The use of this system and
fractionation with acetone, ammonium sulfate, and sephadex
G-50 provided a much more stable enzyme preparation than
the crude tea homogenate. The optimum pH of the enzyme is
known to be 7.5.20 In another study, TS activity was detected in
acetone powder extracts of imbibed pea seeds. The TS activity
of the pea seed enzyme was substantially inhibited by ammonia
at low concentrations (77% inhibition with 0.5 mM ammonia).
Therefore, L-theanine synthesis by pea enzyme preparations
was ascribed to a nonspecific reaction of GS. In contrast, the tea
TS was shown to be a specific enzyme for L-theanine synthesis
from ethylamine.23 Since Sasaoka et al. first reported the acti-
vity of native TS,20 only a few publications have reported
on this enzyme. Li et al. determined the activity of TS
obtained from tea seedlings using capillary electrophoresis with
2,4-dinitrofluorobenzene as a derivative reagent, but these analyses
could not provide information on the properties of the enzyme.32
L-Theanine Synthetases in Plants Other than C. sinensis.
To investigate whether TSs are also present in other plants,
GSs/TSs from A. thaliana were isolated, cloned, sequenced,
and functionally characterized. The phylogenetic analysis
(Figure 2) revealed that the amino acid sequence of CsTS1/2
showed high homology with sequences from Arabidopsis,
tomato, and maize, while GS1 and GS2 were closely clustered.
The complete open reading frames of AtGS/TS1 and
AtGS/TS2 contained 1071 nucleotides with a calculated protein
molecular mass of 39 kDa, and 1293 nucleotides with a calcu-
lated protein molecular mass of 47 kDa, respectively (Figure 3).
The activity of TS was evaluated using a homogenate of
11-day-old etiolated tea seedlings from which the cotyledons
had been removed. The crude tea TS preparation was very
D
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Attempts to purify the native enzyme to apparent homogeneity
have not yet been successful, probably because the enzyme
activity is low and labile in tea extracts.
The TaiyoKagaku Company has a patent on TS genes.38
Two genes encoding TS isoforms, TSl (DD410896) and TS2
(DD410895), have been cloned from the cDNA library of tea.
In a previous study, transcripts of CsTS1 and CsTS2 were found
in all organs, but at lower levels in cotyledons than in roots
and shoots.21 The native CsGSs have also been cloned and
characterized.39 Although TS activity has been studied using
partially purified enzyme preparations and its encoding genes
have been patented, it is still unclear if the reported CsTS1 and
CsTS2 are distinct from CsGSs.14 We analyzed all the CsGS
sequences in the NCBI, and they were homologous to CsGS1.1
(GenBank accession no. AB115183), CsGS1.2 (GenBank acces-
sion no. AB115184), and CsGS1.3 (GenBank accession no.
AB117934). The CsTS sequences were the same as CsTS1
(DD410896) and CsTS2 (DD410895). CsTS1 shared 99%
sequences of CsGS1.3, and CsTS2 shared 97% of CsGS1.1.
The genes encoding GSs have been cloned and characterized
from Arabidopsis,40 Z. mays,41,42 and S. lycopersicum.43 These GSs
Figure 2. Phylogenetic analysis of CsGS/TSs and related functionally
characterized GSs. At, A. thaliana; Zm, Zea mays; Sl, S. lycopersicum;
Cs, C. sinensis. TS, theanine synthetase; GS, glutamine synthetase.
Sequences were aligned using the ClustalX program. The neighbor-
joining phylogenetic tree was generated by MEGA 5 software. The
proteins included in the tree are represented by GenBank accession
number.
Figure 3. Biosynthetic pathways of L-glutamine and L-theanine, and identification of recombinant AtGSs. (A) Biosynthetic pathways of L-glutamine and
L-theanine. (B) SDS-PAGE analysis of AtGSs expressed in E. coli. Black bold frames indicate target proteins. Proteins were resolved by SDS-PAGE on
polyacrylamide gel and stained with Coomassie brilliant blue. Purified His-tagged combined AtGS1 and AtGS2 with a calculated protein molecular mass
of about 57 kDa and about 66 kDa, respectively. (C) GC−MS identification of L-glutamine derived from L-glutamic acid and ammonia under action of
recombinant AtGS1 and AtGS2. m/z 156 for L-glutamine-MSTFA derivate. (D) GC−MS identification of L-theanine derived from L-glutamic acid and
ethylamine under action of recombinant AtGS1 and AtGS2. m/z 159 and 273 for L-theanine-MSTFA derivate.
E
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Article
ORCID
catalyze the conversion of Glu and free ammonium to Gln and
are involved in nitrogen metabolism. However, it is still
unknown whether these GSs catalyze the conversion of Glu and
ethylamine to L-theanine. The present study showed that GSs
from Arabidopsis were able to produce Gln and L-theanine
(Figure 3), which indicated that the GSs in a wide variety of
plant species can synthesize L-theanine if the right substrate is
present.
Ziyin Yang: 0000-0003-3112-3479
Author Contributions
⊥S.C. and X.F. are co-first authors and contributed equally to
this work.
Funding
This study was supported by the financial support from the
Guangdong Natural Science Foundation for Distinguished Young
Scholar (2016A030306039), the Guangdong Special Support
Plan for Training High-Level Talents (2016TQ03N617), and the
Guangdong Innovation Team of Modern Agricultural Industry
Technology System (2016LM1143).
Formation of Ethylamine. The results of this study
indicate that ethylamine availability is the restricting factor for
the differential accumulation of L-theanine between C. sinensis
and other plant species. This led us to investigate why
C. sinensis accumulates ethylamine and other plants do not.
Ethylamine may be derived from soil or it may be synthesized
by the plant. No ethylamine was detected in the soils used to
cultivate the plants in this study (Figure S1), which suggested
that ethylamine is synthesized by the plants themselves.
Amines are compounds with aromatic and aliphatic struc-
tures that are widespread in the plant kingdom. They form
during metabolism and show diverse characteristics and
biological functions.44 Ethylamine occurs in Camellia, Vicia
faba and Z. mays, Ecballium cicutarium, the fruits of apple and
Crataegus, and the flowers of Sambucus nigra and Arum
italicum.45 Alanine and acetaldehyde are the proposed
precursors of ethylamine in plant tissues.45−47 Alanine is closer
than acetaldehyde to L-theanine in the biosynthetic pathway.48
Deng et al. reported the incorporation of radioactivity from
[U−14C]-alanine into theanine.34 The formation of ethylamine
by enzymatic decarboxylation of alanine has not been demon-
strated previously. Since alanine is present in a wide variety of
plant species (Table 1), it would be interesting to investigate if
the enzyme catalyzing the conversion of alanine to ethylamine
specifically accumulates in C. sinensis and whether this enzyme
is crucial for the differential accumulation of ethylamine between
C. sinensis and other plants. Ongoing research in our laboratory is
aimed at isolating, purifying, and characterizing this enzyme from
C. sinensis.
We detected differential accumulation of L-theanine between
C. sinensis and other plant species (C. nitidissima, C. japonica,
Z. mays, A. thaliana, and S. lycopersicum). We used a stable
isotope labeling approach to elucidate the biosynthesis of
L-theanine in C. sinensis and other plant species and measured
immediate metabolites by mass spectrometry. Arabidopsis GSs
were able to produce Gln and L-theanine, which suggested that
GSs in a wide variety of plant species can also function in
L-theanine synthesis. Ethylamine is crucial for the accumulation
of L-theanine in plants. These results help to explain why
C. sinensis produces specialized metabolites that determine the
quality of tea and why C. sinensis is the most suitable material
for tea production.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
Ala, alamine; At, A. thaliana; Cj, Camellia japonica; Cn,
Camellia nitidissima; Cs, Camellia sinensis; ESI, electrospray
ionization; GC−MS, gas chromatography−mass spectrometer;
Glu, glutamic acid; Gln, glutamine; GS, glutamine synthetase;
IBCF, isobutyl chloroformate; IPTG, isopropyl-β-D-thiogalacto-
pyranoside; IS, internal standard; LB, Luria−Bertani; MSTFA,
N-methyl-N-(trimethylsilyl)-trifluoroacetamide; OPA, o-phtha-
laldehyde; SDS, sodium dodecyl sulfate; SIM, selective ion
monitoring; Sl, S. lycopersicum; TMEMD, N,N,N′,N′-tetrame-
thylethylenediamine; TS, theanine synthetase; UPLC−QTOF-MS,
ultraperformance liquid chromatography−quadrupole time-of-
flight mass spectrometry; Zm, Zea mays
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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.7b02437.
Primers of AtGSs used; analysis of ethylamine of soils
used for selected plants by GC−MS (PDF)
AUTHOR INFORMATION
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Corresponding Author
*E-mail: zyyang@scbg.ac.cn. Phone: +86-20-38072989.
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