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N‑Methyltransferases of Caffeine Biosynthetic Pathway in Plants

Meng-zhen Zhou, Chang-yu Yan, Zhen Zeng, Li Luo, Wen Zeng, and Ya-hui Huang*

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ABSTRACT: Caffeine (Cf) is one of the important components of plant-derived drinks, such as tea, coffee, and cola. It can protect
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soft tissues from being infected by pathogens and is also medically beneficial for human health. In this review, we first introduced the
Cf biosynthesis pathways in plants and the related N-methyltransferases (NMTs), with a focus on the current research status of the
substrate specificity, structural basis for substrate recognition, and catalytic mechanism in members of the caffeine synthase gene
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family. In addition, we addressed the expression characteristics and potential regulatory mechanisms of NMTs and also projected the
future research directions. The goal was to summarize the Cf biosynthetic pathway and related NMTs in plants and to provide the
molecular basis for regulating the caffeine biosynthesis, so as to effectively guide future tea and coffee breeding.
KEYWORDS: caffeine biosynthesis, N-methyltransferase, substrate specificity, catalytic

1. INTRODUCTION 2. PATHWAYS FOR THE BIOSYNTHESIS OF CF IN

Caffeine (1,3,7-trimethylxanthine, Cf) is one of the well-known
plant products because it is frequently found in plant-drived
drinks, such as coffee and tea. It has been reported that more
than 100 plants contain Cf; the most common are from the
genera Camellia, Cof fea, Theobroma (cacao), Cola, Paullinia
(guaraná), Ilex (maté), and Citrus.1 On one hand, Cf present in
young leaves, fruits, and flower buds can protect soft tissues
from being infected by herbivores,2−4 bacterial pathogens,2 and
fungi.5 On the other hand, Cf present in the seed coats can be
released into the soil where it inhibits the germination of seeds
of other plants.6 Cf also has a medically beneficial impact on
human health. For example, appropriate intake of Cf can
improve stomach digestion and diuresis7 and reduce high
blood pressure.8 However, excessive intake of Cf can cause
insomnia,9 anxiety,10 and osteoporosis and can also increase
cancer incidence.11 Therefore, how to effectively regulate Cf
biosynthesis in plants is of great significance for the efficient
use of plant resources. Lines of current evidence have indicated
that “Xanthosine (XR) → 7-methylxanthosine (7-mXR) → 7-
methylxanthine (7-mX) → theobromine (3,7-dimethylxan-
thine, Tb) → caffeine (Cf)” is the major Cf biosynthesis route
in both tea (Camellia sinensis)12 and coffee (Cof fea arabica).13
A series of methylation reactions could be critically required
for the conversion of XR to 7-mXR, Tb, and Cf, which all use
S-adenosyl-L-methionine (SAM) as methylation donor.14 Over
the past decade or so, important progress has been made on
the active site, expression patterns, and regulatory mechanism
of NMTs. The knowledge gained is helpful for us to effectively
regulate the Cf biosynthesis at the molecular level. This review
aimed to summarize the Cf biosynthetic pathway and related
NMTs in plants.
PLANTS
2.1. Origin of the Purine Ring of Cf in Plants. Since
Anderson and Gibbs first demonstrated that carbon atoms of
purine skeletons of Cf are delivered from the same precursors
for both purine nucleotides and nucleic acids, there are lots of
studies on the origin of the purine ring.15 Ogutuga et al.
thought that the purine ring more likely comes from nucleic
acid breakdown.16 Later, it was found that the purine ring in Cf
is directly derived from the purine nucleotides in the
nucleotide pool rather than as a degradation product of
nucleic acids.14,17,18 A number of purine precursors to the Cf
biosynthesis have been postulated. There are (i) inosine 5′-
monophosphate (IMP) synthesized by nucleotide biosynthesis
de novo,19 (ii) adenosine 5′-monophosphate (AMP) derived
from adenine nucleotide pools,17 (iii) guanosine-5′- mono-
phosphate (GMP) derived from guanine nucleotide pools,17
and (iv) adenosine derived from the SAM cycle,20 as illustrated
in Figure 1. The available evidence indicates that the most
important routes in young tea leaves are (i) and (iv).12,19,20 In
the SAM cycle, SAM is converted to S-adenosyl-L-homo-
cysteine (SAH) which in turn is hydrolyzed to L-homocysteine
and adenosine. Adenosine was used to synthesize the purine
ring of Cf, and L-homocysteine was used to resynthesize SAM.
S-Adenosyl-L-methionine (SAM) is the active methyl donor for
the methylation steps of Cf.20
2.2. Purine Ring Methylation in Plants. Based on the
results of isotope tracing experiments, the major pathway of Cf
Received: September 25, 2020
Revised: October 20, 2020
Accepted: October 29, 2020
Published: November 18, 2020

© 2020 American Chemical Society https://dx.doi.org/10.1021/acs.jafc.0c06167

15359 J. Agric. Food Chem. 2020, 68, 15359−15372
Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Review

Figure 1. Pathways for the biosynthesis of caffeine in tea and coffee. The solid arrows represent the major biosynthesis routes, and the dotted
arrows indicate minor routes or uncertain routes. Abbreviations: Adenosine 5′-monophosphate (AMP); inosine 5′-monophosphate (IMP); XMP,
xanthosine 5′-monophosphate; 7-methyl-XMP, 7-methyl-xanthosine 5′-monophosphate. Enzymes: (1) SAM synthetase; (2) SAM-dependent N-
methyltransferases; (3) SAH hydrolase; (4) methionine synthase; (5) adenosine nucleosidase; (6) adenine phosphoribosyltransferase; (7)
adenosine kinase; (8) AMP deaminase; (9) IMP dehydrogenase; (10) 5′-nucleotidase; (11) guanosine deaminase; (12) 7-methylxanthosine
synthase; (13) 7-methylxanthosine nucleosidase; (14) theobromine synthase; (15) caffeine synthase.

biosynthesis in Camellia and Cof fea plants was initially
determined as follows: Cf is synthesized through one
nucleoside cleavage reaction and sequential three-step
methylation of xanthosine (XR) derivatives at the positions
7-N, 3-N, and 1-N.21 XR is initially methylated to generate 7-
methylxanthosine (7-mXR) and then to generate 7-methyl-
xanthine (7-mX), followed by two methylation steps to
generate theobromine (Tb) and caffeine (Cf). This is
supported by the purification of caffeine synthase from tea
leaves22 and the in vitro expression of caffeine synthase23−27
and RNAi.28,29 Due to the extensive and broad spectrum of
substrates of some enzymes and the presence of other
secondary enzyme activities, there may also be several
secondary pathways in plants as follows: (1) xanthosine-5′-
monophosphate (XMP) → 7-methyl-XMP → 7-mXR → 7-mX
→ Tb → Cf;30 (2) 7-mX → 1,7-paraxanthine (Px) → Cf;31
(3) xanthine (X) → 7-mX → Tb → Cf;32 (4) X → 3-
methylxanthine (3-mX) → Tb → Cf;29,30,33 (5) X → 3-mX →
theophylline (Tp) → Cf;34 (6) Tp → 3-mX → Tb → Cf.34
These major and minor pathways for the Cf biosynthesis are
shown in Figure 1.
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3. NMTS OF THE CF BIOSYNTHETIC PATHWAY
The three methylation steps in the major Cf biosynthesis
pathway are catalyzed by SAM-dependent NMTs. According
to the substrates specificity, NMTs are divided into three
groups: the first group, 7-methylxanthosine synthase, which
catalyzes the 7-N methylation reaction of XR to generate 7-
mXR; the second group, theobromine synthase, which
catalyzes the 3-N methylation of 7-mX to form Tb; and the
third group, caffeine synthase, which catalyzes the 1-N
methylation of Tb to form Cf.
3.1. NMTs Activity in Cell Free Preparations. Suzuki et
al.14 first reported two N- methyltransferase in extracts of tea
leaves crude, catalyzing 3-N methylation of 7-mX to form Tb
and subsequently 1-N methylation of Tb to form Cf. The
optimal pH values of these two enzymes are almost the same
(8.4), and their activities are similarly affected by metal ions
and indoles. However, whether or not they are the same
enzyme was not determined at that time due to the limited
research conditions. NMTs involved in Cf biosynthesis were
subsequently detected in cell-free extracts of immature
fruits,14,35 callus,36 and coffee cell suspension culture.37 Negishi
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Table 1. Substrate Specificity of Tea Native Caffeine Synthasea

Substrate/methylation position
X/3N XR/7N XMP/7N 1-mX/3N 3-mX/1N 7-mX/3N Px/3N Tp/7N Tb/1N
Relative activity (%) Tr nd nd 4.2 17.6 100 210 tr 26.8
Product 3-mX 7-mXR 7-mXMP Tp Tb Cf Cf Cf Cf
a
The relative activity is indicated as the percentage of the activity relative to the most preferred substrate. Abbreviations are as follows: nd, not
detected; tr, trace; −, not determined; X, xanthine; XR, xanthosine; XMP, xanthosine 5′-monophosphate; 7mXMP, 7-methyl xanthosine 5′-
monophosphate; 1-mX, 1-methylxanthine; 3-mX, 3-methylxanthine; 7-mX, 7-methylxanthine; Px, paraxanthine; Tb, theobromine; Tp,
theophylline; and Cf, caffeine.

Table 2. Cloned NMT Genes in Plants

Gene Origin1 Origin2 Method Accession No. Reference
7-Methylxanthosine synthase
CmXRS1 Cof fea arabica Leaf cDNA library Screening AB034699 27
CaXMT1 Cof fea arabica Immature fruit cDNA RACE AB048793 26
CaXMT1* Cof fea arabica Leaf cDNA RT-PCR JX978521 26
CaXMT2* Cof fea arabica Leaf cDNA RT-PCR JX978522 50
XMT Cof fea canephora EST library BLAST DQ422954 49
CcXMT1* Cof fea canephora Leaf cDNA RT-PCR JX978518 50
Theobromine synthase
PCS1 Camellia ptilophylla Leaf cDNA RACE AB207817 51
ICS1 Camellia irrawadiensis Leaf cDNA RACE AB056108 51
CkTbS Camellia assamica var. kucha Leaf cDNA library Screening MN163830 52
CjCS1 Camellia japonica Leaf cDNA RACE AB297451 53
ClCS1 Camellia lutchuensis Leaf cDNA RACE AB362885 53
CgCS1 Camellia granthamiana Leaf cDNA RACE AB362882 53
CgCS2 Camellia granthamiana Leaf cDNA RACE AB362883 53
CkCS1 Camellia kissi Leaf cDNA RACE AB362884 53
CTS1 Cof fea arabica Leaf cDNA library Screening AB034700 46
CTS2 Cof fea arabica Leaf cDNA library Screening AB054841 46
CaMXMT1 Cof fea arabica L. var. caturra Leaf cDNA library Screening AB048794 46
CaMXMT2 Cof fea arabica Immature fruit cDNA RACE AB084126 26
CaMXMT1* Cof fea arabica Leaf cDNA RT-PCR JX978519 50
CaMXMT2* Cof fea arabica Leaf cDNA RT-PCR JX978520 50
CcMXMT1* Cof fea canephora Leaf cDNA RT-PCR JX978517 50
BCS1 Theobroma cacao Leaf cDNA RACE AB096699 51
Caffeine synthase
TCS1 Camellia sinensis Leaf cDNA RACE AB031280 24
CkCS Camellia assamica var. kucha Leaf cDNA library Screening MN163829 52
CCS1 Cof fea arabica L. Liquid endosperm cDNA RACE AB086414 23
CtCS7 Cof fea arabica L. Liquid endosperm cDNA RACE AB086415 23
CaDXMT1 Cof fea arabica Immature fruit cDNA RACE AB084125 26
CaDXMT1* Cof fea arabica Leaf cDNA RT-PCR KF678863 50
CaDXMT2* Cof fea arabica Leaf cDNA RT-PCR KJ577793 50
DXMT Cof fea canephora EST library BLAST DQ422955 49
CcDXMT* Cof fea canephora Leaf cDNA RT-PCR JX978516 50
PcCS Paullinia cupana Immature seed EST library BLAST BK008796 54
Unknown or no function
TCS2 Camellia sinensis Leaf cDNA library Screening AB031281 51
TCS3 Camellia sinensis Leaf cDNA LA-PCR JX647692 55
TCS4 Camellia sinensis Leaf cDNA LA-PCR JX647693 55
TCS5 Camellia sinensis Leaf cDNA LA-PCR JX647694 55
TCS6 Camellia sinensis Leaf cDNA LA-PCR JX647695 55
PCS2 Camellia ptilophylla Leaf cDNA RACE AB207818 51
ICS2 Camellia irrawadiensis Leaf cDNA RACE AB207816 51
CcCS2 Camellia chrysantha Leaf cDNA RACE AB362886 53
CaMTL1 Cof fea arabica L. var. caturra Leaf cDNA library Screening AB039725 25
CaMTL2 Cof fea arabica L. var. caturra Leaf cDNA library Screening AB048792 25
CtCS3 Cof fea arabica L. Leaf cDNA library Screening AB054842 27
CtCS4 Cof fea arabica L. Leaf cDNA library Screening AB054843 27

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Table 3. Comparison of the Substrate Specificity of NMTsa

Substrate/methylation position
Recombinant enzyme XR/7N XMP/7N 1-mX/3N 3-mX/1N 7-mX/3N Px/3N Tp/7N Tb/1N Reference
7-Methylxanthosine synthase
CaXMT1(GST fusion) 100 ND ND ND ND ND ND ND 26
Theobromine synthase
ICS1(native form) - - ND ND 100 10.9 ND 0.7 51
PCS1(native form) - - ND ND 100 11 ND ND 51
CjCS1(native form) ND ND ND ND 100 ND ND ND 53
CgCS1(native form) ND ND ND ND 100 ND ND ND 53
CgCS2(native form) ND ND ND ND 100 ND ND 1.9 53
ClCS1(native form) ND ND ND ND 100 ND ND ND 53
CkCS1(native form) ND ND ND ND 100 ND ND ND 53
CTS1(native form) ND - ND ND 100 1.4 ND ND 46
CTS2(native form) ND - - - 100 1.1 - ND 46
CaMXMT1(GST fusion) ND ND ND ND 100 15 ND ND 26
CaMXMT1(GST fusion) - - ND ND 100 5 ND ND 26
CaMXMT2(GST fusion) - - ND ND 100 5.3 ND ND 26
BCS1(native form) - - ND ND 100 ND ND ND 51
Caffeine synthase
TCS1(native form) ND ND 12.3 1 100 230 <0.1 18.5 24
CCS1(native form) ND ND 2.2 3.4 100 416 - 102 23
CCS1(Trx fusion) ND ND 2.7 3.5 100 622 - 142 23
CaDXMT1(GST fusion) - - ND ND 1 100 ND 3.8 26
PcCS(6X His-tag) - - ND ND 53 ND ND 100 54
Product 7-mXR 7-mXMP Tp Tb Cf Cf Cf Cf
a
The relative activity is indicated as the percentage of the activity with the most preferred substrate. Abbreviations as follows: ND, not detected; tr,
trace; −, not determined; X, xanthine; XR, xanthosine; XMP, xanthosine 5′-monophosphate; 7-mXMP, 7-methylxanthosine 5′-monophosphate; 1-
mX, 1-methylxanthine; 3-mX, 3-methylxanthine; 7-mX, 7-methylxanthine; Px, paraxanthine; Tb, theobromine; Tp, theophylline; Cf, caffeine.

et al.38 found a NMT from tea-leaf extracts catalyzing the
methylation of XR to produce 7-mXR. The optimal pH of this
enzyme was found to range from 7.5 to 8.0. p-Chloromercur-
ibenzoic acid (PCMB) (0.5 mM), Zn2+ (1 mM), and Cu2+ (1
mM) were shown to impose significant inhibitory effects on
enzyme activity. Schulthess et al.30 detected a NMT catalyzing
the N-7 methylation of purine rings in coffee cell-free extracts.
This enzyme displayed a higher catalytic activity for XMP than
XR, and thus, they proposed that XMP rather than XR was the
first methylated precursor in the Cf biosynthesis pathway.
Subsequently, many researchers attempted to extract and
separated the NMT involved in Cf biosynthesis from both tea
plants and coffee plants. However, one problem was that NMT
activities were very low and disappeared during the purification
process. Mazzafera et al.39 first reported the purification of a
NMT from the endosperm and leaves of coffee and found that
the single enzyme catalyzed 3-N methylation of 7-mX to form
Tb and subsequently 1-N methylation of Tb to form Cf. Kato
et al.31 partially purified NMT from tea-leaf extract with ion
exchange and gel filtration chromatography. They found that
NMTs exhibited catalytic activities toward XR, 7-mX, and Tb,
suggesting that a single enzyme may participate in the three
transmethylation reactions of Cf biosynthesis. Mosli Wald-
hauser et al.40 also partially purified NMTs from coffee leaves
using ion-exchange chromatography, chromatofocusing, and
gel filtration. The partially purified enzyme showed N-7
methylation activities on XMP and XR, which were different
from those of the other two NMTs. In order to obtain the
enzyme with high-purity, Kato et al.22 separated highly purified
(520-fold) Cf synthase (CS) from young tea leaves by
ammonium sulfate precipitation, hydroxyapatite, anion ex-
change, adenosine-agarose, and gel filtration chromatography.
15362
The molecular weight estimated by gel filtration chromatog-
raphy was 61 kDa, and the molecular weight determined by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was 41 kDa. CS exhibited both 3-N and 1-N
catalytic activities, with high activities on Px, 7-mXR, Tb, and
low activities on 3-mX and 1-mX. However, CS did not display
7-N catalytic activities for XR and XMP (Table 1). The Km
values for Px, Tb, 7-mX, and SAM were determined to be 24,
186, 344, and 21 μM, respectively. Among the tested
substrates, the Km value for Px was lowest and Vmax for
this substrate was the highest, suggesting that Px is the best
methyl receptor for CS. However, the synthesis of Px from 7-
mX or 1-mX was limited, Px was not actually an important
methyl receptor.14,26,31 Recently, Teng et al.41 partially purified
theobromine synthase from the leaves of Camellia gymnogyna.
The estimated molecular mass of the native enzyme was
approximately 62 kDa by continuous chromatography. It
displayed catalytic activity on Tb, XR, and Px, with the highest
affinity for Tb.
3.2. Molecular Cloning of NMT Genes and Recombi-
nant Enzymes. Since the beginning of this century, the
research of molecular cloning of NMT genes has also made
some progress. Based on the N-terminal amino acid sequence
of CS, Kato et al.24 cloned the first tea caffeine synthase gene
(TCS1) using rapid amplification of cDNA ends (RACE)
technology, which was also the first NMT cloned from plants.
TCS1 gene (AB031280) consists of 1438 base pairs and
encodes 369 amino acids. Recombinant enzyme mainly
catalyzes 3-N methylation and 1-N methylation of the purine
ring of mono- and dimethylxanthines, confirming that TCS1 is
a gene encoding tea caffeine synthase (CS). Later, a large
number of NMT genes were cloned from purine alkaloid
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Figure 2. Evolutionary relationship of caffeine synthases and its related enzymes. The unrooted tree was created by using ClustalW through
application of the neighbor joining method. (a) Evolutionary relationship of motif B′ methyltransferase family. (b) Distribution of genes encoding
NMTs among Camellia, Coffee, Theobroma, and Paullinia. Substrates of the enzymes are indicated in parentheses. The substrates of ICS2, PCS2,
TCS2, TCS4, TCS5, CcCS2, CaMTL1, CaMTL2, CtCS3, and CtCS4 are unknown. Abbreviations of substrates are as follows: XR, xanthosine; 7-mX,
7-methylxanthine; Tb, theobromine; SA, salicylic acid; BA, benzoic acid; JA, jasmonic acid; GA, gibberellic acid; IAA, indole-3-acetic acid; NA,
nicotinic acid. Sources of the sequences are as follows: AtJAMT, AY008434; CbSAMT, AF133053; AmBAMT, AF198492; AtGAMT1,
NM118775; AtIAMT1, EU375746; CkTcS, MN163831; CTgS1, AB054842, and the others indicate the accession number in Table 2.

Camellia plants, such as kucha (Camellia assamica var. kucha),
cocoa tea (Camellia ptilophylla), and Camellia irrawadiensis and
purine alkaloid-free Camellia plants such as Camellia japonica,
Camellia lutchuensis, Camellia granthamiana, Camellia gran-
thamiana, and Camellia kissi. Genome sequencing of tea (C.
sinensis var. assamica, CSA, and Camellia sinensis var. sinensis,
CSS), cacao (Theobroma cacao), and coffee (Cof fea canephora)
showed that the NMTs were present in plants as a gene family
and have evolved independently in different lineages.42−45
There have been two genome-wide replication events in the
evolutionary history of tea plants. Recent genome-wide
replication events and large-scale segment repetition have
promoted the evolution of the NMTs gene of Cf biosynthesis,
emerging to a large number of paralogous genes.44,45 Although
multiple NMTs have been identified from the tea genome,
their enzymatic properties and functions remain unclear. Based
on information about the conservative amino acids of TCS1,
plant O-methyltransferase, and Arabidopsis putative protein,
NMT genes were cloned from the coffee cDNA library and
EST library through RACR technology, screening, and
isolation. The 7-methylxanthosine synthase genes (CmXRS1,
CaXMT1),26,27 theobromine synthase genes (CaMXMT1,
CaMXMT2, CTS1, CTS2),25,26,46 and caffeine synthase genes
(CtCS7, CCS1, CaDXMT1)26,47 were successfully cloned from
Coffee arabica. CaXMT1 and CTS1 were later found to be
identical with CmXRS1 and CaMXMT1, respectively.26,47,48 7-
Methylxanthosine synthase (XMT) and caffeine synthase
(DXMT) were also cloned from Cof fea canephora and
correspond to CmXRS1 and CCS1, respectively.49 The
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NMTs of coffee are amplified by sequential tandem replication,
and a total of 23 NMTs were annotated on the genome of
Cof fea canephora. Based on genomic data, three genes
encoding NMTs were amplified in Cof fea canephora:
CcXMT1*, CcMXMT1*, and CcDXMT*, and six genes
encoding NMTs, including CaXMT1*, CaXMT2*,
CaMXMT1*, CaMXMT2*, CaDXMT1*, and CaDXMT2*
were amplified in Cof fea arabica.50 Table 2 summarizes the
cloned NMT genes in plants.
The substrate specificities of the three NMTs in the caffeine
synthesis pathway are significantly different (Table 3). 7-
Methylxanthine synthase (CaXMT1) is highly specific and can
only catalyze the 7-N methylation of XR to form 7-mXR and
has no activity on XMP. It was originally believed that TCS2
was an enzyme with unknown function because it did not show
NMT activities when xanthine derivatives were used as
substrate. 51 But Huang et al. 56 reported that TCS2
demonstrated a high methyltransfer activity at 7-N of XR,
indicating that TCS2 is a 7-methylxanthine synthase similar to
CaXMT1. Theobromine synthase (PCS1, ICS1, CjCS1, CgCS1,
CgCS2, ClCS1, CkCS1)51,53 of Camellia plants, theobromine
synthase (CTS1, CTS2, CaMXMT1, CaMXMT2) of coffee,
and theobromine synthase (BCS1)51 of cacao, only catalyzed
3-N methylation of 7-mX and did not display 3-N methylation
activity. The substrate specificity of TCS1 and coffee caffeine
synthases (CCS1, CaDXMT1)23,26 were found to be very
similar to natural enzyme (CS) purified from tea young leaves;
they mainly catalyzed mono- and dimethyl 3-N methylation
and 1-N methylation of the purine ring and did not exhibit
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catalytic activity for 7-N methylation. Of the three mono-

PcCS
47.4
47.4
48.2
47.7
47.1
37.7
37.7
38.3
37.3
38.6
37.3
38.6
39.1
38.8
38.6
50.7
100
methylxanthines studied, 7-mX was found to be the most easily
methylated one. When dimethylxanthines were used as the
substrate, Px was the optimal methyl receptor, followed by Tb.

BCS1
54.7
54.1
55.6
55.8
55.8
40.1
39.8
37.6
37.3
37.2
37.7
37.3
37.0
37.2
36.4
100
As mentioned above, Px is the best methyl receptor for tea
caffeine synthase and coffee caffeine synthase. However, there
is limited synthesis of Px from 7-mX, and it is not an important

DXMT
38.0
37.7
39.1
38.1
38.1
81.6
81.6
80.7
82.3
80.5
82.8
99.0
99.0
94.0
methyl acceptor in vivo.57 CtCS7 is a tentative caffeine

100
synthase because its heterologous protein shows low activity
and has weak catalytic activity for Px but not Tb.23 Unlike

CaDXMT1
38.2
37.7
40.0
39.2
38.7
83.1
82.6
82.6
84.1
82.3
83.9
93.8
93.8
other caffeine synthases, guarana caffeine synthase (PcCS)

100
showed higher affinity for Tb but not for Px.54

4. STRUCTURAL CHARACTERISTICS OF NMTS AND

CtCS7
38.2
37.6
39.9
39.7
38.6
82.1
82.1
81.3
82.8
81.0
83.1
94.5
THE BASIS OF SUBSTRATE RECOGNITION

100
4.1. Characteristic Sequence of Motif B′ Methyl-
transferase Family in Plants. By comparing 56 plant genes/

CCS1
38.0
37.7
39.4
38.4
38.4
81.0
81.0
80.5
82.0
80.2
82.6
100
cDNAs, Joshi and Chiang58 found that the conservative amino
acid motifs of plant methyltransferases were inconsistent with
those derived from animal sources. Thus, they proposed that

CTS2
37.2
37.0
40.0
38.7
38.7
85.2
85.2
93.5
97.7
93.2
100
there were three conserved motifs (A, B, and C) in plant
methyltransferases involved in the binding of methyl donor
SAM. Mizuno et al.58 found that the NMTs genes from tea and

CTS1
37.5
37.0
39.8
38.8
39.9
84.4
84.4
99.7
95.6
100
coffee contained three SAM-binding motifs (motif A, motif B′,
and motif C) and one conserved motif YFFF but lacked motif
B. The motif B′ region is located between motif A and motif C, CaMXMT2
37.5
37.2
40.0
39.0
39.0
85.2
85.2
95.8
and its consensus sequence was defined as “LNDLF/

100
PXNDFN” (X, any amino acid). On the other hand, the
YFFF region is located downstream of motif C, and its
consensus sequence is “AYXXQFXXDFXXFL”. Subsequently,
CaMXMT1
37.5
36.9
39.8
38.8
39.9
84.7
84.7
it was found that not only SAM-dependent NMTs involved in

100
purine alkaloids biosynthesis but also SAM-dependent NMTs
involved in nicotinic acid biosynthesis had four conserved
motifs (motif A, motif B′, motif C, and motif YFFF).50−53 In
XMT
40.2
39.9
41.5
41.3
41.6
99.5
100

addition, several other SAM-dependent methyltransferases

were also found in plants including the four conserved motifs
(motif A, motif B′, motif C, and motif YFFF) such as SAMT
CmXRS1
40.2
39.9
41.5
41.3
41.6

(SAM: salicylic carboxyl methyltransferase),59 JAMT (SAM:

100

jasmonic acid carboxyl methyltransferase),60 BAMT (SAM:

benzoic acid carboxyl methyltransferase),61 GAMT (SAM:
PCS1
90.9
89.8
95.9
98.6

gibberellic acid methyltransferase), and IAMT (SAM: indole-

100

3-acetic acid methyltransferase).59 Since these enzymes contain

Table 4. Percentages of Sequence Identity among NMTs

motif B′ rather than motif B, they are collectively referred to as

92.0
91.2
97.3
ICS1

the motif B′ methyltransferase family. The motif B′ and YFFF

100

regions are the specific regions of this family. Most members of

the motif B′ methyltransferase family catalyze the formation of
CkTbS
91.4
90.3

small-molecule methyl esters by using SAM as a methyl group

100

and substrates with a carboxyl group as methyl acceptors.61

Figure 2a shows a phylogenetic tree analysis of the motif B′
CkCS
96.8

methyltransferase family. Caffeine synthases are more closely

100

related to theobromine synthase and 7-methylxanthine

synthase in the same genu than caffeine synthase from other
TCS1
100

species, suggesting caffeine biosynthesis evolved independently

among plants.
4.2. Structural Basis for Substrate Recognition. On
CaMXMT1
CaMXMT2

CaDXMT1
CmXRS1

one hand, tea caffeine synthases share a low degree of sequence

DXMT
CkTbS

CtCS7
CCS1
TCS1

CTS1
CTS2
CkCS

BCS1
PCS1

identity (<40%) with coffee caffeine synthases, but they exhibit

XMT

PcCS
ICS1

similar substrate selectivity (Table 4). On the other hand

caffeine synthases share a high degree of sequence identity
CaMXMT1
CaMXMT2

(>80%) with 7-methylxanthine synthases and theobromine

CaDXMT1
CmXRS1

synthases in the same genus, but they exhibit distinguishing

DXMT
CkTbS

CtCS7
CCS1
TCS1

CTS1
CTS2
CkCS

BCS1
PCS1

XMT

PcCS
ICS1

substrate selectivity (Table 4). These results indicate that

caffeine biosynthesis evolved independently, in accordance
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Figure 3. Multiple sequence alignment of representative NMTs. The proposed SAM-binding motifs (A, B′, and C) and the conserved YFFF region
are shown by open boxes. The postulated important amino acid residues for the substrate specificity of N-methylation are marked by purple fill.
The nominated amino acids in substrate binding are indicated by closed circles.71 Asterisks indicate tyrosine (Y) or phenylalanine (F) residues in
the region.

with the evolutionary relationship of caffeine (Figure 2a and
Figure 2b). An interesting question is what is the structural
basis for substrate recognition of NMTs and are they the same
among different plants?
Ogawa et al.25 found that the major difference in the amino
acid sequences among TCS1, CaMXMT1, and CaMTLs was
that both TCS1 and CaMXMT1 contained the Val159-His160-
Tyr161 (VHW) motif, whereas CaMTLs did not, so they
speculated that substrate recognition specificity of NMTs
depended on several key amino acids. However, whether or
not the VHW structure is its key amino acid position was not
confirmed yet. Mizuno et al.23 found that the VHW structure
only exists in the CaMXMT(s) and TCS1. However, coffee
caffeine synthases such as CCS1 at this position are LHW
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structures, so that the VHW structure may not be a recognition
site for substrates by NMTs.
McCarthy et al.62 studied the crystallographic structure of 7-
methylxanthosine synthase (XMT) and caffeine synthase
(DXMT) from Cof fea canephora by X-ray diffraction. XMT
and DXMT consist of two domains, the core SAM-dependent
methyltransferase and an α-helical cap domain. Both XMT and
DXMT were nearly identical to SAMT, another member of the
motif B′ family. By comparing the crystal structures of both
XMT and DXMT, it was found that Ser-316 was likely to be
crucial for XR substrate specificity in XMT. The Tyr-321 and
Tyr-356 of XMT help to identify the substrate. His-160 may
have an important role in methylation activity in DXMT. His-
160 is conserved among all theobromine synthases and caffeine
synthases in coffee, but it was not in XMT or SAMT. There are
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Figure 4. Three methylation steps in the major Cf biosynthesis pathway. Figure 4a and Figure 4b show that H160 in DXMT may be used as a
general base/acid catalyst to mediate transfer of N3 and N1 methylated protons, thereby promoting the attachment of the positively charged
methyl groups to the substrate. XR might deprotonate through N3 on the purine ring, making it easy for XR to form a single anion for combining
with the active site of XMT, and its lone pair of electrons on N7 attacks the carbon atom of SAM to transfer the methyl group to the N7 position of
XR.

15 amino acid residues within 5 Å of the Tb binding site in
DXMT, and they proposed that only 3 were important to
discriminate the substrate specificity by comparing MXMT and
DXMT sequences. These three amino acid residues are Phe-27,
Ser-237, and Ile-266 of DXMT, corresponding to Ala-27, Pro-
237, and Phe-266 of MXMT. Ile-266 of DXMT may be a key
site for identifying Tb. However, the Phe-266 at the position
will have a spatial conflict with the methyl group at the N3
position, thereby preventing the binding of Tb and its
subsequent methylation. The function of the other two
amino acid residues is unclear. Although the 266-residue
substitution may be important for substrate recognition in
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coffee, theobromine synthase and caffeine synthase from other
plants are quite different in this region (Figure 3).
Some researchers have studied the structural basis for
substrate recognition of NMTs by the hybrid protein and site-
directed mutations. In order to further understand the
mechanism of substrate specificity, Yoneyama et al.51 found
that the substrate specificity of TCS1 was in the 114−287
amino acid region by constructing a hybrid protein of TCS1
and PCS1. There are only differences in nine amino acid
residues (129, 192, 225, 229, 236, 246, 267, 271, and 272)
between TCS1 and PCS1 in this region, which determine the
difference of substrate specificity between caffeine synthase and
theobromine synthase. The arginine residue located at position
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225 of TCS1 corresponds to histidine at position 221 of PCS1.
The arginine residue located at position 225 of TCS1, locating
in the center of enzyme activity, seems to be important for
substrate recognition.63 The TCS1 mutant (R225H) shows
only 3-N methylation activity, suggesting that R225 in TCS1
plays an important role in 1-N methylation.64,65 However, the
mutant PCS1 (H221R) shows a 5-fold increase in 3-N
methylation activity toward Px but still did not show activity
on 1-N methylation.51 From this point of view, R225 is an
important recognition site and more than one amino acid
residue is involved in the 1-N methylation of TCS1.
Furthermore, three TCS1 mutants (F271P, V317M, and
C269S) produced slightly higher amount of Tb and lower
amount of Cf than the wild-type enzyme, indicating the
residues 271, 317, and 269 contributed to Cf synthase activity
and substrate recognition but not to substrate specificity.64,66
R225 of TCS1 plays an important role in 1-N methylation, but
there is no arginine in the corresponding position of the
caffeine synthases in coffee. Therefore, the substrate specificity
of tea caffeine synthase and coffee caffeine synthase may be
caused by different structures. It was proposed that H160 is
important for methylation in both DXMT and MXMTs,62
while the corresponding residue in CmXRS1 is Gln161.
Mizuno et al.67 found that the CmXRS1 mutant (Q161H)
retained 3-N-methylation activity against XR and newly gained
the 3-N-methylation activity against Px and 7-mX. CmXRS1
mutant has gained higher affinity to Px than 7-mX, suggesting
that H160 is essential for 3-N methylation activity of caffeine
synthase in coffee. However, wild-type theobromine synthase
in coffee also contains a histidine residue corresponding to
H160 of CCS1 and it has low 3-N methylation activity against
Px. It is possible that theobromine synthase has a specific
amino acid residue(s) which interferes with the 3-N
methylation activity against Px. Another reason is that Tb
contains specific amino acid residue(s) contributing to the
enhancement of 3-N methylation activity against 7-mX.67 In
addition, the 3-N methylation activity of CmXRS1 in the
H161Q mutant appears to be enhanced by the additional
mutations of P104Q and L191Q in CmXRS1. There is a high
degree of homology (80%) between CmXRS1 and CtCSs, but
only CmXRS1 has 7-N methylation activity against XR.67 The
region with the largest sequence difference between CtCSs and
CmXRS1 is the region harboring 13 extra amino acid residues
in the C-terminus of CtCSs. Mizuno et al.68 speculated that the
lack of 13 residue sequences of CmXRS1 formed a XR ribose
binding space, which may be closely related to substrate
binding. To further understand the role of these 13 amino acid
residues on CCS1, they constructed corresponding deletion
mutants of CCS1. The deletion mutants of CCS1 lacking 13-
residues did not have 7-N methylation activity, indicating that
7-N methylation activity may be related to several other limited
amino acid residues rather than these 13 amino acid residues.68
The above research revealed that the substrate specificity of
NMTs is determined only by a few amino acid residues and
the spatial conformation of the protein amino acid sequence.
Although orthologous genes in different plants share the same
methylation activity, the structural basis of the substrate
specific recognition may be quite different. Some important
sites of NMTs involved in substrate recognition and catalytic
activity have been found in Camellia and Coffee plants, but
many important sites are at the theoretical stage or have not
yet been discovered. Further research is needed to clarify the
structure and function-relationship of NMTs.
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4.3. Catalytic Mechanism of the Enzyme-Catalyzed
Reactions. NMTs may need some additional assistants to
transfer the methyl group of SAM to the N position on the
purine ring because nitrogen is not as electronegative as
oxygen.62 Through quantum mechanics/molecular mechanics
and free energy simulation, Yue et al.69 speculated that the
H160 in DXMT may be used as a general base/acid catalyst to
mediate transfer of N3 and N1 methylated protons, thereby
promoting the attachment of the positively charged methyl
groups to the substrate (Figure 4a and Figure 4b) The catalytic
mechanism of XMT is different from that of DXMT because
the corresponding residue in XMT is Gln161, which may have
hydrogen bonding potential with the substrate, but it is
unlikely to assume the role of a general acid−base catalyst, so it
does not catalyze 7-mX and Tb. Qian et al.70 showed that XR
might deprotonate through N3 on the purine ring, making it
easy for XR to form a single anion for combining with the
active site of XMT, and its lone pair of electrons on N7 attacks
the carbon atom of SAM to transfer the methyl group to the
N7 position of XR (Figure 4c).
5. REGULATION OF EXPRESSION OF NMTS
5.1. The Expression Level of N-Methyltransferase is
Regulated by Variety/Species Differences. It has also
been shown that caffeine synthase (TCS1) plays a crucial role
in Cf biosynthesis72 in tea plants and the amount of TCS1
transcripts positively correlated with the Cf content.73 Xia et al.
analyzed the expression level of TCS1 in 24 Camellia species
based on comparative transcriptomic and phytochemical data,
finding that the level of TCS1 transcripts was 12.17-fold on
average higher in section Thea species than in non-Thea
sections.44 Similarly, Cf in section Thea is higher (∼9.50-fold
on average) compared with species from non-Thea sections.44
According to the insertion/deletion mutations in the upstream
regulatory region of TCS1 5′-UTR, six allelic variations of
TCS1 including TCS1a (TCS1, AB031280), TCS1b (ICS1,
AB056108), TCS1c (PCS1, AB207817), TCS 1d (KT215399),
TCS1e (KT215397), and TCS 1f (KT215398) were identified
from 60 accessions of section Thea plants.72 TCS1 transcripts
were found in almost all tea germplasms, while TCS1b-f
transcripts were only found in a few wild tea germplasms.
Xia et al. found that the expression level of TCS1 was higher
in tea plants with high Cf content than in tea plants with low
Cf content by RT-PCR.74 The transcription level of 7-
methylxanthosine synthase (CmXRS1) in Coffea arabica AC1
with low Cf content was similar to Cof fee arabica cultivar
Mundo Novo with conventional Cf content, while levels of
theobromine synthase (CTS2) and caffeine synthase (CCS1)
transcripts were lower in Cof fee arabica AC1 than in Coffee
arabica cultivar Mundo Novo.75
5.2. Tissue Specific Distribution and Expression of
NMTs. Using semiquantitative reverse transcription PCR, Li et
al. reported that the transcripts of genes involved in Cf
biosynthesis were observed in all tissues of 4-month-old tea
seedlings, but the level of TCS1 transcripts was much higher in
young leaves than other tissues and declined markedly during
leaf development.76 Kato et al.77 confirmed this result by
Northern blot analysis, and he also found the amount of TCS1
transcripts remained constant throughout the development of
the flower. Furthermore, tea fruits have lower levels of CS
expression compared to apical bud, leaves, and young stem.
Using an RNA in situ hybridization technique, Li et al.78
reported TCS1 was mainly expressed in the cell cytoplasm and
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nucleus of palisade tissue in tea leaves and weakly expressed in
the spongy parenchyma, vascular bundle, and hypoderm.
Cof fea arabica possesses multiple enzymes or isoforms to
catalyze the first, second, and last steps of methylation in the
Cf biosynthesis. Although these enzymes have the same
catalytic activity, their expression profiles in various tissues are
quite different. For example, CmXRS1 was highly expressed in
the first leaves, young leaves, flower buds, and immature fruits,
but not in mature fruits.78 CaXMT2* was expressed in a low
level in young leaves and coffee beans.50 Both CaMXMT1 and
CaMXMT2 were highly expressed in expanded leaves, flower
buds, and immature fruits but were weakly expressed in early
leaves and were not expressed in mature fruits.26 CTS2 was
weakly expressed in young leaves and fruits but strongly
expressed in flower buds.23,79 CaDXMT1 was mainly expressed
in immature fruits, while both CCS1 and CtCS7 were highly
expressed in young leaves and fruits but weakly expressed in
flower buds.26 The transcripts of CmXRS1, CTS2, CCS1, and
methionine synthase (MS) were detected at every stage of
whole fruits of Cof fea arabica cv. Mokka and C. canephora,
although these transcripts were lower in later stages of fruit
ripening. The amounts of the transcripts of the three genes
encoding N-methyltransferases were higher in seeds of
developing fruits than in pericarp. Furthermore, in situ
biosynthesis of purine alkaloids from [14C] adenine is
demonstrated in seeds of developing fruits of C. arabica,
suggesting that Cf accumulated in coffee seeds is mainly
synthesized in seed.79
In Guarana, caffeine synthase gene (PcCS) is expressed in all
tissues. The amount of PcCS transcripts was much higher in
young leaves and the apical stem than in mature leaves and
roots. In fruits, expression was observed in both the seeds and
pericarp. The expression level of PcCS was 7−20 times higher
in seeds from green fruit than other tissues and stages. The
results obtained from RT-qPCR were confirmed by in situ
hybridization analysis, as PcCS was expressed in the cotyledon
of seeds of immature and intermediate stages of fruit
maturation but not in mature fruits.54
5.3. Characterization of NMTs Expression in Re-
sponse to Environmental and Chemical Factors. An
early study suggested Cf biosynthesis occurs in young tea
leaves during April to June.80 However, caffeine synthase
expression was found in both nondormant (April−September)
and dormant (October−March) growth phases, although the
amounts of transcripts of TCS1 as well as Cf content were
higher in nondormant growth phases than in dormant growth
phases.81 The XMT, MXMT, and DXMT transcripts were
several-fold higher within 6 h of light exposure before falling
back to an amount comparable to leaves of the control plants
by the 12 h of light exposure.82 Plants respond to osmotic
stresses, such as drought, high salinity, and low temperature,
via regulation of the expression of genes involved in ABA-
dependent and ABA-independent pathways.83,84 The pro-
moters of CcXMT and CcMXMT genes contain both ABA-
dependent motif (ABRE) and ABA-independent motifs (DRE,
CE3 and Hex3) in silico analysis of 3 kb upstream regions, but
CcDXMT promotor does not’s.85 Salt and drought stresses
changed the Cf content in young coffee leaves by changing the
Cf degradation pathway and perhaps to an extent even
changing the transcription regulation of three NMTs (XMTs,
MXMTs and DXMTs) involved in Cf biosynthesis to a smaller
extent.85 Computer simulation analysis of the promoter of the
MXMT1-like gene (putative theobromine synthase gene) from
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Cof fea canephora showed that there are some light responsive
elements (GT box and GATA), salicylic acid responsive
elements (T/G box), and methyl jasmonate responsive
element (W-box). Since NMT genes are regulated by
developmental stage of tissues as well as in response to stress
signals, it should be considered whether stress stimulation can
overcome the developmental suppression of NMTs.86,87
External application of salicylic acid and methyl jasmonate
could increase the expression levels of NMTs in young coffee
leaves, resulting in a net increase in the Cf content in young
leaves. Furthermore, salicylic acid or methyl jasmonate could
alleviate the repression of XMTs and MXMTs caused by
maturation, resulting in a slight increase in Cf content. Bailey
et al. reported that ethylene and ethylene/methyl jasmonate
treatment reduced TcCaf-1 (a putative caffeine synthase)
transcript levels in young leaves by at least 74% and methyl
jasmonate induced accumulation of TcCaf-1 transcript in
young leaves to levels 2.56 times higher than controls, but
TcCaf1 did not respond to methyl jasmonate or ethylene in
mature leaves.86,87 Besides, the amounts of transcripts of
TcCaf-1 were not affected by mechanical wounding, contrary
to Aneji’s findings.88 There are many differences in TCS1 in
response to environmental and chemical factors as compared
to NMTs from other plants. Kato et al. reported that drought
stress significantly altered the expression of TCS1 transcripts,
while low temperature, salt stress, light, wounding, plant
hormone treatments, and nitrogen source have little or no
effect on TCS1 gene expression.77
In tea and coffee, the expression level of NMTs is higher in
high-caffeine containing germplasms than in low-caffeine
containing germplasms. From this perspective, the expression
level of caffeine synthase is mainly affected by the variety. In
addition, the expression level of N-methyltransferase is also
regulated by the developmental stage of tissues. For example,
in tea, the amount of TCS1 transcripts in recently emerged
developing leaves was the highest among all the developing
stages and fell by around 90% in mature leaves.77 In C. arabica,
transcripts for CaDXMT1 were predominantly found in
immature fruits.26 Environmental factors also affect the
expression level of NMTs. According to previous studies,
light is an important environmental factor that affects the
expression of NMTs in coffee leaves; drought stress is an
important factor that affects the expression of TCS1; other
environmental stresses have little effect on the expression of
NMTs. In addition, plant hormone treatments have little effect
on the expression of NMTs. Since NMT genes are regulated
by developmental stage of tissues as well as in response to
stress signals, it should be considered whether stress
stimulation can overcome the developmental suppression of
NMTs. Bailey showed that MeJ and ethylene induced an
increase in TcCaf-1 expression in young cocoa leaves, while
TcCaf-1 in mature leaves did not respond to MeJ or ethylene,
further proving that the effect of hormone on NMTs is limited
to the developmental stage of tissues.86
The expression profiles of homologous genes or paralogous
genes among species and the environmental response are
different, which is related to post-transcriptional regulation. In
order to elucidate the potential regulatory mechanisms, it is
necessary to further study the upstream regulatory elements in
the promotors of NMTs and verify its function.
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6. PERSPECTIVES AND EXPECTATIONS
Cf catabolism has been investigated through tracer experi-
ments using 14C-labeled purine alkaloids. Cf is mainly
metabolized via Tp and 3-mX to X which is degraded by the
purine catabolism pathway to CO2 and NH3 in coffee and
tea.89,90 Studies also demonstrated that Cf conversion to Tp is
the rate limiting step in young tissues, which is one reason for
their high Cf content.91 Furthermore, the biosynthesis and
catabolism of Cf have been compared in leaves of C. arabica
and three low-caffeine-containing species of coffee.92 The
result showed that the low Cf accumulation in C. salvatrix, C.
eugenioides, and C. bengalensis is a consequence of a slow rate of
Cf biosynthesis as compared to C. arabica, whereas rapid
degradation of Cf also contributes to the low endogenous Cf
pool in C. eugenioides. It can be concluded that the Cf content
in coffee has been attributed to the ratio of biosynthetic
activity to the degradation dynamics. The rate of Cf
biosynthesis depends upon the amount and activity of enzymes
and the availability of substrates such as XR and SAM.93 In the
previous study, the activity of all three NMTs was only found
in the period of Cf biosynthesis of tea leaves, suggesting that
the capacity for Cf biosynthesis is mainly dependent upon the
levels of N-methyltransferases.93 Further, Cf content and CS
expression showed direct relation in various tissues except
stems of sectional varieties, suggesting caffeine synthase as one
of the regulatory enzymes of Cf biosynthesis.81 Mohanpuria et
al.29 suppressed expression of caffeine synthase by the double
stranded RNA interference (RNAi) method. Transgenic
embryonic tissues exhibited a remarkable reduction in
transcripts for caffeine synthase and a significant reduction of
44−61% in Cf and 46−67% in Tb contents in comparison with
the controls. Ogita et al.28 constructed the RNAi structure
according to the 3′-untranslated region and coding regions of
the CaMXMT1, and then transferred it into the embryonic
tissues of coffee plants through the Agrobacterium-mediated
transformation techniques. It was found that the CaMXMT1-
RNAi sequence suppressed expression of not only CaMXMT1
but also CaXMT1 and CaDXMT1. The content of Tb and Cf
in both embryonic tissues and plantlets was reduced by 30%−
50% as compared to the controls. To investigate whether the
fine control of Cf biosynthesis involves the availability of
purine compounds and a methyl-donor, Deng et al. added
precursors of purine nucleotides to the culture medium.
Results showed that precursors of purine nucleotides influence
the growth of culture medium but not the accumulation of Cf,
suggesting that the availability of purine precursors is not a
rate-limiting factor of Cf biosynthesis. But, feeding with Px
causes a double increase of Cf content. This indicates SAM is
not the principal factor in the control of Cf biosynthesis and
points to 7-methylxanthosine synthase being the most
plausible rate-limiting factor of Cf biosynthesis.94 More work
is needed to prove this conclusion.
At present, the structural basis for substrate recognition in
the gene family of NMT is still at a preliminary stage, and some
active sites for substrate specificity and catalytic activity have
not yet been discovered. Therefore, it is necessary to reveal the
spatial structure and function relationship and the structural
recognition substrates of NMTs in different plants for
clarifying their catalytic mechanism. Furthermore, there have
been few studies on transcriptional regulation of NMTs, and
some factors affecting transcriptional regulation are still in the
basic experimental stage. It is necessary to analyze the
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upstream regulatory elements of NMTs, clarify the regulatory
mechanism of NMTs in plants, and provide a molecular basis
for improving the Cf biosynthetic pathway, so as to effectively
guide both tea and coffee breeding.
■
AUTHOR INFORMATION
Corresponding Author
Ya-hui Huang − Department of Tea Science, College of
Horticulture, South China Agricultural University,
Guangzhou 510642, China; Guangdong Provincial Key
Laboratory of Nutraceuticals and Functional Foods,
Guangzhou 510642, China; orcid.org/0000-0002-0247-
7959
Authors
Meng-zhen Zhou − Department of Tea Science, College of
Horticulture, South China Agricultural University,
Guangzhou 510642, China
Chang-yu Yan − Department of Tea Science, College of
Horticulture, South China Agricultural University,
Guangzhou 510642, China
Zhen Zeng − Department of Tea Science, College of
Horticulture, South China Agricultural University,
Guangzhou 510642, China
Li Luo − Department of Tea Science, College of Horticulture,
South China Agricultural University, Guangzhou 510642,
China
Wen Zeng − Department of Tea Science, College of
Horticulture, South China Agricultural University,
Guangzhou 510642, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c06167
Funding
This work was supported by the Fu Jian Province
“2011Collaborative Innovation Center” Chinese Oolong Tea
Industry Innovation Center Special Project (J2015-75) and
Guangdong Tea Industry Research System (2019LM1117).
Notes
The authors declare no competing financial interest.
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