Biosynthesis of methyl (E)-cinnamate in the liverwort Conocephalum salebrosum and evolution of cinnamic acid methyltransferase

Article abstract of DOI:10.1016/j.phytochem.2019.04.013

Methyl (E)-cinnamate is a specialized metabolite that occurs in a variety of land plants. In flowering plants, it is synthesized by cinnamic acid methyltransferase (CAMT)that belongs to the SABATH family. While rarely reported in bryophytes, methyl (E)-cinnamate is produced by some liverworts of the Conocephalum conicum complex, including C. salebrosum. In axenically grown thalli of C. salebrosum, methyl (E)-cinnamate was detected as the dominant compound. To characterize its biosynthesis, six full-length SABATH genes, which were designated CsSABATH1-6, were cloned from C. salebrosum. These six genes showed different levels of expression in the thalli of C. salebrosum. Next, CsSABATH1-6 were expressed in Escherichia coli to produce recombinant proteins, which were tested for methyltransferase activity with cinnamic acid and a few related compounds as substrates. Among the six SABATH proteins, CsSABATH6 exhibited the highest level of activity with cinnamic acid. It was renamed CsCAMT. The apparent Km value of CsCAMT using (E)-cinnamic acid as substrate was determined to be 50.5 μM. In contrast, CsSABATH4 was demonstrated to function as salicylic acid methyltransferase and was renamed CsSAMT. Interestingly, the CsCAMT gene from a sabinene-dominant chemotype of C. salebrosum is identical to that of the methyl (E)-cinnamate-dominant chemotype. Structure models for CsCAMT, CsSAMT and one flowering plant CAMT (ObCCMT1)in complex with (E)-cinnamic acid and salicylic acid were built, which provided structural explanations to substrate specificity of these three enzymes. In phylogenetic analysis, CsCAMT and ObCCMT1 were in different clades, implying that methyl (E)-cinnamate biosynthesis in bryophytes and flowering plants originated through convergent evolution.

Full text of DOI:10.1016/j.phytochem.2019.04.013

Phytochemistry164(2019)50–59
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Biosynthesis of methyl (E)-cinnamate in the liverwort Conocephalum
salebrosum and evolution of cinnamic acid methyltransferase
Chi Zhanga, Xinlu Chena, Barbara Crandall-Stotlerb, Ping Qianc, Tobias G. Köllnerd, Hong Guoe,f,
Feng Chena,∗
a Department of Plant Sciences, University of Tennessee, Knoxville, TN 37996, USA
b Department of Plant Biology, Southern Illinois University, Carbondale, IL 62901, USA
c Shandong Agricultural University, Chemistry and Material Science Faculty, Tai'an 271018, Shandong, China
d Department of Biochemistry, Max Planck Institute for Chemical Ecology, Hans-Knöll-Strasse 8, D-07745 Jena, Germany
e Department of Biochemical, Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA
f UT/ORNL Center for Molecular Biophysics, Oak Ridge National Laboratory, Oak Ridge, TN 37830, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Conocephalum salebrosum
Conocephalaceae
Methyl (E)-cinnamate is a specialized metabolite that occurs in a variety of land plants. In flowering plants, it is
synthesized by cinnamic acid methyltransferase (CAMT) that belongs to the SABATH family. While rarely re-
ported in bryophytes, methyl (E)-cinnamate is produced by some liverworts of the Conocephalum conicum
complex, including C. salebrosum. In axenically grown thalli of C. salebrosum, methyl (E)-cinnamate was detected
as the dominant compound. To characterize its biosynthesis, six full-length SABATH genes, which were desig-
nated CsSABATH1-6, were cloned from C. salebrosum. These six genes showed different levels of expression in the
thalli of C. salebrosum. Next, CsSABATH1-6 were expressed in Escherichia coli to produce recombinant proteins,
which were tested for methyltransferase activity with cinnamic acid and a few related compounds as substrates.
Among the six SABATH proteins, CsSABATH6 exhibited the highest level of activity with cinnamic acid. It was
renamed CsCAMT. The apparent Km value of CsCAMT using (E)-cinnamic acid as substrate was determined to be
50.5 μM. In contrast, CsSABATH4 was demonstrated to function as salicylic acid methyltransferase and was
renamed CsSAMT. Interestingly, the CsCAMT gene from a sabinene-dominant chemotype of C. salebrosum is
identical to that of the methyl (E)-cinnamate-dominant chemotype. Structure models for CsCAMT, CsSAMT and
one flowering plant CAMT (ObCCMT1) in complex with (E)-cinnamic acid and salicylic acid were built, which
provided structural explanations to substrate specificity of these three enzymes. In phylogenetic analysis,
CsCAMT and ObCCMT1 were in different clades, implying that methyl (E)-cinnamate biosynthesis in bryophytes
and flowering plants originated through convergent evolution.
Liverworts
SABATH methyltransferase
Specialized metabolism
Convergent evolution
1. Introduction
1990). In flowers and fruits, methyl (E)-cinnamate is probably involved
in attracting pollinators and seed dispersers, respectively (Dodson et al.,
Plants produce diverse volatile specialized metabolites that include
carboxyl methyl esters. These methyl esters play important roles in
various biological processes (Chen et al., 2003a; Knudsen et al., 1993;
Seo et al., 2001). Methyl (E)-cinnamate, methyl ester of (E)-cinnamic
acid, is one such compound. Its odor is defined as “balsamic, straw-
berry, fruity, cherry”. Methyl (E)-cinnamate occurs as a scent com-
pound in many flowers such as orchids (Kaiser, 1993) and Narcissus
species (Arai, 1994). It is also made by some fruits such as strawberry
(Gomes da Silva and Chaves das Neves, 1999) and plum (Ismail et al.,
1980). Its occurrence in leaves of some plants is also known, such as in
basils (Viña and Murillo, 2003) and Eucalyptus species (Curtis et al.,
1969; Eltz and Lunau, 2005). In leaves, this compound most likely
functions as a chemical defense (Hattori et al., 1992). It has been shown
to inhibit the growth of pathogenic fungi and bacteria (Ali et al., 2010).
The molecular basis of methyl (E)-cinnamate biosynthesis has so far
only been studied in basil (Kapteyn et al., 2007), where cinnamic acid/
p-coumaric acid methyltransferases (ObCCMTs) catalyze the formation
of methyl (E)-cinanmate using S-adenosyl-L-methionine (SAM) as me-
thyl donor (Kapteyn et al., 2007).
ObCCMTs belong to the family of methyltransferases known as
SABATH (D'Auria et al., 2003). Most substrates of SABATH methyl-
transferases are important phytohormones including indole-3-acetic
∗ Corresponding author.
E-mail address: fengc@utk.edu (F. Chen).
https://doi.org/10.1016/j.phytochem.2019.04.013
Received 11 February 2019; Received in revised form 16 April 2019; Accepted 24 April 2019
0031-9422/©2019ElsevierLtd.Allrightsreserved.

C. Zhang, et al.
Phytochemistry164(2019)50–59
acid, gibberellins, salicylic acid and jasmonic acid (Chen et al., 2003a;
Qin et al., 2005; Seo et al., 2001; Varbanova et al., 2007; Zhao et al.,
2007). Other substrates of SABATH methyltransferases include benzoic
acid (Murfitt et al., 2000), p-methoxybenzoic acid (Koeduka et al.,
2016), farnesoic acid (Yang et al., 2006), anthranilic acid (Köllner et al.,
2010), nicotinic acid (Hippauf et al., 2010) and theobromine (Kato
et al., 2000; McCarthy and McCarthy, 2007). Such diverse substrates
within a same protein family make SABATH proteins useful models for
studying substrate specificity evolution (Huang et al., 2012). Within the
SABATH family, indole-3-acetic acid methyltransferase gene (IAMT)
has been proposed to be an ancient member in seed plants (Zhao et al.,
2008). ObCCMTs are closely related to IAMT (Kapteyn et al., 2007),
making it tempting to speculate that ObCCMTs may have evolved from
IAMT through gene duplication and functional divergence.
Liverworts are considered extant plants that are most closely related
to ancestral land plants (Bowman et al., 2017). Like flowering plants,
liverworts produce a diverse array of specialized metabolites, especially
terpenoids (Chen et al., 2018). While rarely reported, methyl (E)-cin-
namate is known to be made by liverworts within the Conocephalum
conicum complex (Ghani et al., 2016; Harinantenaina et al., 2007;
Toyota, 2000; Wood et al., 1996). Species of the Conocephalum conicum
(L.) Underw. (Conocephalaceae) complex, known as great scented li-
verworts, are widely distributed in North America, Europe and East
Asia (Szweykowski et al., 2005). They can be easily recognized by their
snake skin-like surface and aromatic odor (Atherton et al., 2010). The
C. conicum complex is comprised of two morphologically distinct spe-
cies, namely C. conicum and C. salebrosum Szweyk., Buczkowska &
Odrzykoski and multiple cryptic species (Szweykowski et al., 2005).
Although when Szweykowski et al. (2005) named C. salebrosum, they
indicated that it mostly corresponded to C. conicum genotype S, North
American specimens with genotype A from North Carolina were also
included. This species level relationship between genotypes A and S
(=C. salebrosum) was further confirmed in Ludwiczuk et al. (2013). In
an early study of presumed C. conicum populations in the in United
States, methyl (E)-cinnamate was identified as a major constituent from
a sample of unknown natural origin as well as a population from
Southern Illinois, but was not detected in the extracts of wild C. conicum
from coastal northern California (Wood et al., 1996). In subsequent
studies by a Japanese group, three chemotypes of C. conicum were
identified: methyl (E)-cinnamate-dominant, sabinene-dominant and
bornyl acetate-dominant (Toyota, 2000; Toyota et al., 1997). In this
study, we aim at elucidating the molecular basis of methyl (E)-cinna-
mate biosynthesis in the North American member of the C. conicum
complex (i.e., C. salebrosum), comparing it to that in flowering plants
and gaining understanding about the molecular basis for methyl (E)-
cinnamate variations among C. conicum populations.
Fig. 1. Chemical analysis of C. salebrosum. (A) GC chromatogram of the organic
extraction of C. salebrosum. 1*, sabinene; 2, 1-octanol; 3, 1-octenyl acetate; 4*,
methyl (E)-cinnamate; 5, unidentified sesquiterpene hydrocarbon; 6, 10-epi-
cubebol; 7, 1-epi-cubenol; 8, 1-octadecyne; 9, (E)-2-tetradecen-1-ol; 10, phytol.
(B) GC chromatogram of headspace collection of C. salebrosum. 1*, sabinene;
4*, methyl (E)-cinnamate; 5, unidentified sesquiterpene hydrocarbon. “*” in-
dicates compounds that were verified by authentic standards.
determine whether C. salebrosum grown axenically on culture medium
emits volatiles, we performed headspace collection of explants of C.
salebrosum, which was analyzed by GC-MS. Three dominant compounds
were detected (Fig. 1B). All of them were also detected by organic ex-
traction. Similar to organic extraction, methyl (E)-cinnamate was the
most abundant compound in the volatile bouquet of C. salebrosum
(Fig. 1B).
2.2. Isolation of SABATH genes from C. salebrosum and gene expression
analysis
We hypothesized that in the C. salebrosum methyl (E)-cinnamate is
synthesized by the action of SABATH methyltransferase. To identify
SABATH genes, we analyzed the transcriptome of C. conicum prepared
from vegetative thalli (https://sites.google.com/a/ualberta.ca/onekp),
which was produced by the 1 KP consortium (Matasci et al., 2014). Via
a HMMR search (Finn et al., 2011) using Methyltransf_7 Pfam profile
(Finn et al., 2015) as query, a total of nine unigenes encoding SABATH
proteins were identified. Six of them appear to be in full-length when
compared to other SABATH proteins and their corresponding orthologs
in C. salebrosum were designated CsSABATH1-6 (Table S1). Using RT-
PCR, full-length cDNAs for all six CsSABATH genes were cloned from
the thallus of the axenically grown C. salebrosum. The lengths of six
proteins range from 383 to 443 amino acids (Fig. 2). Sequence simila-
rities among CsSABATHs range from 37% to 52%. Their similarities to
ObCCMT1, one known CAMT from basil, are between 36% and 48%.
The amino acid residues for binding the methyl donor SAM among
CsSABATHs and ObCCMT1 are identical. The residues that interact
with the carboxyl moiety of the substrate are also conserved. However,
the residues that interact with the aromatic moiety are significantly
different (Fig. 2).
2. Results
2.1. Production of axenic culture of C. salebrosum and chemical profiling
The sample collected from Illinois (IL) morphologically matched C.
salebrosum, a segregate species of C. conicum (Szweykowski et al.,
2005). This population is the chemotype with methyl (E)-cinnamate as
the dominant compound described by Wood et al. (1996), and was
therefore chosen as the model chemotype for most of the experiments in
this study. Live plants were collected in the field and grown in axenic
culture. Chemical profiling analysis was performed using gas chroma-
tography–mass spectrometry (GC-MS) to determine the volatile com-
ponents in the axenically grown gametophytes of this species of the C.
conicum complex. From organic extraction, a total of 10 volatiles were
detected (Fig. 1A), of which methyl (E)-cinnamate was the dominant
component, accounting for 50.5% of the total volatiles. The mono-
terpene sabinene was the second most abundant with a concentration of
20% of that of methyl (E)-cinnamate. The thallus of C. salebrosum, when
growing in the wild, possesses a characteristic aromatic odor. To
With six SABATH genes cloned from C. salebrosum, next we com-
pared their expression levels using semi-quantitative reverse tran-
scription polymerase chain reaction (RT-PCR) analysis. At 25 cycles of
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C. Zhang, et al.
Phytochemistry164(2019)50–59
Fig. 2. Sequence alignment of CcSABATHs with ObCCMT1 and CbSAMT. Conserved residues are in shade with the more conserved the darker. Residues indicated
with “&” are SAM/SAH-binding residues. Residues indicated with “*” are residues that interact with the carboxyl moiety of substrate. Residues indicated with “#”
interact with the aromatic moiety of substrate and are important for substrate selectivity. CcSABATHs, C. conicum sensu latu SABATHs; ObCCMT1, Ocimum basilicum
cinnamate/p-coumarate carboxyl methyltransferase 1; CbSAMT, Clarkia breweri salicylic acid methyltransferase.
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Phytochemistry164(2019)50–59
properties using a purified CsCAMT containing a N-terminal His-tag.
The optimum pH for CsCAMT was determined to be 6.5 (Fig. 4A). For
temperature stability, CsCAMT was relatively stable when the assay
temperatures were below 42 °C (Fig. 4B). CsCAMT activity can be af-
fected by various ions (Fig. 4C). Na+ and Mg2+ ions slightly stimulated
the activity of CsCAMT. K+, NH4+, Ca2+ and Mn2+ had a mild in-
hibitory effect. Fe2+, Zn2+, Cu2+ or Fe3+ completely inhibited the
activity of CsCAMT (Fig. 4C). The kinetic property of CsCAMT was also
measured. The apparent Km value of CsCAMT using cinnamic acid as
substrate was determined to be 50.5 μM (Fig. 4D). The product of
CsCAMT was verifed to be methyl (E)-cinnamate (see Fig. 5).
Fig. 3. Expression of six CsSABATHs genes in sterile thalli measured by semi-
quantitative RT-PCR. Same amount of cDNA was used in each reaction. This
experiment was repeated three times with similar results. “M” indicates mole-
cular marker.
2.5. Comparison of CAMT genes in different chemotypes of the C. conicum
complex
PCR reactions, transcripts for five of the six genes could be detected
from thallus tissue, the exception being CsSABATH1 (Fig. 3). CsSA-
BATH4 showed the highest level of expression. While CsSABATH2 and
CsSABATH6 showed also higher expression levels. CsSABATH3 and
CsSABATH5 were found to be expressed at relatively low levels (Fig. 3).
As previously reported, some populations of C. conicum showed a
monoterpene-dominant chemical profile (Craft et al., 2016; Ludwiczuk
et al., 2013; Toyota, 2000). To gain some understanding into the mo-
lecular basis of such variations, we collected a population of C. sale-
brosum from Tennessee (TN). This population exhibited sabinene-
dominant chemotype (Fig. S1). Next, a putative ortholog of CsCAMT
gene was cloned from the sabinene-dominant plants and fully se-
quenced. It is identical to the CsCAMT gene isolated from methyl (E)-
cinnamate-dominant plants.
2.3. Biochemical characterization of CsSABATHs expressed in E. coli
Gene expression analysis (Fig. 3) suggested that CsSABATH2,
CsSABATH4 and CsSABATH6 are the most probable candidates as
CAMT. To validate this prediction, we characterized the proteins en-
coded by all six CsSABATH genes using a biochemical approach. Full-
length cDNA for each of the six CsSABATH genes was cloned by RT-PCR
into a protein expression vector without any tag and expressed in E. coli
to produce recombinant proteins. Each of the recombinant CsSABATH
enzymes was assayed with (E)-cinnamic acid and an additional nine
carboxyl acids: indole-3-acetic acid, gibberellin A3, salicylic acid, jas-
monic acid, anthranilic acid, nicotinic acid, benzoic acid, p-coumaric
acid and abscisic acid (Table 1). Among the six CsSABATHs, CsSA-
BATH6 was highly active using (E)-cinnamic acid as substrate. It also
showed activity with benzoic acid and p-coumaric acid, which was
about 32% and 18% of the activity with (E)-cinnamic acid. CsSABATH4
also showed some activity with (E)-cinnamic acid, but the activity was
very low. Instead, CsSABATH4 was most active with salicylic acid as
substrate. Its activity with (E)-cinnamic acid was only about 6% of that
with salicylic acid. CsSABATH6 and CsSABATH4 were renamed
CsCAMT (GenBank accession no. MK673137) and CsSAMT (GeneBank
accession no. MK673138), respectively. CsSABATH1, CsSABATH2,
CsSABATH3 and CsSABATH5 did not show activity with any of the
substrates tested.
2.6. Structural modeling of CsCAMT, CsSAMT and ObCCMT1
The structure models for CsCAMT, CsSAMT and one basil CAMT
(ObCCMT1) (Kapteyn et al., 2007) were built using the X-ray structure
of CbSAMT (Zubieta et al., 2003) as the template. The active sites of the
models for CsCAMT and CsSAMT in complex with (E)-cinnamic acid are
plotted in Fig. 6A and B, respectively. In each of these cases, the model
was first superposed with the protein structure of CbSAMT that is
complexed with salicylic acid and S-adenosyl-L-homocysteine (SAH) at
the active site. (E)-cinnamic acid was then docked into the active site
such that its cinnamate carboxylate group was superposed with that of
salicylic acid to form the reactive configuration for the methyl transfer.
Fig. 6A shows that (E)-cinnamic acid can fit well into the active site of
CsCAMT with its carboxyl moiety located at the suitable position for
accepting the methyl group from SAM. The result is consistent with the
experimental observation that (E)-cinnamic acid is the preferred sub-
strate for CsCAMT. In contrast, when (E)-cinnamic acid was docked into
the active site of CsSAMT (Fig. 6B) in the similar fashion, the ring of (E)-
cinnamic acid becomes too close to some of the residues of CsSAMT. For
instance, the distances between two of the carbon atoms on the ring are
only about 2.4 Å and 2.1 Å to the Cα and Cβ carbons of Ile237, re-
spectively. Such close contacts would create significant repulsions be-
tween the enzyme and (E)-cinnamic acid and is likely to destroy the
reactive configuration for the methyl transfer.
2.4. Biochemical properties of CsCAMT
With the identification of CsCAMT in C. salebrosum, next, we per-
formed biochemical assays to determine its detailed biochemical
The active sites of the models for CsCAMT and CsSAMT in complex
with salicylate are shown in Fig. 6C and D. Fig. 6D shows that, unlike
(E)-cinnamic acid, salicylic acid can fit into the active site of CsSAMT
without the close contacts observed for (E)-cinnamic acid. This is ex-
pected, as salicylic acid is significantly smaller than (E)-cinnamic acid.
Trp159 of CsCAMT (Fig. 6C) and Trp157 of CsSAMT (Fig. 6D) are
equivalent to Trp151 of CbSAMT, one of the two key residues for en-
suring proper orientation and proximity of salicylic acid to SAM for the
methylation reaction in CbSAMT (Zubieta et al., 2003). It is of interest
to note from Fig. 6C that, unlike in CbSAMT (Trp151) and CsSAMT
(Trp157), Trp159 in CsCAMT seems to have the potential to interact not
only with the carboxyl group of salicylic acid, but also with the 2-OH
group. Such additional interaction with the 2-OH group might distort
the optimal arrangement between the methyl acceptor and donor and
therefore interferes with the methyl transfer. Consistent with this sug-
gestion, Table 1 shows that CsCAMT does not have activity on salicylic
acid, even though it is active on benzoic acid.
Table 1
The relative activities of CsSABATH4 and CsSABATH6 from C. salebrosum with
ten carboxyl acids.
CsSABATH4 (CsSAMT)
CsSABATH6 (CsCAMT)
(E)-cinnamic acid
p-Coumaric acid
Nicotinic acid
Anthranilic acid
Benzoic acid
Salicylic acid
Abscisic acid
Jasmonic acid
Gibberellic acid
Indole-3-acetic acid
6%a
4%
0%
12%
51%
100%
0%
1%
0
5%
100%
18%
3%
2%
32%
0%
0%
0%
0%
3%
a
Values are averages of three independent measurements using radio-
chemical assays. The final concentration of each substrates tested was 1 mM.
The highest level of activity was arbitrarily set at 100%.
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C. Zhang, et al.
Phytochemistry164(2019)50–59
Fig. 4. Biochemical properties of CsCAMT using (E)-
cinnamic acid as substrate. (A) pH optimum. Level of
CsCAMT activity in the buffer of pH 6.5 was arbi-
trarily set at 1.0. (B) Thermostability. The activity of
CsCAMT incubated at 4 °C for 30 min was arbitrarily
set at 1.0. (C) Effect of ions. Individual ions were
added to reactions in the form of chloride salts at
final concentration of 5 mM. Level of activity without
any ion added served as a control (Ctr) and was ar-
bitrarily set at 1.0. (D) Steady-state kinetic mea-
surement of CsCAMT. Each value was the mean of
three independent measurements and bar indicates
standard deviation.
Fig. 5. Authentic methyl (E)-cinnamate and the
product of CsCAMT methylation. (A) GC chromato-
gram of authentic methyl (E)-cinnamate; (B) Mass
spectrum of authentic methyl (E)-cinnamate; (C) GC
chromatogram of the product of CsCAMT; (D) Mass
spectrum of the product of CsCAMT; (E) Schematic
methylation reaction catalyzed by CsCAMT. SAM
stands for S-adenosyl-L-methionine. SAH stands for S-
adenosyl-L-homocysteine.
In Fig. 6E, the structure model for ObCCMT1 complexed with (E)-
2.7. Relatedness of CsCAMT with other SABATH methyltransferases
cinnamic acid is plotted on the left, and the superposition of the active
sites for CsCAMT and ObCCMT1 is shown in Fig. 6F. Similar to the case
of CsCAMT, (E)-cinnamic acid can fit into the active site of ObCCMT1
without close contacts with the active site residues (Fig. 6E), in
agreement with an earlier study on this enzyme (Kapteyn et al., 2007).
It is of interest to note from Fig. 6F that a number of the active site
residues for ObCCMT1/CsCAMT are conserved in these two enzymes
(e.g., Tyr29/Tyr20, Gln36/Gln27, Asp68/Asp59, Ser72/Ser63, Asn76/
Asn67, Asp108/Asp98, Leu109/Leu99, Tyr141/Tyr139, Phe157/
Phe155, His160/His158, Trp161/Trp159 and Tyr272/Tyr268).
An approximately-maximum-likelihood phylogenetic tree was con-
structed using the six CsSABATHs, three ObCCMTs and SABATH pro-
teins from four sequenced plants that include Arabidopsis thaliana,
Oryza sativa, Physcomitrella patens and Selaginella moellendorffii to un-
derstand their evolutionary relatedness (Fig. 7). All SABATHs in seed
plant except gibberellic acid methyltransferases from A. thaliana (At-
GAMTs) resolved in a single clade. ObCCMTs from basil resolved in a
clade with IAMTs from A. thaliana (AtIAMT) and O. sativa (OsIAMT).
CsCAMT and CsSAMT resolved with other uncharacterized CsSABATHs
from C. salebrosum and together they are resolved in a clade with
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C. Zhang, et al.
Phytochemistry164(2019)50–59
Fig. 6. Active sites of the structure models of CsCAMT, CsSAMT and ObCCMT1 with (E)-cinnamic acid or salicylic acid docked into the active sites. (A) CsCAMT
(orange) complexed with (E)-cinnamic acid (green); the sulfur atom from SAH is shown as a yellow ball. Distances between the substrate and enzyme/SAH atoms are
given in Å. (B) CsCAMT complexed with (E)-cinnamic acid. (C) CsCAMT complexed with salicylic acid. (D) CsSAMT complexed with salicylic acid. (E) ObCCMT1
complexed with (E)-cinnamic acid. (F) Superposition of ObCCMT1 (orange) and CsCAMT (cyan) with some active-site residues shown. (E)-cinnamic acid and SAH are
shown in line except for the sulfur atom. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
AtGAMTs. Surprisingly, liverwort SABATHs do not resolve with the
other major SABATHs in seed plants, including IAMTs and CCMTs.
with the substrate (E)-cinnamic acid than ObCCMT1 from basil
(Kapteyn et al., 2007). Its Km value (50.5 μM) is about the half of that
for ObCCMT1 (124 μM). CsCAMT and ObCCMT1 also showed different
selectivity towards p-coumaric acid and benzoic acid. ObCCMT1 had
about 30% and 10% of activities of (E)-cinnamic acid with p-coumaric
acid and benzoic acid, respectively (Kapteyn et al., 2007). CsCAMT
exhibited the opposite trend: its activities with p-coumaric acid and
benzoic acid were 18% and 32% respectively of that with (E)-cinnamic
acid (Table 1). The identification of CsCAMT and CsSAMT (CsSA-
BATH4) together with the availability of basil ObCCMT1 makes it
possible to understand the structural basis underlying their substrate
3. Discussion
With the isolation and characterization of six SABATH genes from C.
salebrosum, this is the first report on functional study of SABATH genes
in liverworts. Particularly, we successfully identified CsCAMT
(CsSABATH6) as the gene with the capability for synthesis of methyl
(E)-cinanmate in C. salebrosum (Table 1). In addition, CsSABATH4 was
determined to encode SAMT (Table 1). CsCAMT has a higher affinity
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C. Zhang, et al.
Phytochemistry164(2019)50–59
methyltransferases and emerged independently from ObCCMT in basil.
The phylogeny in combination with structural modeling implies that
the evolution of methyl (E)-cinnamate synthesis in the liverwort and
flowering plants is the result of convergent evolution.
C. conicum and C. salebrosum usually inhabit shady moist environ-
ments, which can be hotbeds for microbe proliferation. Several studies
showed that methyl (E)-cinnamate has antimicrobial activity (Gilles
et al., 2010; Padalia et al., 2017). This could be a good reason for some
populations of C. salebrosum steadily producing volatile methyl (E)-
cinnamate in relatively large amounts (Fig. 1B). Aside from the cap-
ability of inhibiting the growth of fungi and bacteria, methyl (E)-cin-
namate has larvicidal activity (Fujiwara et al., 2017). Larvae of the
moth Epimartyri pardella have been reported to feed on C. conicum
(Davis and Landry, 2012). This can be another rational cause for li-
verwort to defend itself from herbivores. Ghani et al. (2016) have
shown that production of methyl (E)-cinnamate increases in popula-
tions of C. conicum from Japan when they are exposed to stressful
growth conditions, suggesting that gene expression may be influenced
by environment, while Szweykowski et al. (2005) note that C. sale-
brosum often grows in habitats that vary from flooded to seasonally dry
conditions.
The in vitro product of CsSAMT, methyl salicylate, has been shown
to be a critical signaling molecule in plant defenses, particularly as the
mobile signal in systematic acquired resistance against pathogens (Park
et al., 2007; Shulaev et al., 1997). It may also affect plant defense
against insects in both direct and indirect ways (Ament et al., 2010; Zhu
and Park, 2005). The identification of CsSAMT indicates that methyl
salicylate might play similar roles liverworts. As in the case of CsCAMT
in the phylogenetic analysis, CsSAMT is more closely related to other
CsSABATH members rather than to other characterized SAMTs or
BSMTs (benzoic acid/salicylic acid MT) in seed plants. This observation
implies that SAMTs might have evolved independently in liverworts
and in flowering plants as well.
Besides methyl (E)-cinnamate, C. salebrosum produces a number of
terpenoids (Fig. 1). Some progress has been made recently in our un-
derstanding of terpenoid biosynthesis in several species of bryophytes
(Jia et al., 2016; Kumar et al., 2016a; Xiong et al., 2018), in which
monoterpenes and sesquiterpenes are synthesized by a newly identified
type of terpene synthase genes called MTPSL (Jia et al., 2018). It will be
interesting to determine whether the same type of enzyme is re-
sponsible for the formation of monoterpenes and sesquiterpenes in C.
salebrosum and what biological function these terpenoids have.
Fig. 7. Phylogenetic tree of the SABATH family of methyltransferases in C.
salebrosum, Arabidopsis thaliana, Oryza sativa, Physcomitrella patens, Selaginella
moellendorffii and ObCCMTs. The SABATH proteins from each species were
color-coded. CCMTs: Cinnamate/p-coumarate methyltransferases; CAMT: cin-
namic acid methyltransferase; IAMT: indole-3-acetic acid methyltransferase;
SAMT: salicylic acid methyltransferase. AtIAMT, AtGAMTs, CsCAMT, CsSAMT,
OsIAMT and ObCCMTs were indicated with arrows. (For interpretation of the
references to color in this figure legend, the reader is referred to the Web
version of this article.)
specificities.
We demonstrated from computer modeling that while the active site
of CsCAMT can accommodate (E)-cinnamic acid and generate the re-
active configuration for the methyl transfer (Fig. 6A), CsSAMT may not
be able to do so. This is because some of the active site residues are too
close to the ring of (E)-cinnamic acid (Fig. 6B), and such close contacts
would create significant repulsions between CsSAMT and (E)-cinnamic
acid, leading to the non-reactive configuration for the methyl transfer.
This suggestion is supported by the lack of the activity of CsSAMT on
(E)-cinnamic acid (see Table 1). Similar discussion has been made in
our earlier quantum mechanical/molecular mechanical (QM/MM)
study for understanding the substrate specificity of CbSAMT (Yao et al.,
2010). Interestingly, as observed in other characterized SABATH me-
thyltransferases, our results on ObCCMT1/CsCAMT (Fig. 6F) show that
both SAM/SAH-binding residues and carboxyl moiety interacting re-
sidues are very conservative, but residues that interact with the aro-
matic moiety of the substrate are different (Fig. 2). This indicates that
similar interaction between active sites and substrate is achieved by the
combination of different residues.
As indicated by our phylogenetic study (Fig. 7), ObCCMTs share a
common ancestor with IAMTs from Arabidopsis and rice. Although
IAMT in basil has not been identified, our results suggest that ObCCMTs
and IAMTs, which belong to an ancient SABATH group in seed plants
evolved from a common ancestral type. However, CsCAMT together
with other CsSABATH members share a common ancestor with Arabi-
dopsis GAMTs, which is beyond our expectation. This demonstrates that
CsCAMT may have evolved from other uncharacterized SABATH
4. Conclusions
In this study, we identified and characterized the enzyme CsCAMT
that is capable for the biosynthesis of methyl (E)-cinnamate in the li-
verwort C. salebrosum, a species in the C. conicum complex. CsCAMT
belongs to the SABATH family of methyltransferases. The SABATH fa-
mily in C. salebrosum has at least nine members based on transcriptome
analysis, six of which were judged to be full-length and selected for
characterization in this study. It is evident that members of the SABATH
family in C. salebrosum have different substrate specificities, which is
similar to the SABATH family in flowering plants such as Arabidopsis
(Varbanova et al., 2007; Yang et al., 2006), rice (Zhao et al., 2008) and
gymnosperms (Chaiprasongsuk et al., 2018). This implies repeated gene
duplication followed by functional divergence of the SABATH family.
Selected members of the C. salebrosum SABATH family are not con-
served with their counterparts in flowering plants, evidenced by CAMT
and SAMT in this study (Fig. 7). Particularly for CAMT, we can infer
that this enzyme in C. salebrsum and in flowering plants evolved in-
dependently. There are strong similarities between the active site of
CsCAMT and that of ObCCMT1, despite their relatively distant relat-
edness among the SABATH family, indicating similarities in catalytic
mechanisms. There are a number of interesting future directions. With
CsCAMT and ObCCMT1 resulting from convergent evolution, it will be
56

C. Zhang, et al.
Phytochemistry164(2019)50–59
interesting to ask whether they evolved from similar or different an-
cestral activities. Because the two chemotypes of C. salebrosum contain
the same CAMT gene, it will be interesting to ask what causes the dif-
ference in the contents of methyl (E)-cinnamate: different expression
levels of CsCAMT or the different concentrations of its substrate cin-
namic acid or both? With methyl (E)-cinnamate accumulated in the
thallus and also emitted as a volatile compound, it will also be inter-
esting to ask what are the biological functions CsCAMT and its product
methyl (E)-cinnamate. In addition, for the four CsSABATHs that did not
show activity with any of the ten carboxylic acids tested, their in vivo
substrates and biological functions can be an important future study.
trees (Price et al., 2010). The trees were further edited using MEGA
(version 7.0.21) (Kumar et al., 2016b).
5.5. Cloning full length cDNA of CsSABATHs
Total RNA was extracted from vegetative thalli using the RNeasy
Plant Mini Kit (Qiagen, Valencia, CA) and reverse-transcribed into first
strand cDNA in a 15 μL reaction volume using the First-strand cDNA
Synthesis Kit (Amersham Biosciences, Piscataway, NJ) as previously
described (Chen et al., 2003a). CsSABATH full-length cDNAs were
amplified using forward primer and reverse primers corresponding with
the 5′ and 3′ ends of the CsSABATH coding region (Table S2), respec-
tively. The PCR was carried out using the following program: 94 °C for
3 min followed by 32 cycles at 94 °C for 30 s, 58 °C for 30 s, 72 °C for
1 min 30 s, and followed by a final extension at 72 °C for 10 min. PCR
products were isolated on 1.0% agarose gel and purified using QIAquick
Gel Extraction kit (Qiagen, Valencia, CA). The cDNAs were then cloned
into the vector pEXP-5-CT/TOPO following the vendor's protocol (In-
vitrogen, Carlsband, CA). The cloned cDNAs were fully sequenced.
5. Experimental
5.1. Plant culture and axenic culture
Two populations of C. salebrosum Szweyk., Buczkowska
&
Odrzykoski (Conocephalaceae) were used this study. One population
was collected from Illinois: Williamson Co., Rocky Bluff Nature
Preserve, nr. Devils Kitchen Lake, growing over moist sandstone rocks;
37°38′32.34″ N, 89°05′51.25″ W; elevation 157 m, 16 Oct. 2017, J.
Henry s.n. [F]). This population is methyl (E)-cinnamate-dominant. The
other population was collected from Tennessee: Knox Co., Campbell
station park, nr. North Fork Turkey Creek, growing over moist rocks;
35°53′13.5″N 84°10′02.4″W and is sabinene-dominant. The Illinois
plants collected from the field were rinsed with running water until
thalli were clean. Explants were sterilized using 70% ethanol solution
for 2 min, then rinsed 3 times with sterilized distilled water. The ex-
plants were further surface-sterilized using 10% bleach (v/v) for 5 min,
then rinsed 3 times using sterilized distilled. The sterilized explants
were transferred onto Hatcher medium which was prepared following
the protocol of Dr. Crandall-Stotler's Lab (http://bryophytes.plant.siu.
edu).
5.6. Semi-quantitative reverse-transcription PCR
Semi-quantitative reverse-transcription PCR (RT-PCR) was per-
formed as previously described (Chen et al., 2003a). The cDNA from a
single reverse-transcription reaction was used as template and 6 pairs of
specific primers (Table S2) were used in the PCR reaction. PCR was
performed using the same program as described in Section 5.5 except
that the cycle number was 25. 30 μL PCR products and 3 μL 1 kb ladder
marker were loaded in the agarose gel.
5.7. Purification of CsSABATHs expressed in E. coli
CsSABATHs were subcloned into the vector of pET32a (Invitrogen,
Carlsband, CA). To express the CsSABATH proteins, the protein ex-
pression constructs were transformed into the E. coli strain BL21 (DE3)
CodonPlus (Stratagene, La Jolla, CA) and cultured under 25 °C.
Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added into E. coli
cultures at a concentration of 500 μM to induce protein expression.
After 18 hours of cultivation at 25 °C, the bacterial cells were harvested
and lysed by sonication. His-tagged CsSABATH proteins were enriched
from the E. coli cell lysate using Ni-NTA (Invitrogen, Carlsband, CA)
following the manufacturer's protocol. An empty pET32a vector
without any insert was set to be a negative control.
5.2. Organic extraction and headspace collection
Thallus materials of axenically grown and field-collected C. sale-
brosum were ground in liquid nitrogen and subject to organic extraction
using ethyl acetate as solvent as previously described (Chen et al.,
2003b). For volatile profiling, a solid phase microextraction (SPME)
fiber coated with 100-μm polydimethylsiloxane was inserted into the
headspace of a C. salebrosum culture plate to start volatile collection.
After 1.5 h, the SPME fiber was retracted and inserted into the injector
port of GC for compound separation and identification.
5.8. Radiochemical methyltransferase activity assay
5.3. GC-MS analysis
CsSABATH enzyme assays including the measurements of optimum
pH, thermostability, the effect of cations and kinetic parameters were
conducted using a radiochemical protocol as previously described
(Zhao et al., 2013). A non-radiochemical assay followed by extraction
and GC-MS analysis was performed to determine the chemical identity
of the product of CsSABATH methylation.
Samples from either organic extraction or headspace collection were
analyzed with a Shimadzu 17A gas chromatograph coupled to a
Shimadzu QP5050A quadrupole mass selective detector. The GC con-
ditions included splitless injection, Restek R xi-5Sil MS column (Restek,
Bellefonte, PA), Helium as the carrier gas and a temperature gradient of
5 °C/min from 40 °C to 240 °C after an initial 3-min hold. Compounds
were identified based on comparisons to authentic standards when
available or the NIST library.
5.9. Protein structure modeling
Based on the crystallographic structure (PDB code: 1M6E) of
CbSAMT, the homology models of CsCAMT, CsSAMT and ObCCMT1
were built by using the tools in the MOE program [Molecular Operating
Environment (MOE), version 2013.08 (2015) Chemical Computing
Group Inc., Montreal]. SAH in each of the models was built based on the
superposition of the model with the X-ray structure of the CbSAMT
complex containing both SAH and SA. (E)-cinnamic acid molecule was
manually docked into the active site in each case based on the super-
position of the (E)-cinnamic acid carboxylate group with that of SA in
the CbSAMT complex that is close to the sulfur atom of SAH. Such
correct binding mode of the carboxyl moiety in the enzyme-substrate
5.4. Database search and phylogenetic analysis
To identify putative SABATH genes from the C. conicum complex,
the protein sequences of sample ID ILBQ from OneKP database (Matasci
et al., 2014) were searched using Pfam profile PF03492 (Finn et al.,
2015) via the hmmsearch (Finn et al., 2011). For phylogeny re-
construction, MAFFT (version 7.369b, under L-INS-I strategy) was used
to perform multiple protein sequence alignments (Katoh and Standley,
2013). FastTree (version 2.1.10, under the JTT + CAT model with 1000
resamples) was used to construct Approximately-Maximum-Likelihood
57

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Phytochemistry164(2019)50–59
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Acknowledgements
Chi Zhang has been partly supported by a scholarship from the
China Scholarship Council. We thank the 1 KP initiative for the avail-
ability of Conocephalum conicum transcriptome data. Ping Qian has been
supported in part by the Natural Science Foundation of Shandong
Province of China (No. ZR2017MB048).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2019.04.013.
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