Cloning and functional characterization of two 4-coumarate: CoA ligase genes from selaginella moellendorffii

Article abstract of DOI:10.3390/molecules23030595

Selaginella is an extant lycopodiophyte genus, which is representative of an ancient lineage of tracheophytes. The important evolutionary status makes it a valuable resource for the study of metabolic evolution in vascular plants. 4-coumarate: CoA ligase

Full text of DOI:10.3390/molecules23030595

molecules
Article
Cloning and Functional Characterization of Two
4-Coumarate: CoA Ligase Genes from
Selaginella moellendorffii
Xin-Yan Liu, Ping-Ping Wang, Yi-Feng Wu, Ai-Xia Cheng * and Hong-Xiang Lou *
Key Laboratory of Chemical Biology of Natural Products, Ministry of Education, School of Pharmaceutical
Sciences, Shandong University, Jinan 250012, China; qqbailiu@163.com (X.-Y.L.);
wangpingp1234@163.com (P.-P.W.); wendywu.12@163.com (Y.-F.W.)
* Correspondence: aixiacheng@sdu.edu.cn (A.-X.C.); louhongxiang@sdu.edu.cn (H.-X.L.);
Tel.: +86-531-8838-2012 (A.-X.C.); Fax: +86-531-8838-2019 (A.-X.C.)
Received: 6 February 2018; Accepted: 26 February 2018; Published: 7 March 2018
Abstract: Selaginella is an extant lycopodiophyte genus, which is representative of an ancient lineage
of tracheophytes. The important evolutionary status makes it a valuable resource for the study
of metabolic evolution in vascular plants. 4-coumarate: CoA ligase (4CL) is the pivotal enzyme
that controls the flow of carbon through the phenylpropanoid metabolic pathway into the specific
lignin, flavonoid, and wall-bound phenolics biosynthesis pathways. Although 4CLs have been
extensively characterized in other vascular plants, little is known of their functions in Selaginella.
Here, we isolated two 4CL genes (Sm4CL1 and Sm4CL2) from Selaginella moellendorffii. Based on the
enzymatic activities of the recombinant proteins, both of these genes encoded bona fide 4CLs. The
4CL isoforms in S. moellendorffii have different activities: Sm4CL2 was more active than Sm4CL1. The
enzymatic properties and gene expression patterns indicated that the 4CL genes have been conserved
in the evolution of vascular plants.
Keywords: Selaginella moellendorffii; 4-coumarate: CoA ligase; biochemical characterization
1. Introduction
Phenylpropanoids are plant-specific natural products that serve important functions during
growth, development, and environmental interactions [1]. Phenylpropanoids are synthesized from
phenylalanine via the central phenylpropanoid pathway, which is mediated by phenylalanine
ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate: CoA ligase (4CL).
4-coumarate: CoA ligase (EC 6.2.1.12) catalyzes the conversion of several hydroxycinnamic acids
into their corresponding CoA esters, enabling the biosynthesis of a diverse array of natural products
(Figure 1), including lignin, flavonoids, and wall-bound phenolics [
of 4CL might explain why it is encoded by a gene family present in plants. The 4CL gene family
has four members in Arabidopsis thaliana [ ], five members in Oryza sativa [ ], and four members in
Physcomitrella patens [ ]. The catalytic properties of 4CL protein isoforms have been studied extensively.
In some species, such as potato and parsley, cloned genes encode identical, or nearly identical, proteins
that possess similar substrate affinities [ ]. In other plants, such as aspen trees and Arabidopsis,
2–4]. The multiple functions
5
6
7
8,9
structurally- and functionally-divergent protein isozymes have been isolated [5,10].
Selaginella, also known as spikemoss, is the only surviving genus within the Selaginellaceae. This
genus first appears in the fossil record some 400 million years ago. Selaginellaceae, together with
two other extant families (Lycopodiaceae, the clubmosses, and Isoetaceae, the quillworts), comprise
the division Lycopodiophyta, the oldest extant lineage of vascular plants [11]. Over the last several
decades, the isolation and structural elucidation of phenylpropanoids from the Selaginella genus has
Molecules 2018, 23, 595; doi:10.3390/molecules23030595
www.mdpi.com/journal/molecules

Molecules 2018, 23, 595
2 of 11
expanded. In vascular plants, deposition of lignin is of significance because it reinforces plant cell
walls, facilitates water transport, provides compressive strength to conducting tissues, and acts as a
mechanical barrier to pathogens. Additionally, previous investigations revealed the genus Selaginella
to be a rich source of flavonoids, including monomeric flavonoids and biflavonoids [12]. However,
little is known about the genes in the phenylpropanoid pathways of Selaginella species. Recently,
the genome of S. moellendorffii was sequenced by the Joint Genome Institute (JGI) because of its
important evolutionary position among land plants and its small genome size; S. moellendorffii is,
therefore, an important new model species [13,14]. Here, we isolated and identified two 4CL genes
(Sm4CL1 and Sm4CL2) from S. moellendorffii. The activities of these two enzymes were substantially
different. We also measured the organizational expression patterns of the two genes.
Figure 1. The proposed synthesis pathway of phenylpropanoids in Selaginella moellendorffii. PAL:
phenylalanine ammonia lyase, C4H: cinnamate 4-hydroxylase, 4CL: 4-coumarate: CoA ligase, C3H:
4-coumarate 3-hydroxylase, COMT: caffeic acid O-methyl transferase, HCT: p-hydroxy cinnamoyl
transferase, CCoAOMT: caffeoyl-CoA O-methyltransferase, CHS: chalcone synthase.
2. Results
2.1. Identification of 4CL Genes from S. moellendorffii
We identified and cloned two putative 4CL genes (Sm4CL1: GenBank accession number
XP_002969881; Sm4CL2: XP_002979073) from the S. moellendorffii genome. The ORF of Sm4CL1
was 1614 bp, predicting a protein of 537 residues, with a molecular mass of 57.34 kDa and a pI of
5.88. The ORF of Sm4CL2 was 1695 bp, predicting a protein of 564 residues, with a molecular mass of
61.56 kDa and a pI of 5.87. Sm4CL1 contained five exons and four introns, while Sm4CL2 contained six
exons and five introns (Figure 2). Sm4CL genes had multiple introns and exons, consistent with 4CL
genes in other species [7].

Molecules 2018, 23, 595
3 of 11
Figure 2. Genome structure of Sm4CLs and other plant 4CL genes. Schematic diagrams with
exons (black arrow) and introns (gray box). The gene accession numbers are as follows: Pp4CL1
(Physcomitrella patens): EU167552; Pp4CL2 (P. patens): EU167553; Pp4CL3 (P. patens): EU167554; Pp4CL4
(P. patens): EU167555.
2.2. Protein Sequence and Phylogenetic Analysis of Sm4CLs
The predicted proteins Sm4CL1 and Sm4CL2 had 55–60% sequence identity with 4CL proteins
in other species of plants (At4CL2 and Pt4CL). Our multiple sequence alignment indicated that
both Sm4CLs contained two motifs that are conserved across the 4CL family in plants: a putative
AMP-binding motif (box I) and a conserved box II “GEICIRG” domain (Figure 3). The central cysteine
residue of box II might be involved in catalysis [15].
Figure 3. Sequence alignment of Sm4CLs with other plant 4CLs. Box I represents the putative
AMP-binding domain, and box II represents the conserved “GEICIGR” putative catalytic site. Sequence
accession numbers are given in Supplementary Data Table S2.

Molecules 2018, 23, 595
4 of 11
Our neighbor-joining phylogenetic analysis divided the 4CL proteins into four clades: one clade of
Sm4CL1, Sm4CL2, and 4CL proteins from mosses and gymnosperms; and three angiosperm 4CL clades
(class I, class II, and class III) (Figure 4). Angiosperm class I 4CLs are more closely associated with lignin
biosynthesis, as demonstrated by antisense and RNAi suppression studies [
class II 4CLs are likely involved the biosynthesis of other phenolic compounds, based on their
compartmentalized expression in various plant organs and tissues [10 18]. Angiosperm class I and
6,16,17]. Angiosperm
,
II 4CLs are predominantly present in dicots, whereas class III 4CLs are largely restricted to monocots.
Figure 4. Neighbor-joining phylogeny of Sm4CLs and previously published 4CL gene sequences from
other species. Sequence accession numbers are given in Supplementary Data Table S2.
2.3. Biochemical Characterization of Recombinant Sm4CL In Vitro
We examined the biochemical properties of the Sm4CLs by synthesizing these enzymes in
Escherichia coli. Following purification of the corresponding recombinant 4CLs, fractionation with
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the molecular
masses were between 75 and 79 kDa (including the tags) (Figure 5). We tested whether the purified
proteins utilized p-coumaric acid, dihydro-p-coumaric acid, trans-cinnamic acid, caffeic acid, ferulic
acid, and sinapic acid. Both Sm4CL1 and Sm4CL2 had distinct CoA ligation activity with all
substrates except sinapic acid (Table 1). Reaction products were identified by HPLC using p-coumaroyl
CoA, dihydro-p-coumaroyl CoA, caffeoyl CoA, and feruloyl CoA as product standards (Figure 6).
We determined the effects of pH and temperature on the enzyme activity with p-coumaric acid as
substrate. The enzymatic activities of the Sm4CLs were pH-dependent and temperature-sensitive. The
optimum pH for both Sm4CL1 and Sm4CL2 was about 7.0. The optimum temperature for Sm4CL1
◦
◦
was 40 C, while for Sm4CL2, it was 50 C. Under optimal conditions, recombinant Sm4CL1 showed a
high affinity for p-coumaric acid (Km = 11.89 M) and caffeic acid (Km = 10.87 M) (Table 2). However,
µ
µ

Molecules 2018, 23, 595
5 of 11
the substrate turnover rate (kcat) was higher with p-coumaric acid than with caffeic acid, resulting in a
higher catalytic efficiency (kenz) with p-coumaric acid. Thus, p-coumaric acid was the best substrate
for Sm4CL1.
Similarly, Sm4CL2 showed a higher affinity for p-coumaric acid (Km = 19.67
µM) and caffeic
acid (Km = 18.96 M), and a higher substrate turnover rate with p-coumaric acid, ferulic acid,
µ
and dihydro-p-coumaric acid (Table 2). Based on the Km value and the substrate turnover rate,
we considered p-coumaric acid the best substrate for Sm4CL2.
We also found that the turnover rate of Sm4CL2 was much higher than that of Sm4CL1: the kcat
of p-coumaric acid for Sm4CL2 was 42.91 min−1, and while that of Sm4CL1 was only 4.09 min−1
.
Figure 5. SDS-PAGE analysis of purified recombinant proteins from E. coli. Lane M: molecular mass
standards; lane 1: empty pET32a vector; lane 2: pET32a-Sm4CL1; lane 3: pET32a-Sm4CL2.
Table 1. The substrate specificity of Sm4CLs.
−1
−1
)
Specific Activity (nmol mg min
Substrate
Sm4CL1
Sm4CL2
p-coumaric acid
caffeic acid
cinnamic acid
ferulic acid
dihydro-p-coumaric acid
sinapic acid
71.77 ± 0.31
16.92 ± 0.2
2.29 ± 0.08
1.14 ± 0.00
16.71 ± 0.45
ND
557.5 ± 15.58
239.71 ± 5.6
283.08 ± 5.22
494.83 ± 3.61
271.77 ± 29.57
ND
ND: No detectable activity.
Table 2. Kinetic parameters of Sm4CLs.
Vmax
(nmol mg min
−1
−1
−1
)
Enzyme
Substrate
Km (µM)
kcat (min
)
kenz (M min
−1
−1
)
p-coumaric acid
caffeic acid
di-p-coumaric acid
11.89 ± 1.56
10.87 ± 1.6
71.37 ± 2.58
25.40 ± 0.94
43.73 ± 1.34
4.09 ± 0.15
343,986.54
134,314.63
5673.6
1.46 ± 0.05
2.51 ± 0.08
Sm4CL1
442.4 ± 53.74
p-coumaric acid
caffeic acid
cinnamic acid
ferulic acid
19.67 ± 2.92
18.96 ± 2.61
126.9 ± 11.57
95.99 ± 13.39
220.1 ± 36.86
697.1 ± 32.06
268.1 ± 9.74
382 ± 9.31
42.91 ± 1.97
16.50 ± 0.60
23.52 ± 0.57
32.33 ± 1.3
41.44 ± 1.43
2,181,494.66
870,253.16
185,310.64
336,819.59
188,278.06
Sm4CL2
525.2 ± 21.18
673.1 ± 23.19
di-p-coumaric acid

Molecules 2018, 23, 595
6 of 11
Figure 6. HPLC profiles of reaction products generated by recombinant Sm4CLs. Recombinant Sm4CL
enzymes were provided with p-coumaric acid, dihydro-p-coumaric acid, caffeic acid, ferulic acid, and
cinnamic acid as substrates. We used a bacterial clone inoculated with an empty pET32a vector to
produce the control reaction product.
2.4. Determination of Gene Expression Patterns in Tissues
Transcription of Sm4CL1, Sm4CL2 was detected in roots, stems and leaves, but the expression
level of the two genes was different. Sm4CL1 was most highly expressed in roots, then stems, then
leaves. Sm4CL2 was also the most highly expressed in roots like Sm4CL1, but its expression level in the
leaves was higher than in the stems (Figure 7).
Figure 7. Expression of Sm4CL genes in roots, stems, and leaves of S. moellendorffii. Values represent
the mean ± SD, measured from at least three biological replicates.

Molecules 2018, 23, 595
7 of 11
3. Discussion
Selaginella species are evolutionarily important among land plants: these are the oldest vascular
plants and are distinguished from primitive bryophytes by the development of vascular tissue capable
of transporting fluids throughout the plant body. 4CL is the pivotal enzyme that controls the flow
of carbon through the phenylpropanoid metabolic pathway into the specific lignin, flavonoid, and
coumarin biosynthesis pathways. The 4CL genes have been widely investigated in other lineages of
vascular plants, but little is known about 4CL genes in Selaginella. A better understanding of the 4CL
genes in Selaginella will inform our understanding of the evolution of 4CLs across all vascular plants.
We cloned two 4CL genes from S. moellendorffii, based on the enzymatic activities of the
recombinant proteins, both of these genes encoded bona fide 4CLs. This was consistent with previous
reports that the 4CL genes exist as a gene family in most vascular plants [10,19–21]. We noticed that
the properties of the Sm4CL genes we investigated were similar to those of 4CLs in other vascular
plants: their genetic structure was made up of multiple introns and exons, they contained conserved
putative catalytic domains, and they had close phylogenetic relationships with other vascular plant
4CLs. Sm4CL1 and Sm4CL2 were more highly expressed in the roots than in the stems or the leaves; this
is consistent with previous studies [22–24]. These results indicated the 4CL genes are evolutionarily
conserved, underlining the importance of 4CL as a key enzyme in the phenylpropanoid metabolism of
all vascular plants.
Although the Sm4CL1 and Sm4CL2 isoforms clustered together in our phylogeny, the activities
of these two enzymes were substantially different. The catalytic efficiency of Sm4CL2 toward
hydroxycinnamic acids was similar to that of other plant 4CLs [22,23,25,26], but the enzymatic
activity of Sm4CL1 toward hydroxycinnamic acids was weak. Our kinetic analysis indicated that
Sm4CL2 efficiently catalyzed the conversions of p-coumaric acid, caffeic acid, and ferulic acid into
their corresponding CoA esters. Meanwhile, lignin in Selaginella species is comprised of three major
types of aromatic units: p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S), which are derived from
p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, respectively [3,27–31]. Therefore, it is likely
that Sm4CL2 plays a role in the biosynthesis of the p-hydroxyphenyl and guaiacyl lignin subunits.
The lack of any detectable conversion of sinapate by each Sm4CL isoforms, and the existence of
ferulate 5-hydroxylation and O-methylation pathway which operates on feruloyl-CoA to produce
sinapoyl-CoA [32,33], suggested that the biosynthesis of sinapyl alcohol and syringyl lignin occurs via
an independent 4CL pathway in S. moellendorffii.
In summary, we identified two 4CL genes from the S. moellendorffii genome. These genes were
expressed in all tested organs. Our bioinformatic characterization and in vitro enzyme assay indicated
that typical 4CL genes were evolutionarily conserved across the vascular plants. The enzyme activities
of Sm4CLs were interesting, which included a highly-active enzyme and a very weakly active enzyme.
This paper provides a framework for future work on the phenylpropanoid metabolic pathway in S.
moellendorffii.
4. Materials and Methods
4.1. Plant Material, Nucleic Acid Extraction, and Reagents
◦
S. moellendorffii was grown in a greenhouse under controlled conditions: 25 C with a 12 h
photoperiod. Genomic DNA was extracted from fresh ferny foliage using the CTAB method [34]. Total
RNA was extracted from foliage using the modified CTAB method [35], and was used as a template for
cDNA synthesis using PrimeScript RT Master Mix (Takara, Otsu, Japan), following the manufacturer’s
protocols. All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA),
unless otherwise indicated. p-Coumaroyl CoA, dihydro-p-coumaroyl CoA, caffeoyl CoA, and feruloyl
CoA were enzymatically synthesized following previously-published procedures [36–38].

Molecules 2018, 23, 595
8 of 11
4.2. DNA Isolation and Sequence Analysis
We identified two putative 4CL genes in the genome of S. moellendorffii v.1.0 (http://phytozome.
jgi.doe.gov/pz/portal.html) [39] using the TblastN algorithm and designated these putative genes
Sm4CL1 and Sm4CL2. The full-length cDNA sequences of these genes were amplified using the
corresponding primer pairs (Sm4CL1-F/R, Sm4CL2-F/R; Supplementary Data Table S1). The Sm4CL
genomic DNA sequences were also obtained from genomic DNA with the same corresponding primer
pairs (Supplementary Data Table S1). Predicted polypeptide sequences were aligned using DNAMAN
v.7.0.2 (Lynnon Corp., Vaudreuil-Dorion, QC, Canada). Neighbor-joining phylogenetic trees were
constructed using MEGA v5.0 [40]. We tested tree robustness by running 1000 bootstrap replicates.
4.3. Recombinant Protein Expression and Purification
The open reading frames (ORFs) of Sm4CL were amplified with pET primer pairs (Table S1), and
subcloned into a pET32a vector. After the sequences were confirmed, each construct was transformed
into Escherichia coli strain BL21 (DE3) for heterologous expression. The transgenic cultures were
◦
incubated at 37 C until the OD600 reached 0.4–0.6, then the recombinant proteins were induced
◦
at 16 C for 16 h after adding 0.5 mM isopropyl
β-D-1-thiogalactopyranoside (IPTG). N-terminal
hexahistidine-tagged proteins were purified by passing through a Ni-NTA Sefinose His-bind column
(Bio Basic Inc., Markham, ON, Canada), and then were exchanged through an Ultrafiltration tube
(Millipore, Billerica, MA, USA) in the presence of binding buffer (20 mM Tris–HCl, 500 mM NaCl,
pH 8.0). Protein concentrations were determined with the Bradford reagent (Beyotime, Shanghai,
China), using BSA as a standard. The resulting purified proteins were monitored on SDS-PAGE, using
Coomassie Blue R250 staining. The migration of standard molecular weight markers (10–170 kDa)
(Fermentas, Waltham, MA, USA) was used to estimate the molecular masses of the target proteins.
4.4. Enzyme Assays
We performed Sm4CLs enzyme assays to detect the formation of the CoA esters of various
cinnamic acid derivatives. Each 200
ATP, 300
µL assay contained 10 µg purified protein, 200 µM substrate, 5 mM
M CoA, and 5 mM MgCl2, made up in 200 mM Tris-HCl buffer (pH 7.5). Enzymatic reactions
were incubated for 30 min at 30 ◦C, and the reaction products were analyzed using a HPLC device
µ
(1260 Infinity Binary LC system, Agilent, Santa Clara, CA, USA), equipped with a multi wavelength
detector. The samples were separated through a 5-µm reverse-phase XDB-C18 column with a flow rate
of 1 mL/min. A linear gradient of solvent A (1% H3PO4 in H2O) and solvent B (CH3CN) were applied
as follows: 0–5 min, 5% B isocratic; 5–35 min, 5–25% B linear; 35–36 min, 25–100% B linear. Standard
solutions of reference compounds were used for calibration.
The effects of pH and temperature on the enzyme activity were examined using p-coumaric
acid as substrate. To determine optimal pH, enzymatic activity was assessed in 200 mM Tris-HCl
buffer (pH 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0), while the optimal temperature was determined by
◦
measuring enzymatic activity at 10, 20, 30, 40, 50, and 60 C. All experiments were performed in
triplicate. Kinetic parameters were determined using different substrate concentrations. We performed
this experiment in triplicate, with 3 µg purified enzyme in a final volume of 200 µL of 200 mM Tris-HCl
buffer at the optimal pH and temperature for 10 min. The level of UV absorption was recorded at
1 min intervals. Relevant wavelengths were 311 nm (cinnamoyl CoA), 333 nm (p-coumaroyl CoA),
346 nm (caffeoyl CoA), 346 nm (feruloyl CoA), and 352 nm (sinapoyl CoA) [37,41,42]. For determining
kinetic properties with dihydro-p-coumaric acid, reactions were incubated at optimal temperature for
10 min and analyzed using HPLC, and then the quantity of the reaction product present was estimated
from a standard calibration curve.

Molecules 2018, 23, 595
9 of 11
4.5. Expression Profile Analysis
For real-time quantitative PCR, various S. moellendorffii tissues (roots, stems, and leaves) were
collected and snap-frozen in liquid N2. Total RNA was extracted and cDNA was synthesized as
described above. Transcript abundance was quantified using real-time quantitative PCR based on the
corresponding RT primer pairs (sequences given in Table S1), using an Eppendorf Mastercycler ep
realplex RealTime PCR System (Eppendorf, Hamburg, Germany). Each 10
µL reaction contained 1 µg
cDNA, 0.5 M of each primer, and 1 SYBR Green PCR Master mix. A fragment of the S. moellendorffii
µ
×
geneome encoding an actin gene was used as the internal reference sequence and was amplified using
primers Smactin-RTF/R (Table S1).
Supplementary Materials: The following are available online. Table S1: Primer sequences used in this research,
Table S2: Accession numbers of amino acid sequences used for phylogenetic reconstruction.
Acknowledgments: We are grateful for the support of the National Natural Science Foundation of China (no.
81630093 and no. 31370330).
Author Contributions: Ai-Xia Cheng and Hong-Xiang Lou designed the research. Xin-Yan Liu performed most
of the experiments with the help of Ping-Ping Wang and Yi-Feng Wu. Ai-Xia Cheng and Xin-Yan Liu wrote the
paper. All authors discussed the results and edited the article and approved the final manuscript.
Conflicts of Interest: The authors declare that there is no conflict of interest in the present investigation.
References
1.
2.
Vogt, T. Phenylpropanoid biosynthesis. Mol. Plant 2010, 3, 2–20. [CrossRef] [PubMed]
Dixon, R.A.; Achnine, L.; Kota, P.; Liu, C.J.; Reddy, M.S.; Wang, L. The phenylpropanoid pathway and plant
defence-a genomics perspective. Mol. Plant Pathol. 2002, 3, 371–390. [CrossRef] [PubMed]
Boerjan, W.; Ralph, J.; Baucher, M. Lignin biosynthesis. Annu. Rev. Plant Biol. 2003, 54, 519–546. [CrossRef]
[PubMed]
Vassão, D.G.; Kim, K.-W.; Davin, L.B.; Lewis, N.G. Lignans (neolignans) and allyl/propenyl phenols:
Biogenesis, structural biology, and biological/human health considerations. In Comprehensive Natural
Products II Chemistry and Biology; Mander, L., Lui, H.-W., Eds.; Elsevier: Oxford, UK, 2010; Volume 1,
pp. 815–928.
3.
4.
5.
Hamberger, B.; Hahlbrock, K. The 4-coumarate:CoA ligase gene family in Arabidopsis thaliana comprises one
rare, sinapate-activating and three commonly occurring isoenzymes. Proc. Natl. Acad. Sci. USA 2004, 101,
2209–2214. [CrossRef] [PubMed]
6.
7.
8.
Gui, J.; Shen, J.; Li, L. Functional characterization of evolutionarily divergent 4-coumarate:coenzyme A
ligases in rice. Plant Physiol. 2011, 157, 574–586. [CrossRef] [PubMed]
Silber, M.V.; Meimberg, H.; Ebel, J. Identification of a 4-coumarate:CoA ligase gene family in the moss,
physcomitrella patens. Phytochemistry 2008, 69, 2449–2456. [CrossRef] [PubMed]
Becker-Andre, M.; Schulze-Lefert, P.; Hahlbrock, K. Structural comparison, modes of expression, and putative
cis-acting elements of the two 4-coumarate:CoA ligase genes in potato. J. Biol. Chem. 1991, 266, 8551–8559.
[PubMed]
9.
Lozoya, E.; Hoffmann, H.; Douglas, C.; Schulz, W.; Scheel, D.; Hahlbrock, K. Primary structures and catalytic
properties of isoenzymes encoded by the two 4-coumarate:CoA ligase genes in parsley. Eur. J. Biochem. 1988
176, 661–667. [CrossRef] [PubMed]
,
10. Hu, W.J.; Kawaoka, A.; Tsai, C.J.; Lung, J.; Osakabe, K.; Ebinuma, H.; Chiang, V.L. Compartmentalized
expression of two structurally and functionally distinct 4-coumarate:CoA ligase genes in aspen
(Populus tremuloides). Proc. Natl. Acad. Sci. USA 1998, 95, 5407–5412. [CrossRef] [PubMed]
11. Banks, J.A. Selaginella and 400 million years of separation. Annu. Rev. Plant Biol. 2009, 60, 223–238.
[CrossRef] [PubMed]
12. Weng, J.K.; Noel, J.P. Chemodiversity in Selaginella: A reference system for parallel and convergent metabolic
evolution in terrestrial plants. Front. Plant Sci. 2013, 4, 119. [CrossRef] [PubMed]

Molecules 2018, 23, 595
10 of 11
13. Wang, W.; Tanurdzic, M.; Luo, M.; Sisneros, N.; Kim, H.R.; Weng, J.K.; Kudrna, D.; Mueller, C.;
Arumuganathan, K.; Carlson, J.; et al. Construction of a bacterial artificial chromosome library from
the spikemoss Selaginella moellendorffii: A new resource for plant comparative genomics. BMC Plant Biol.
2005, 5, 10. [CrossRef] [PubMed]
14. Banks, J.A.; Nishiyama, T.; Hasebe, M.; Bowman, J.L.; Gribskov, M.; dePamphilis, C.; Albert, V.A.; Aono, N.;
Aoyama, T.; Ambrose, B.A.; et al. The Selaginella genome identifies genetic changes associated with the
evolution of vascular plants. Science 2011, 332, 960–963. [CrossRef] [PubMed]
15. Stuible, H.P.; Buttner, D.; Ehlting, J.; Hahlbrock, K.; Kombrink, E. Mutational analysis of 4-coumarate:CoA
ligase identifies functionally important amino acids and verifies its close relationship to other
adenylate-forming enzymes. FEBS Lett. 2000, 467, 117–122. [CrossRef]
16. Kajita, S.; Hishiyama, S.; Tomimura, Y.; Katayama, Y.; Omori, S. Structural characterization of modified
lignin in transgenic tobacco plants in which the activity of 4-coumarate:coenzyme A ligase is depressed.
Plant Physiol. 1997, 114, 871–879. [CrossRef] [PubMed]
17. Wagner, A.; Donaldson, L.; Kim, H.; Phillips, L.; Flint, H.; Steward, D.; Torr, K.; Koch, G.; Schmitt, U.; Ralph, J.
Suppression of 4-coumarate-CoA ligase in the coniferous gymnosperm Pinus radiata. Plant Physiol. 2009, 149,
370–383. [CrossRef] [PubMed]
18. Ehlting, J.; Buttner, D.; Wang, Q.; Douglas, C.J.; Somssich, I.E.; Kombrink, E. Three 4-coumarate:coenzyme A
ligases in Arabidopsis thaliana represent two evolutionarily divergent classes in angiosperms. Plant J. 1999, 19,
9–20. [CrossRef] [PubMed]
19. Lee, D.; Meyer, K.; Chapple, C.; Douglas, C.J. Antisense suppression of 4-coumarate:coenzyme A ligase
activity in Arabidopsis leads to altered lignin subunit composition. Plant Cell 1997, 9, 1985–1998. [CrossRef]
[PubMed]
20. Koutaniemi, S.; Warinowski, T.; Karkonen, A.; Alatalo, E.; Fossdal, C.G.; Saranpaa, P.; Laakso, T.;
Fagerstedt, K.V.; Simola, L.K.; Paulin, L.; et al. Expression profiling of the lignin biosynthetic pathway in
norway spruce using EST sequencing and real-time RT-PCR. Plant Mol. Biol. 2007, 65, 311–328. [CrossRef]
[PubMed]
21. Kumar, A.; Ellis, B.E. 4-coumarate:CoA ligase gene family in Rubus idaeus: cDNA structures, evolution,
and expression. Plant Mol. Biol. 2003, 51, 327–340. [CrossRef] [PubMed]
22. Wang, C.H.; Yu, J.; Cai, Y.X.; Zhu, P.P.; Liu, C.Y.; Zhao, A.C.; Lu, R.H.; Li, M.J.; Xu, F.X.; Yu, M.D.
Characterization and functional analysis of 4-coumarate:CoA ligase genes in Mulberry. PLoS ONE 2016, 11,
e0155814. [CrossRef] [PubMed]
23. Rastogi, S.; Kumar, R.; Chanotiya, C.S.; Shanker, K.; Gupta, M.M.; Nagegowda, D.A.; Shasany, A.K.
4-Coumarate:CoA ligase partitions metabolites for eugenol biosynthesis. Plant Cell Physiol. 2013, 54,
1238–1252. [CrossRef] [PubMed]
24. Liu, T.T.; Yao, R.L.; Zhao, Y.C.; Xu, S.; Huang, C.L.; Luo, J.; Kong, L.Y. Cloning, functional characterization
and site-directed mutagenesis of 4-coumarate:coenzyme A ligase (4CL) involved in coumarin biosynthesis
in Peucedanum praeruptorum dunn. Front. Plant Sci. 2017, 8. [CrossRef]
25. Chen, H.Y.; Babst, B.A.; Nyamdari, B.; Hu, H.; Sykes, R.; Davis, M.F.; Harding, S.A.; Tsai, C.J. Ectopic
expression of a loblolly pine class II 4-coumarate:CoA ligase alters soluble phenylpropanoid metabolism but
not lignin biosynthesis in populus. Plant Cell Physiol. 2014, 55, 1669–1678. [CrossRef] [PubMed]
26. Li, Z.B.; Li, C.F.; Li, J.; Zhang, Y.S. Molecular cloning and functional characterization of two divergent
4-coumarate:coenzyme A ligases from Kudzu (pueraria lobata). Biol. Pharm. Bull. 2014, 37, 113–122. [CrossRef]
[PubMed]
27. Logan, K.J.; Thomas, B.A. Distribution of lignin derivatives in plants. New Phytol. 1985, 99, 571–585.
[CrossRef]
28. Jin, Z.F.; Matsumoto, Y.; Tange, T.; Akiyama, T.; Higuchi, M.; Ishii, T.; Iiyama, K. Proof of the presence of
guaiacyl-syringyl lignin in Selaginella tamariscina. J. Wood Sci. 2005, 51, 424–426. [CrossRef]
29. Weng, J.K.; Li, X.; Stout, J.; Chapple, C. Independent origins of syringyl lignin in vascular plants. Proc. Natl.
Acad. Sci. USA 2008, 105, 7887–7892. [CrossRef] [PubMed]
30. Yoon, J.; Choi, H.; An, G. Roles of lignin biosynthesis and regulatory genes in plant development. J. Integr.
Plant Biol. 2015, 57, 902–912. [CrossRef] [PubMed]
31. Rogers, L.A.; Campbell, M.M. The genetic control of lignin deposition during plant growth and development.
New Phytol. 2004, 164, 17–30. [CrossRef]

Molecules 2018, 23, 595
11 of 11
32. Weng, J.K.; Akiyama, T.; Bonawitz, N.D.; Li, X.; Ralph, J.; Chapple, C. Convergent evolution of syringyl
lignin biosynthesis via distinct pathways in the lycophyte Selaginella and flowering plants. Plant Cell 2010
,
22, 1033–1045. [CrossRef] [PubMed]
33. Weng, J.K.; Akiyama, T.; Ralph, J.; Chapple, C. Independent recruitment of an o-methyltransferase for
syringyl lignin biosynthesis in Selaginella moellendorffii. Plant Cell 2011, 23, 2708–2724. [CrossRef] [PubMed]
34. Porebski, S.; Bailey, L.G.; Baum, B.R. Modification of a CTAB DNA extraction protocol for plants containing
high polysaccharide and polyphenol components. Plant Mol. Biol. Rep. 1997, 15, 8–15. [CrossRef]
35. Gambino, G.; Perrone, I.; Gribaudo, I. A rapid and effective method for RNA extraction from different tissues
of grapevine and other woody plants. Phytochem. Anal. PCA 2008, 19, 520–525. [CrossRef] [PubMed]
36. Beuerle, T.; Pichersky, E. Enzymatic synthesis and purification of aromatic coenzyme A esters. Anal. Biochem.
2002, 302, 305–312. [CrossRef] [PubMed]
37. Meng, H.B.; Campbell, W.H. Facile enzymic synthesis of caffeoyl CoA. Phytochemistry 1997, 44, 605–608.
[CrossRef]
38. Obel, N.; Scheller, H.V. Enzymatic synthesis and purification of caffeoyl-CoA, p-coumaroyl-CoA, and
feruloyl-CoA. Anal. Biochem. 2000, 286, 38–44. [CrossRef] [PubMed]
39. Phytozome v12.1: Home. Available online: https://phytozome.jgi.doe.gov/pz/portal.html (accessed on 12
February 2018).
40. Tamura, K.; Dudley, J.; Nei, M.; Kumar, S. Mega4: Molecular evolutionary genetics analysis (MEGA) software
version 4.0. Mol. Biol. Evol. 2007, 24, 1596–1599. [CrossRef] [PubMed]
41. Luderitz, T.; Schatz, G.; Grisebach, H. Enzymic synthesis of lignin precursors. Purification and properties
of 4-coumarate:CoA ligase from cambial sap of spruce (Picea abies L.). Eur. J. Biochem. 1982, 123, 583–586.
[CrossRef] [PubMed]
42. Stockigt, J.; Zenk, M.H. Chemical syntheses and properties of hydroxycinnamoyl-coenzyme a derivatives.
Z. Naturforsch. C 1975, 30, 352–358. [PubMed]
Sample Availability: Samples of the compounds p-Coumaroyl CoA, dihydro-p-coumaroyl CoA, caffeoyl CoA,
and feruloyl CoA are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).