Identification of a 4-coumarate:CoA ligase gene family in the moss, Physcomitrella patens

Article abstract of DOI:10.1016/j.phytochem.2008.06.014

Since the early evolution of land plants from primitive green algae, phenylpropanoid compounds have played an important role. In the biosynthesis of phenylpropanoids, 4-coumarate:CoA ligase (4CL; EC 6.2.1.12) has a pivotal role at the divergence point from general phenylpropanoid metabolism to several major branch pathways. Although higher plant 4CLs have been extensively studied, little information is available on the enzymes from bryophytes. In Physcomitrella patens, we have identified a 4CL gene family consisting of four members, taking advantage of the available EST sequences and a draft sequence of the P. patens genome. The encoded proteins of three of the genes display similar substrate utilization profiles with highest catalytic efficiency towards 4-coumarate. Interestingly, the efficiency with cinnamate as substrate is in the same range as with caffeate and ferulate. The deduced proteins of the four genes share sequence identities between 78% and 86%. The intron/exon structures are pair wise similar. Pp4CL2 and Pp4CL3 each consists of four exons and three introns, whereas Pp4CL1 and Pp4CL4 are characterized each by five exons and four introns. Pp4CL1, Pp4CL2 and Pp4CL3 are expressed in both gametophore and protonema tissue of P. patens, unlike Pp4CL4 whose expression could not be demonstrated under the conditions employed. Phylogenetic analysis suggests an early evolutionary divergence of Pp4CL gene family members. Using Streptomyces coelicolor cinnamate:CoA ligase (ScCCL) as an outgroup, the P. patens 4CLs are clearly separated from the spermatophyte proteins, but are intercalated between the angiosperm 4CL class I and class II. A comparison of three P. patens subspecies from diverse geographical locations shows high sequence identities for the four 4CL isoforms.

Full text of DOI:10.1016/j.phytochem.2008.06.014

Phytochemistry 69 (2008) 2449–2456
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Identification of a 4-coumarate:CoA ligase gene family in the moss,
Physcomitrella patens q
Martina V. Silber a, Harald Meimberg b, Jürgen Ebel a,
*
a Department Biologie I – Botanik, Ludwig-Maximilians-Universität, Menzinger Strasse 67, D-80638 München, Germany
b CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão, R. Monte-Crasto, 4485-661 Vairão, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Since the early evolution of land plants from primitive green algae, phenylpropanoid compounds have
played an important role. In the biosynthesis of phenylpropanoids, 4-coumarate:CoA ligase (4CL; EC
6.2.1.12) has a pivotal role at the divergence point from general phenylpropanoid metabolism to several
major branch pathways. Although higher plant 4CLs have been extensively studied, little information is
available on the enzymes from bryophytes. In Physcomitrella patens, we have identified a 4CL gene family
consisting of four members, taking advantage of the available EST sequences and a draft sequence of the
P. patens genome. The encoded proteins of three of the genes display similar substrate utilization profiles
with highest catalytic efficiency towards 4-coumarate. Interestingly, the efficiency with cinnamate as
substrate is in the same range as with caffeate and ferulate. The deduced proteins of the four genes share
sequence identities between 78% and 86%. The intron/exon structures are pair wise similar. Pp4CL2 and
Pp4CL3 each consists of four exons and three introns, whereas Pp4CL1 and Pp4CL4 are characterized each
by five exons and four introns. Pp4CL1, Pp4CL2 and Pp4CL3 are expressed in both gametophore and pro-
tonema tissue of P. patens, unlike Pp4CL4 whose expression could not be demonstrated under the condi-
tions employed. Phylogenetic analysis suggests an early evolutionary divergence of Pp4CL gene family
members. Using Streptomyces coelicolor cinnamate:CoA ligase (ScCCL) as an outgroup, the P. patens
4CLs are clearly separated from the spermatophyte proteins, but are intercalated between the angio-
sperm 4CL class I and class II. A comparison of three P. patens subspecies from diverse geographical loca-
tions shows high sequence identities for the four 4CL isoforms.
Received 11 October 2007
Received in revised form 7 May 2008
Available online 21 August 2008
Keywords:
Physcomitrella patens
Funariaceae
Moss
Enzyme evolution
Phenylpropanoid metabolism
4-Coumarate:CoA ligase
Multigene family
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Fig. 1) and a number of related substrates to the corresponding CoA
thioesters catalyzed by 4-coumarate:CoA ligase (4CL; EC 6.2.1.12).
The bryophytes contain a large number of secondary com-
pounds, several of which are bioactive constituents which protect
the organisms from herbivores, fungi, bacteria, slugs, and snails.
Among the secondary constituents, major phenylpropanoid-de-
rived classes of compounds are flavonoids, bibenzyls, and bis(bib-
enzyl)s with widespread occurrence in liverworts and mosses
(Markham, 1988; Asakawa, 2001). Central to many of phenylprop-
anoid biosynthetic pathways is the activation of 4-coumarate (2 in
The CoA thioester products constitute common C9 building units
for proposed specific branch pathways subsequent to general phe-
nylpropanoid metabolism.
Because of the importance of phenylpropanoid-derived com-
pounds in plants, 4CL has been the subject of extensive study for
many years, mainly in higher plants. It has been shown to occur
in the form of multiple isoenzymes with either similar (e.g. Lozoya
et al., 1988) or distinct (e.g. Lindermayr et al., 2002) substrate affin-
ities. More recently, a gene encoding a 4CL-like enzyme with more
than 40% identity in amino acid sequence to higher plant 4CLs was
found in the genome of the filamentous bacterium, Streptomyces
coelicolor A3(2) (Kaneko et al., 2003). The recombinant enzyme
showed distinct 4CL activity with highest catalytic efficiency to-
wards cinnamate (1). However, only limited information is avail-
able on 4CL and other enzymes of phenylpropanoid biosynthesis
in bryophytes. Recently, a multigene family of chalcone synthase,
the enzyme catalyzing the committed step of flavonoid biosynthe-
sis, was characterized in the moss, Physcomitrella patens (Jiang
et al., 2006).
Abbreviations: 4CL, 4-coumarate:CoA ligase; EST, expressed sequence tag
q
Data deposition: EU167552 (Pp4CL1, P. patens ssp. patens), EU167553 (Pp4CL2, P.
patens ssp. patens), EU167554 (Pp4CL3, P. patens ssp. patens), EU167555 (Pp4CL4, P.
patens ssp. patens), EU179312 (Pp4CL1, P. patens ssp. californica), EU179313
(Pp4CL2, P. patens ssp. californica), EU179314 (Pp4CL3, P. patens ssp. californica),
EU179315 (Pp4CL4, P. patens ssp. californica), EU180599 (Pp4CL1, P. patens ssp.
magdalenae), EU180600 (Pp4CL2, P. patens ssp. magdalenae), EU180601 (Pp4CL3, P.
patens ssp. magdalenae), EU180602 (Pp4CL4, P. patens ssp. magdalenae).
* Corresponding author. Tel.: +49 (0)89 17861285; fax: +49 (0)89 1782274.
E-mail address: j.ebel@lrz.uni-muenchen.de (J. Ebel).
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.06.014

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M.V. Silber et al. / Phytochemistry 69 (2008) 2449–2456
Table 1
O—
O
Gene specific sequences of oligonucleotides to amplify putative Physcomitrella patens
4CL1–4 genes and transcripts
R1, R2, R3 = H, cinnamate (1)
Gene
Designation
Sequence
R1 = OH, R2, R3 = H, 4-coumarate (2)
R1, R2 = OH, R3 = H, caffeate (3)
R1 = OH, R2 = OCH3, R3 = H, ferulate (4)
R1 = OH, R2, R3 = OCH3, sinapate (5)
Pp4CL1
Pp4CL1_50UTR
Pp4CL1_30UTR
Pp4CL1RT_PCRfor
Pp4CL1RT_PCRrev
50-TATTGTAGAGTCCGATAGCGTC
50-TAAACCCTAAGTTGCAGGATCTG
50-TGGAATGACAGAAGCTGGTCC
50-CCTAAGTTGCAGGATCTGGCTC
R3
R2
Pp4CL2
Pp4CL3
Pp4CL4
Pp4CL2_50UTR
Pp4CL2_30UTR
Pp4CL2RT_PCRfor
Pp4CL2RT_PCRrev
50-GGAAGCAAGAGTTTCGGAG
50-ACATTGTGGGAGGATACCGC
50-AGCATCAGGATGGTGATGTCCG
50-ACATTGTGGGAGGATACCGC
R1
Fig. 1. Chemical structures of the five naturally occurring 4CL substrates investi-
gated in this study.
Pp4CL3_50UTR
Pp4CL3_30UTR
Pp4CL3RT_PCRfor
Pp4CL3RT_PCRrev
50-ACAGATCGGTGAGCACATTGG
50-TGTTTCAGGAATGGATGCGTC
50-CCAGGATCTTGTGGCACTGTC
50-AGGCTCACGATTCCATCCGAC
P. patens has drawn much attention because of its evolutionary
position among land plants and of its exceptional genetic proper-
ties (Schaefer and Zrÿd, 2001). Thus, P. patens may represent an
ideal case for studies on the evolution of metabolic pathways
including phenylpropanoid biosynthesis. As a result of multiple
sequencing projects, nucleic acid sequences of >100,000 expressed
sequence tags (ESTs) and >16,000 annotated transcripts are avail-
able (Nishiyama et al., 2003; Lang et al., 2005). In addition, the first
draft sequence of the P. patens genome has recently been released
(http://shake.jgi-psf.org/Phypa1/Phypa1.home.html; Rensing et al.,
2008). In this study, by exploiting the various public resources, the
existence of a family of four closely related 4CL genes in the moss,
P. patens, was demonstrated. For three of the encoded proteins, we
verified the identity as 4CL isoenzymes and demonstrated that the
respective genes are expressed in moss tissues of different devel-
opmental stages. Phylogenetic reconstructions indicated that the
four Pp4CL proteins are closely related and may have resulted from
comparably recent gene duplication events. They are forming to-
gether with the gymnosperm proteins the neighbour group of
the angiosperm 4CL proteins of class II. This indicates that the split
between 4CL class I and II occurred very early in the evolution of
green land plants.
Pp4CL4_50UTR
Pp4CL4_30UTR
Pp4CL4RT_PCRfor
Pp4CL4RT_PCRrev
50-TAGGTTCGGTAATCCTCCACC
50-TGGTATGGTTCCCGTATCGAG
50-TGGCAAGGAGCTTGAGGATGC
50-TCTTGAAACCTACGGATGAAGACTC
3 cDNAs were synthesized as N-terminally His6-tagged proteins in
Escherichia coli and their immunogenic cross-reactivity was dem-
onstrated by using an antiserum raised against parsley 4CL (Ragg
et al., 1981; data not shown). After immobilized metal affinity
chromatography, the three recombinant proteins revealed the rel-
ative molecular masses in SDS/polyacrylamide gels of 65 kDa for
Pp4CL1 and Pp4CL2, respectively, and 64 kDa for Pp4CL3.
The enzyme-kinetic analyses of the purified recombinant pro-
teins (Table 2) verified their biochemical function as bona fide
4CLs. All of the enzymes displayed Michaelis–Menten kinetics,
however with the inherent problem of instability of the catalytic
activity of the affinity-purified proteins. We therefore decided to
estimate Km and relative Vmax values which limits to some extent
the comparison of the data for the different iosoenzymes in terms
of their molar activities. Pp4CL1–3 efficiently converted cinnamate
(1), 4-coumarate (2), caffeate (3), and ferulate (4) to the corre-
sponding CoA esters. According to the Km and Vmax/Km values, cin-
namate (1), which is poorly activated by many higher plant 4CLs,
was converted at a rate similar to that for caffeate (3) and ferulate
(4), whereas 4-coumarate (2) proved to be the most efficient of the
four tested naturally occurring 4CL substrates (Table 2). Sinapate
(5) was not accepted as a substrate under the experimental condi-
tions, in line with most previously characterized 4CLs from a wide
range of higher plant species.
2. Results
2.1. Isolation of cDNAs and genes, heterologous expression, and
biochemical characterization of Pp4CL isoforms
Analysis of the publicly available P. patens ESTs (Physcomitrella
GenBank EST databases) was performed using the following selec-
tive criteria for the identification of putative 4CL sequences: (i) the
presence and identity of 4CL-specific nucleotide sequences which
encode peptides representing the putative AMP-binding domain
(Box I) and the conserved GEICIRG motif (Box II) (Stuible and Kom-
brink, 2001) within candidate proteins; (ii) sequences with signif-
icant homology to the soybean Gm4CL1 cDNA. The search revealed
the presence of one putative Pp4CL cDNA which was designated
Pp4CL1. The presence of putative Pp4CL2–4 genes was indicated
by either screening of a bacterial artificial chromosome (BAC) li-
brary (Centre of Plant Sciences, LIBA, Leeds University, UK)
(Pp4CL2) or analysis of the publicly accessible P. patens genome se-
quence data (Joint Genome Institute of US Department of Energy,
USA) (Pp4CL3 and Pp4CL4). Full-length cDNA clones for Pp4CL1
and Pp4CL3 were obtained from RIKEN BioResource Center, Japan
(Nishiyama et al., 2003), whereas the Pp4CL2 cDNA was cloned
from total RNA isolated from P. patens gametophores by RT-PCR
using gene specific primers as detailed in Table 1. No EST clone ex-
isted for Pp4CL4 and no expression of this gene could be detected
under the conditions used in this study (see below). We therefore
did not attempt to isolate a cDNA representing this isoform. Clon-
ing of the Pp4CL1–4 genes was performed by amplifying genomic
DNA products using Pfu DNA polymerase and pairs of oligonucleo-
tide primers as listed in Table 1. The enzymes encoded by Pp4CL1–
2.2. Comparative analysis of Pp4CL gene family
The nucleotide and amino acid sequence similarities within the
Pp4CL gene family share a percentage of identity between 78% and
86% (amino acid sequences) and 77–83% (nucleotide sequences of
the central part of the coding region of the genes omitting about
180 base pairs of the N-terminus). The overall identity of the Pp4CL
isoforms versus 4CLs from higher plants is about 62%. When com-
pared with 4CLs from higher plants, Pp4CL1–4 contained an exten-
sion of about 30 amino acids at the N-terminal part of the proteins.
At present it is not known whether this N-terminal extension has
any functional impact on the Pp4CLs.
Fig. 2 illustrates the exon/intron structures of the four Pp4CL
genes. Pp4CL2 and Pp4CL3 each contain four exons and three in-
trons of similar sizes, respectively. For both, Pp4CL1 and Pp4CL4,
the major difference to the former pair is represented by an addi-
tional intron in the first exon.
To describe the expression patterns of each member of the
Pp4CL gene family, specific oligonucleotides were designed from
the particular 30 untranslated and 50 coding region of the cDNAs.

M.V. Silber et al. / Phytochemistry 69 (2008) 2449–2456
2451
Table 2
Kinetic properties of P. patens Pp4CL1–3 in vitro
Substrate
Km
(
l
M)
Specific activity relative Vmax
Relative Vmax/Km
Pp4CL1
(l )
Mꢀ1
Pp4CL1
Pp4CL2
Pp4CL3
Pp4CL1
Pp4CL2
Pp4CL3
Pp4CL2
Pp4CL3
Cinnamate (1)
4-Coumarate (2)
Caffeate (3)
50
4
25
66
16
45
73
64
29
725
800
61
100
24
59
100
46
57
100
190
195
1.19
25.97
0.9
0.89
6.25
1.0
0.89
3.5
0.26
0.25
Ferulate (4)
187
114
108
0.61
1.4
All data are mean values from three independent experiments with affinity-purified proteins. Relative Vmax values were obtained by setting Vmax of 4-coumarate for each
isoform to 100%. Sinapate (5) was not accepted as a substrate. See Fig. 1 for chemical structures of substrates.
Pp4CL1
Pp4CL4
Pp4CL2
Pp4CL3
trophoresis was enhanced four-fold compared to the standard
conditions (Fig. 3). This modification again did not result in the
detection of any Pp4CL4 transcripts. Using semi-quantitative PCR
conditions, an apparent gradual strength of expression was evi-
dent: the highest level was visible for Pp4CL2 and the lowest for
Pp4CL3 in both tissues. Similar results were obtained when analyz-
ing RNA from three independent preparations.
2.3. Phylogenetic analysis of the plant 4CL family
Phylogenetic reconstructions were performed to evaluate the
relationship of the different P. patens 4CLs with all available amino
acid sequences of the bona fide plant 4CL protein family (Fig. 4).
The Pp4CL isoenzymes form a highly supported monophyletic
group and are thus separated from higher plant isoforms. They
are positioned as a neighbour group to the gymnosperm se-
quences, the pine isoforms Pt4CL1 and Pt4CL2. In accordance with
earlier studies (Ehlting et al., 1999; Lindermayr et al., 2002), angio-
sperm 4CLs are divided in two groups, designated 4CL class I and
class II as defined earlier (Ehlting et al., 1999). Using the microbial
enzyme, S. coelicolor A3(2) cinnamate:CoA ligase (ScCCL), as out-
group, 4CL isoenzymes of class II appear more closely related to
the Pp4CL isoforms than to the 4CL isoforms of class I.
500bp
Exon
Intron
Fig. 2. Comparison of exon/intron structures of bona fide Pp4CL1–3 and putative
Pp4CL4 of Physcomitrella patens.
A) Gametophore
Tub 4CL1 4CL2 4CL3 4CL4
M
4CL1 4CL2 4CL3 4CL4 Tub
Although the overall sequence similarity between all isolated
Pp4CL family members at the nucleotide and protein level is rela-
tively high (78–86% of amino acid sequence identity; 77–83% of
nucleotide sequence identity among the Pp4CL1–4 isoforms), the
most recent gene duplication event might have occurred between
Pp4CL1 and Pp4CL4. In addition, genome duplication has been pro-
posed to have occurred in P. patens between 30 and 60 million
years ago (Rensing et al., 2007). Both events thus might in part ex-
plain the versatility of metabolism including secondary metabo-
lism, as described for P. patens and other mosses, in comparison
to other land plants (Rensing et al., 2007).
35 cycles
39 cycles
B) Protonema
Tub 4CL1 4CL2 4CL3 4CL4
M
4CL1 4CL2 4CL3 4CL4 Tub
Three P. patens subspecies have been analyzed to detect evolu-
tionary relationships of 4CL within the species. In particular, sub-
species patens (ecotype Gransden 2004 from the UK which has
been the basis for the EST and the genome sequencing projects),
ssp. magdalenae (ecotype Bisoke, Africa), and ssp. californica (ecotype
Okayama, Japan) were used for comparison. Using sequence infor-
mation of the isolated 4CL genes of P. patens ssp. patens, the two
sets of 4CL genes were isolated by PCR from genomic DNA of the
additional subspecies.
39 cycles
44 cycles
Fig. 3. Expression patterns of bona fide and putative 4CL genes in two develop-
mental stages of P. patens. Total RNA isolated from 2-week-old gametophore and 4-
day-old protonema tissue was used for cDNA synthesis. cDNA was analyzed by
semi-quantitative PCR using gene-specific primers and 35 (39) amplification cycles
for gametophore (A) and 39 (44) amplification cycles for protonema (B). Lane M,
size marker; lane Tub, P. patens b-tubulin gene.
P. patens ssp. magdalenae as well as P. patens ssp. californica con-
tain a family of four 4CL genes with an exon/intron structure com-
parable to that of P. patens ssp. patens. The lengths of the exons
were identical except for exon one of 4CL2 from P. patens ssp. mag-
dalenae being six nucleotides shorter than the respective exons
from the other two subspecies, whereas the size of the introns ap-
peared to be more variable. The percentages of identity of the
nucleotide and amino acid sequences within each 4CL family of
the additional subspecies closely matched those of P. patens ssp.
patens. A comparison of the amino acid similarities for each of
As detailed under Experimental, isogene specificity of each primer
pair was proved by PCR using cloned DNA as template. The gene for
b-tubulin, earlier shown to be constitutively expressed (Jost et al.,
2004), was used as a control. As illustrated in Fig. 3, the genes
Pp4CL1–3, but not Pp4CL4, were transcribed in both gametophore
and protonema tissue under the experimental conditions em-
ployed. To enhance the detection limit for low amounts of Pp4CL4
transcripts, the number of amplification cycles was further in-
creased to 47 and the amount of reaction mixture applied to elec-

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M.V. Silber et al. / Phytochemistry 69 (2008) 2449–2456
ScCCL A3(2)
bacteria
Os4CL1
Lp4CL2
1.00
83
1.00
100
Lp4CL3
Lp4CL1
Os4CL2
At4CL3
1.00
100
angiosperms
class II
1.00
92
1.00
76
Popt4CL2
1.00
83
Le4CL2
Ri4CL3
1.00
0.88
1.00
100
Gm4CL4
Gm4CL3
0.95
51
Pt4CL1
Pt4CL2
1.00
100
gymnosperms
mosses
0.95
64
Pp4CL3
1.00
100
Pp4CL2
1.00
85
Pp4CL4
Pp4CL1
1.00
81
0.94
Gm4CL1
0.83
At4CL4
At4CL1
1.00
53
1.00
97
At4CL2
1.00
Ri4CL2
0.83
66
angiosperms
class I
Poph4CL1
Poph4CL2
Popt4CL1
1.00
97
0.83
1.00
Gm4CL2
Ri4CL1
1.00
84
57
0.83
Le4CL1
Pc4CL1
1.00
100
1.00
75
Pc4CL2
Nt4CL
0.96
1.00
99
Nt4CL2
1.00
Nt4CL1
1.00
100
St4CL1
St4CL2
50 changes
1.00
100
Fig. 4. Phylogram of published members of the bona fide 4CL family from higher plants in comparison to 4CL isoforms, Pp4CL1–4, determined for the moss P. patens. Bayesian
analysis was in general accordance to a maximum parsimony analysis. Bayesian posterior probabilities are indicated above branches, corresponding bootstrap values of the
maximum parsimony analysis from 1000 replicates are given below branches. As outgroup, the bacterial cinnamate:CoA ligase (ScCCL) from Streptomyces coelicolor A3(2) was
chosen.
the 4CL isoforms from the three ecotypes revealed a percentage of
identity between 95 (Pp4CL4) and 97 (Pp4CL1).
constitute a central function in the biosynthesis of a host of both
primary and secondary compounds in various pathways. Among
these, the biosynthesis of many phenylpropanoid compounds in
green land plants involves the activation of 4-coumarate and a
number of structurally related substrates to the corresponding
CoA thioesters which is catalyzed by 4CL. As 4CL has been stud-
ied until present mainly in higher plants, the prime objective of
this study was to extend the molecular survey of 4CL to
bryophytes.
The moss 4CL family reported here shows similar enzymatic
characteristics when compared to higher plant 4CLs. Considering
the similarities in substrate specificity and in the protein se-
quence, the Pp4CL isoforms are representing one of the evolu-
tionary oldest 4CL families of land plants and are linking the
phylogeny of S. coelicolor ScCCL to 4CL isoforms of higher plants.
The close relationship of the Pp4CL isoforms to the higher plant
The phylogenetic analysis of the 4CL isoforms from P. patens
ssp. patens, magdalenae and californica (data not shown) supported
(i) a relatively large genetic distance between the Japanese (e.g.
ssp. californica) and the European (e.g. ssp. patens) subspecies
based on internal transcribed spacer regions as concluded earlier
(von Stackelberg and Reski, unpublished results), (ii) a proposed
closer relationship between the Japanese and the African subspe-
cies (e.g. ssp. magdalenae), and in addition (iii) the largest diver-
gence of 4CL4 within the 4CL gene family of P. patens.
3. Discussion
The acyl-activating capacity of CoA ligases, which belong to
the superfamily of adenylate-forming enzymes, is believed to

M.V. Silber et al. / Phytochemistry 69 (2008) 2449–2456
2453
4CL family in the phylogenetic tree together with the catalytic
preference within the group of 4-coumarate (2) and related sub-
strates of the Pp4CL isoforms underlines the importance of 4CL
as a central enzyme of phenylpropanoid metabolism of all plants.
The position of Pp4CL isoforms as a neighbour group to the
angiosperm 4CL isoforms of class II indicates that the split be-
tween class I and class II isoforms occurred at a very early stage
in the evolution of land plants, before the divergence of the lin-
eages that lead to mosses on the one hand and to higher plants
on the other. This illustrates further the high importance of phe-
nylpropanoid pathways for plants.
Pp4CL likely consists of a family of isoforms which exhibits
largely similar structures and enzyme-kinetic properties. In this
regard, Pp4CL resembles Solanum tuberosum St4CL, Petroselinum
crispum Pc4CL, and Nicotiana tabacum Nt4CL (Lozoya et al.,
1988; Becker-Andre et al., 1991; Lee and Douglas, 1996). The
opposite is exemplified by 4CL families which exhibit large struc-
tural, substrate specificity, and apparent evolutionary diversity,
such as Arabidopsis thaliana At4CL, Glycine max Gm4CL, Lithosper-
mum erythrorhizon Le4CL, Populus tremuloides Popt4CL, and Rubus
idaeus Ri4CL (Yazaki et al., 1995; Hu et al., 1998; Lindermayr
et al., 2002; Kumar and Ellis, 2003; Hamberger and Hahlbrock,
2004; Costa et al., 2005). To further advance our knowledge of
the molecular evolution of the 4CL family, the analyses have to
be extended to 4CL members from the pteridophytes and
microorganisms.
The overall amino acid sequence identity between the four
Pp4CL isoforms and higher plant 4CLs is about 62%, as opposed
to 42–43% when compared with the ScCCL from S. coelicolor
A3(2) whose catalytic function has been described as cinna-
mate:CoA ligase (Kaneko et al., 2003). Other 4CL-like sequences
from higher plants have been identified. For example, the genome
of A. thaliana has 14 genes, annotated as putative 4CL isoforms
(Costa et al., 2003). One of the closest relatives to At4CL1–4,
At1g20510, has recently been identified as 12-oxo-phytodienoic
acid (OPDA):CoA ligase (OPCL1) and proposed to be involved in
the biosynthesis of jasmonic acid in Arabidopsis (Koo et al.,
2006). OPCL1 is a member of clade V of the phylogenetic tree
of the 4CL-like subfamily of acyl-activating enzymes in Arabidop-
sis, all of which are characterized by a peroxisomal targeting sig-
nal (Koo et al., 2006). Two further members of this group were
previously shown to encode peroxisomal enzymes that activate
jasmonic acid precursors in vitro as well (Schneider et al.,
2005). The sequence relationship between Pp4CLs and the clade
V member, OPCL1 (36–37% identity with Pp4CL1–4), appears
more remote than between Pp4CLs and S. coelicolor 4CL (42–
43%) which is in agreement with the divergent biochemical func-
tions of the respective enzymes. As the homology criteria used for
the identification of Pp4CLs likely precluded the identification of
putative Pp4CL-like proteins, no conclusion can be drawn at pres-
ent as to the existence and properties of the latter proteins in P.
patens. For other 4CL-like homologues of higher plants, beyond
database annotations, there is a need for demonstrating any spe-
cific enzyme function. To better understand the catalytic specific-
ity of 4CLs and 4CL-like enzymes, however, specificity-
determining amino acids of the substrate-binding pockets will
have to be identified (Kaneko et al., 2003; Lindermayr et al.,
2003; Schneider et al., 2003). Such studies could also reveal
why Pp4CLs accept cinnamate much better than many higher
plant 4CLs. Comprehensive in silico studies, in the same context,
could identify functionally undefined 4CL-like proteins in P. pat-
ens involved in various acyl-activating reactions.
At4CL1, six introns in At4CL3; see Hamberger and Hahlbrock, 2004)
or for the Gm4CL genes (four introns in Gm4CL1, five introns in
Gm4CL4; unpublished results). The position of the first intron in high-
er plant 4CLs, however, appears to match the position of intron one of
Pp4CL2. Whereas the exon/intron organisation of the known four 4CL
genes in soybean is more closely related to that of Pp4CL2 and Pp4CL3,
that of At4CL3 is structurally more similar to Pp4CL1 and Pp4CL4.
Three of the four P. patens 4CL genes were expressed under the
experimental conditions used in our studies and in those employed
for collecting ESTs. The encoded proteins displayed a substrate uti-
lization capacity similar to higher plant 4CLs, however with the
added increase in activity towards cinnamate. Isoforms 1–3 thus
represent bona fide 4CLs. At least one biochemical function of
Pp4CL is obvious, namely its provision of activated C9 precursors
for flavonoid biosynthesis in P. patens (Jiang et al., 2006). Compar-
ative genome analyses demonstrated the presence in P. patens
(Rensing et al., 2008) of orthologues of phenylalanine ammonia-
lyase and of CYP73, cinnamate 4-hydroxylase, which, together
with 4CL, comprise enzymes of core phenylpropanoid metabolism,
and of CYP98, coumaroyl-shikimate/quinate 30-hydroxylase, an
additional member of the pathway assumed to be the major 3-
hydroxylase in phenylpropanoid metabolism of higher plants (Ehl-
ting et al., 2006). More refined functional assignments of the seem-
ingly similar Pp4CL isoforms may require comparative studies of
the family members, including gene-specific knock-out mutations
combined with histochemical, physiological, and biochemical anal-
yses. In addition, some of the higher plant 4CL genes are constitu-
tively expressed while others are transcriptionally induced or
repressed in response to environmental factors such as UV light
and pathogen attack. Little is known at present concerning the con-
tribution of phenylpropanoid compounds in the adaptation of
mosses to environmental challenges. It would be of particular
interest to learn about the regulation of Pp4CLs in response to
external factors and to unravel the evolutionary relationships of
regulation and possible functional divergence of the isoenzymes.
4. Conclusions
The Pp4CL family of Physcomitrella was characterized and its
enzymatic properties in vitro were determined to be similar to
4CL families of those higher plants that contain isoenzymes of lar-
gely similar structures and catalytic properties. The substrate pref-
erences suggested that the Pp4CL family members play a central
role in phenylpropanoid biosynthesis of the moss comparable to
that of higher plant 4CLs. Of the four isolated genes, three were ex-
pressed under the experimental growth conditions used in the
studies. Future investigations are required to yield clues on the
regulation of the Pp4CL family members and on their contribution
to growth and adaptation to environmental challenges of the moss.
5. Experimental
5.1. Plant material and growth conditions
P. patens (Hedw.) B.S.G., kindly provided by Drs. R. Reski and
M. von Stackelberg, was grown under sterile conditions in minimal
medium (Ashton et al., 1979), supplemented with 0.5 g/l ammo-
nium tartrate and 5 g/l glucose. Protonema tissues were propa-
gated by fragmentation of gametophytes with a blender (MICCRA
D-8Si, ART-Labortechnik, Müllheim, Germany) and subcultured
weekly in 500 ml Erlenmeyer flasks containing 200 ml of medium.
Gametophore tissues, grown on agar plates (0.7%, w/v; agar A-7921
from SIGMA), were subcultured at 4-week intervals. Plants were
grown at 16 h light/day (Philips TL-D 36W/33; photon fluence of
The exon/intron structures of 4CL genes from higher plants, in
most cases, have not been analyzed in great detail. The total number
and relative position of the introns within the genes can vary to some
extent, as found for example for the At4CL genes (three introns in
ca. 40
l
mol sꢀ1 mꢀ2) and at 25 °C.

2454
M.V. Silber et al. / Phytochemistry 69 (2008) 2449–2456
5.2. Data retrieval
5.5. Expression of Pp4CL genes
Similarity searches were performed by using the coding region
of the soybean Gm4CL1 cDNA (Genbank accession no. AF279267)
against the Physcomitrella GenBank EST database (http://moss.nib-
b.ac.jp/), allowing the retrieval of a candidate Pp4CL gene (GenBank
accession no. BI437532) which was designated Pp4CL1. A full-
length cDNA clone of Pp4CL1 (pdp11012) was obtained from RIKEN
BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074,
Japan (Nishiyama et al., 2003).
Pp4CL3 and Pp4CL4 were identified within the Community
Sequencing Program at the Joint Genome Institute of US Depart-
ment of Energy and made accessible for BLAST searches (Altschul
et al., 1997) through PHYSCObase (http://moss.nibb.ac.jp) and
the Pp4CL1 cDNA as query. A full-length Pp4CL3 cDNA clone
(pdp35373) was obtained from RIKEN BioResource Center.
For semi-quantitative PCR, gene-specific primers listed in Table
1 were used. Specific downstream/reverse primers were located
within the corresponding 30UTRs. Upstream/forward primers were
designed specific to the coding region of Pp4CL genes (Table 1).
Length, G/C content, calculated annealing temperature and the
expected lengths of the PCR products were in the same range for
all primer pairs. Gene specificity for each primer pair was shown
by PCR using cloned DNA as template (data not shown). At least
three independent RNA preparations were analyzed. All PCR exper-
iments were repeated at least once using 1.25 Units Taq recombi-
nant DNA polymerase (QIAGEN GmbH, Hilden, Germany),
10 pmol of each primer and 100 ng (gametophore tissue) or
200 ng (protonema tissue) of initial total RNA in a volume of
50 ll for one reaction. Samples were loaded onto 1% (w/v) ethi-
dium bromide-stained agarose gels in TAE buffer and visualized
by illumination at k = 254 nm. As an internal standard, P. patens
b-tubulin gene 3 was used which was shown to be constitutively
expressed in the tissues tested here (Jost et al., 2004). Polymerase
chain reactions were carried out under the following conditions:
initial denaturation for 3 min at 94 °C; 30 s at 94 °C, 1 min at
60 °C, 3 min at 68 °C as a second step with as many repetitions
as required (specified under results), and a final extension for
10 min at 68 °C.
5.3. Molecular cloning of Pp4CL genes
Total cellular DNA was extracted using the cetyltrimethylam-
monium bromide (CTAB)-based method (Doyle and Doyle, 1990).
Total RNA was prepared from protonema and gametophore tissues
using the TriFast Reagent (peqlab, Biotechnologie GmbH, Erlangen,
Germany) according to the instructions of the supplier. For reverse
transcription, 1 lg of total RNA (gametophore tissue) and 2 lg of
total RNA (protonema tissue) were incubated with RNasin and ran-
dom primers (Invitrogen, Karlsruhe, Germany) for 7 min at 70 °C
and then kept on ice. Reverse transcription was performed with
M-MLV (Invitrogen, Karlsruhe, Germany) reverse transcriptase as
directed by the manufacturer.
Pp4CL2 was identified by hybridizing a BAC filter (Centre of
Plant Sciences, LIBA, Leeds University, UK) under high stringency
conditions for 24 h at 60 °C using 32P-labeld 400 bp of the C-termi-
nal coding region of Pp4CL1. One clone was identified. Sequence
information was obtained by direct sequencing of the correspond-
ing BAC plasmid.
5.6. Analysis of DNA
DNA sequencing was carried out by the didesoxynucleotide
chain termination method (Sanger et al., 1977) using an ABI PRISM
377 DNA Sequencer (Applied Biosystems, Rodgau-Jügesheim,
Germany, at Department I – Botanik, LMU München). Nucleotide
sequences were analyzed and compared with the Sequencher 4.2
program (Gene Codes Cooperation, Ann Arbor, MI, USA).
5.7. Expression in E. coli and isolation of recombinant enzymes
Genomic PCR products were amplified using Pfu DNA polymer-
ase (Promega GmbH, Mannheim, Germany) with primers
Pp4CL1_50UTR/Pp4CL1_30UTR (Pp4CL1), Pp4CL2_50UTR/Pp4CL2_
30UTR (Pp4CL2), Pp4CL3_50UTR/Pp4CL3_30UTR (Pp4CL3), and
Pp4CL4_50UTR/Pp4CL4_30UTR (Pp4CL4) (For a list of primers see
Table 1. All oligonucleotides were obtained from MWG Biotech,
Ebersberg, Germany) as follows: 1 cycle of 3 min at 94 °C, 34 cycles
of 30 s at 94 °C, 1 min at 55 °C, and 5 min at 68 °C for amplification,
and 1 cycle of 5 min at 68 °C for final extension. The resulting PCR
products were cloned into the pDRIVE vector using the QIAGEN
PCR Cloning Kit (Qiagen GmbH, Hilden, Germany).
Full-length cDNAs were cloned into a modified pT7-7 vector to
give fusion proteins containing N-terminal His6-tags. The resulting
plasmids were transformed into E. coli BL21(DE3) cells. Cultures
were grown until OD ꢁ 1 was reached, induced with 0.3 mM iso-
propyl-b- -thiogalactopyranoside, and incubated for 5 h at 27 °C.
D
Recombinant enzymes were purified by immobilized metal chelate
affinity chromatography using the Ni-NTA metal affinity matrix
(Qiagen) according to the instructions of the manufacturer. The
affinity matrix was washed with buffer containing 20 mM imidaz-
ole followed by elution of adsorbed proteins with buffer containing
250 mM imidazole.
5.4. Cloning of Pp4CL2 cDNA
5.8. Enzyme assay
To obtain a full-length cDNA of Pp4CL2, total RNA isolated
from gametophore tissues was used for cDNA synthesis with
the gene specific primer Pp4CL2_30UTR. The same primer com-
bined with primer Pp4CL2_50UTR were used to amplify the cod-
ing region by PCR. The PCR product was inserted into the vector
pDRIVE. Sequence data from this article have been deposited at
GenBank under accession numbers EU167552 (Pp4CL1, P. patens
ssp. patens), EU167553 (Pp4CL2, P. patens ssp. patens),
EU167554 (Pp4CL3, P. patens ssp. patens), EU167555 (Pp4CL4, P.
patens ssp. patens), EU179312 (Pp4CL1, P. patens ssp. californica),
EU179313 (Pp4CL2, P. patens ssp. californica), EU179314 (Pp4CL3,
P. patens ssp. californica), EU179315 (Pp4CL4, P. patens ssp. cali-
fornica), EU180599 (Pp4CL1, P. patens ssp. magdalenae),
EU180600 (Pp4CL2, P. patens ssp. mahgdalenae), EU180601
(Pp4CL3, P. patens ssp. magdalenae), EU180602 (Pp4CL4, P. patens
ssp. magdalenae).
Enzyme activity was determined spectrophotometrically
according to the method of Knobloch and Hahlbrock (1975). The
change in absorbance caused by CoA-ester formation in the pres-
ence of purified recombinant 4CLs was monitored at k = 311 nm
for cinnamic (1) acid, 333 nm for 4-coumaric acid (2), 346 nm for
caffeic (3) and ferulic acids (4), and at 352 nm for sinapic acid (5)
(Gross and Zenk, 1966). The Km and Vmax values of recombinant
4CL1, 4CL2, and 4CL3 were determined using Linewaever–Burk
plots with at least 10 data points. Relative Vmax values were ob-
tained by setting Vmax of 4-coumarate for each isoform to 100%.
5.9. Phylogenetic analysis
4CL amino acid sequences for the phylogenetic reconstruction
were retrieved from Genbank (http://www.ncbi.nlm.nih.gov). Gen-

M.V. Silber et al. / Phytochemistry 69 (2008) 2449–2456
2455
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Bank accession nos. for amino acid sequences analyzed in addition
to those from P. patens were: U18675 (At4CL1, Arabidopsis thali-
ana), AF106086 (At4CL2, A. thaliana), AF106088 (At4CL3, A. thali-
ana), AAM19949 (At4CL4, A. thaliana), AF279267 (Gm4CL1,
Glycine max), AF002259 (Gm4CL2, G. max), AF002258 (Gm4CL3, G.
max), X69955 (Gm4CL4, G. max), D49366 (Le4CL1, Lithospermum
erythrorhizon), D49367 (Le4CL2, L. erythrorhizon), AF052221
(Lp4CL1, Lolium perenne), AF052222 (Lp4CL2, L. perenne),
AF052223 (Lp4CL3, L. perenne), D43773 (Nt4CL, Nicotiana tabacum),
U50845 (Nt4CL1, N. tabacum), U50846 (Nt4CL2, N. tabacum),
X52623 (Os4CL1, Oryza sativa), L43362 (Os4CL2, O. sativa),
X13324 (Pc4CL1, Petroselinum crispum), X13325 (Pc4CL2, P.
crispum), U12012 (Pt4CL1, Pinus taeda), U12013 (Pt4CL2, P. taeda),
AF008184 (Poph4CL1, Populus hybrida), AF008183 (Poph4CL2, P.
hybrida), AF041049 (Popt4CL1, Populus tremuloides), AF041050
(Popt4CL2, P. tremuloides), AF239687 (Ri4CL1, Rubus idaeus),
AF239686 (Ri4CL2, R. idaeus), AF239685 (Ri4CL3, R. idaeus),
M62755 (St4CL1, Solanum tuberosum), AF150686 (St4CL2, S. tubero-
sum), AL939119 (ScCCL, Streptomyces coelicolor A3(2)).
Amino acid sequences were aligned using Clustal W under man-
ual control as implemented in BioEdit (Hall, 1999). The resulting
data matrix was subsequently analyzed using PAUP version 4.0
(Swofford, 1998) as reported earlier (Lindermayr et al., 2002).
Bayesian analysis (Ronquist and Huelsenbeck, 2003) was per-
formed using two analyses of 10,000,000 generations each and
sampling trees every 1000 generations, a ‘‘burn-in” of 25%, and
equal state frequencies and mutation rates. The 50% majority rule
consensus tree was in accordance to the maximum parsimony
analysis. The topology is shown in Fig. 4, indicating posterior prob-
ability values (Bayes) and bootstrap values (PAUP), respectively.
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the Phylip program package (Felsenstein, 1989; http://evolu-
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Acknowledgements
The Physcomitrella BAC library was constructed at Southern Illi-
nois University (SIU), USA by Khalid Meksem for the Leeds Univer-
sity/Washington University Physcomitrella EST program (PEP). We
thank PEP and SIU for providing EST and BAC samples. This work
was supported by the Deutsche Forschungsgemeinschaft within
the priority program 1152 ‘‘Evolution of Metabolic Diversity”
(Grants EB 62/15-1 and EB 62/15-2).
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