Tài liệu Báo cáo Y học: Divergent members of a soybean (Glycine max L.) 4-coumarate:coenzyme A ligase gene family potx

Divergent members of a soybean (
Glycine max
L.)
4-coumarate:coenzyme A ligase gene family
Primary structures, catalytic properties, and differential expression
Christian Lindermayr
1
, Britta Mo¨ llers
1
, Judith Fliegmann
1
, Annette Uhlmann
1
, Friedrich Lottspeich
2
,
Harald Meimberg
3
and Ju¨ rgen Ebel
1
1
Botanisches Institut der Universita
¨
t, Mu
¨
nchen, Germany;
2
Max-Planck-Institut fu
¨
r Biochemie, Martinsried, Germany;
3

Institut fu
¨
r Systematische Botanik der Universita
¨
t, Mu
¨
nchen, Germany
4-Coumarate:CoA ligase (4CL) is involved in the formation
of coenzyme A thioesters of hydroxycinnamic acids that are
central substrates for subsequent condensation, reduction,
and transfer reactions in the biosynthesis of plant phenyl-
propanoids. Previous studies of 4CL appear to suggest that
many isoenzymes are functionally equivalent in supplying
substrates to various subsequent branches of phenylpropa-
noid biosyntheses. In contrast, divergent members of a 4CL
gene family were identified in soybean (Glycine max L.). We
isolated three structurally and functionally distinct 4CL
cDNAs encoding 4CL1, 4CL2, and 4CL3 and the gene
Gm4CL3. A fourth cDNA encoding 4CL4 had h igh s imi-
larity with 4CL3. The recombinant proteins expressed in
Escherichia coli possessed highly d ivergent catalytic e ffi-
ciency with various hydroxycinnamic acids. Remarkably,
one isoenzyme (4CL1) was able to convert sinapate; thus the
first cDNA en coding a 4CL that accepts highly substituted
cinnamic acids is available for further studies on branches of
phenylpropanoid metabolism that p robably lead to the
precursors of lignin. Surprisingly, the activity levels of the
four isoenz ymes and steady-state levels of their transcripts
were differently affected after elicitor treatment of soybean
cell cultures with a b-glucan elicitor of Phytophthora sojae,

revealing t he down-regulation of 4CL1 vs. up -regulation of
4CL3/4. A similar regulation of the transcript levels of the
different 4CL isoforms was observed in soybean seedlings
after infection with Phytophthora sojae zoospores. Thus,
partitioning of cinnamic acid building units between
phenylpropanoid branch pathways in s oybean could be
regulated at t he level of catalytic specificity and the level of
expression of the 4 CL isoenzymes.
Keywords: 4-coumarate:CoA ligase; differential regulation;
heterologous expression; plant defence; soybean (Glycine
max L.).
Phenylpropanoid compounds are major constituents of
higher plants. They can serve as flower pigments, UV
protectants, d efence chemicals,signalling c ompounds, a llelo -
pathic agen ts, and as building units of the phenolic support
polymer, lignin. Their synthesis is r egulated both by
developmental processes and by environmental cues and it
proceeds via the general phenylpropanoid pathway and
subsequent specialized branches of phenylpropanoid meta-
bolism. Central t o many of t he biosynthetic pathways is the
activation of differently substituted cinnamic acids to the
corresponding CoA t hioesters. This r eaction is catalyzed by
4-coumarate:CoA ligase (4CL; EC 6.2.1.12), a member of
general phenylpropanoid metabolism.
The central position of 4CL, linking the general with
specialized branches of phenylpropanoid metabolism, led to
the suggestion that 4CL could play a pivotal role in
regulating the flux of the activated CoA ester intermediates
into subsequent biosynthetic pathways. This idea was
substantiated by the observation that isoenzymes of 4CL

in soybean (Glycine max), petunia ( Petunia hybrida), pea
(Pisum sativum), oat ( Avena sativa), and poplar (Populus ·
euramericana) d isplayed different substrate a ffinities and/or
tissue distribution [1–5]. In contrast, other plants apparently
contain only a single 4CL isoenzyme or isoforms that
exhibit similar substrate specificities [6–10]. In these cases,
the r ing-modifications o n the cinnamic acid derivatives,
which precede the partitioning into different pathways, may
proceed at the level of the activated esters, as well as the
aldehydes and alcohols, as proposed recently [11–14].
Therefore, the physiological relevance of the occurrence o f
multiple 4CL in the former plants remains largely unknown.
4CL genes have been studied in a large variety of plants,
where the y c omprise s mall gene families in most cases. I n a
number o f p lants, including parsley (Petroselinum crispum),
loblolly pine (Pinus taeda), and potato (Solanum tuberosum),
the g enes encode id entical or very similar p roteins [ 7,15,16],
whereasinotherplants,suchastobacco(Nicotiana
Correspondence to J. Ebel, Botanisches Institut der Universita
¨
t
Mu
¨
nchen, Menzinger Strasse 67, D-80638 Mu
¨
nchen, Germany.
E-mail:
Abbreviation: 4CL, 4-Coumarate:coenzyme A ligase.
Enzyme: 4 -coumarate:CoA ligase ( EC 6.2.1.12).
Note: the nucleotide sequence data reported w ere deposited under

GenBank accession nos AF279267 for Gm4CL1 cDNA, A F002259 for
4CL14 (Gm4CL2 cDNA), AF002258 for 4CL13 ( Gm4CL3 genomic),
and X69955 for 4CL16 ( Gm4CL 4 cDNA).
(Received 2 3 October 2 001, revised 2 8 December 2001, accepted
9 January 2002)
Eur. J. Biochem. 269, 1304–1315 (2002) Ó FEBS 2002
tabacum), Arabidopsis thaliana,aspen(Populus tremuloides),
hybrid poplar (Populus trichocarpa · P. deltoides), and
soybean structurally divergent isoforms have been identified
[9,17–20]. In only a few plants have functionally divergent
4CL gene family members been correlated with specific
phenylpropanoid branch pathways, e.g. with plant tissues
actively producing t ypical phenylpropanoids, o r w ith p ath-
ways that are a ffected by environmental factors. In aspen,
Pt4CL1 has been associated with lignin biosynthesis
because of substrate preference and expression of the
corresponding gene in lignifying xylem tissues [17]. Con-
versely, aspen Pt4CL2 is thought to be involved in the
biosynthesis of phenylpropanoids in lignin-deficient epider-
mal layers. In Arabidopsis, three functionally divergent
At4CL forms have been hypothesized to be involved in
different phenylpropanoid biosyntheses of lignifying and
lignin-free tissues as well as to exhibit different physiological
roles against environmental challenges [ 18].
An unresolved question concerns the ability of 4CL to
catalyse the activation of sinapate. Sinapoyl-CoA was
previously proposed to be a p recursor for syringyl units of
angiosperm lignin. Nevertheless, r ecent findings indicate
that the activated esters of sinapate, f erulate, and
5-hydroxyferulate are not likely to participate in monolig-

nol biosynthesis [11,12,14]. However, 4CL isoforms from
soybean, petunia, pea, and poplar have been fo und to
convert s inapate to sinapoyl-CoA [1–3,5], whereas enzymes
from many other plants apparently lack this activity (see,
for example, [7,17–19]). Reactions obviating the CoA-
activation of highly ring-modified cinnamic acids have
been described, including cytochrome P450-dependent
hydroxylations [11,21] as well as O-methylations that
operate on caffeoyl-CoA and 5-hydroxyconiferyl aldehyde
[12,22,23]. Despite its central position at a branch po int of
phenylpropanoid metabolism in plants, the precise func-
tion of 4CL isoforms in p roviding CoA ester precursors
for t he synthesis o f different classes of phenolic compounds
with specialized functions remains, thus, largely controver-
sial.
In soybean, the production of phenylpropanoid com-
pounds comprises one of t he b iochemical d efence reactions
that are activated upon challenge with the oomycete
pathogen Phytophthor a sojae or treatment with a b-glucan
elicitor derived from the pathogen. The phenolic com-
pounds that accumulate around infection sites or in elicitor-
treated cell c ultures inc lude pterocarpan phytoalexins [24],
isoflavone conjugates [25,26], and wall-bound phenylpro-
panoid compounds [27]. Phytoalexin accumulation is pre-
ceded by the induced expression of many of the enzymes
involved in the biosynthetic p athway, including 4CL [20].
Previous biochemical [1] and molecular studies [20]
indicated that 4CL in soybean is enco ded by a small gene
family. In this study, we report on the isolation and
functional assignment of four soybean cDNAs as well as

one of the encoding 4CL genes. Pronounced differences in
catalytic efficiencies o f t he e ncoded isozymes f or differently
substituted cinnamic a cid substrates were found. Com bined
with differential expression patterns of the isoforms and
corresponding transcripts in different tissues of seedlings as
well as in both elicited cell cultures and infected seedlings,
these studies substantiate earlier conc lusions t hat t he
4CL isoenzymes in soybean serve different physiological
functions. Phylogenetic c omparison based on amino a cid
sequences extends the recent classification [18] of 4CL
isoforms within angiosperms.
EXPERIMENTAL PROCEDURES
Plant material
Soybean seeds (Glycine max L. cv. H arosoy 63) w ere from
R. I. Buzzell and V. Poysa (Agriculture Canada, Research
Station, Harrow, Canada); G. max L. cv. 9007 from Pioneer
Hi-Bred (Buxtehude, Germany). Seedlings were grown on
vermiculite under aseptic conditions as described previously
with minor modifications [28]. For infection e xperiments,
the taproots of 3-d ay-old seedlings were treated with a
suspension of % 10
4
zoospores in 200 lL sterile distilled
water by dip inoculation; control see dlings were placed in
water [28].
Cell suspension cultures of soybean (G. max L. cv.
Harosoy 63) were grown in the dark as described previously
[29] and treated with Phytophthora sojae crude elicitor
(80 lg glucose equivalentsÆmL
)1

medium) obtained by
partial a cid hydrolysis of purified cell walls of the oomycete
[30], as described previously [31].
4CL activity assay
Protein extracts were prepared from cell suspension cultures
of soybean according t o previously reported procedures
[29]. Enzyme activity was determined spectrophotometri-
cally acco rding to th e method of Knobloch and Hahlbrock
[1]. The analysis of activity levels of individual 4CL forms in
isoenzyme mixtures in cell culture extracts was based on
relative conversion rates (V values) for differently substi-
tuted cinnamic acids a t substrate c oncentrations of 500 l
M
according to Knobloch and Hahlbrock [1]. The change in
absorbance caused by CoA-ester production was monitored
at 311 nm for cinnamic acid, 333 nm for 4-coumaric acid,
346 nm for caffeic ac id, ferulic acid, and 3,4-dimetho xycin-
namic acid, and at 352 nm for sinapic acid [32]. Whereas
4-coumarate served as a s ubstrate f or all four isoenzymes,
ferulate was a substrate for isoenzymes 1 and 2 under the
conditions used, and 3,4-dimethoxycinnamate was conver-
ted exclusively by isoenzyme 1. By measuring relative
V-values, the activity level of each 4CL i soenzyme could
be estimated indirectly by using the above i ndicated
substrates and according to the scheme given in Table 1.
Because of highly similar conversion rates of differently
substituted cinnamic acids, isoenzymes 3 and 4 could not be
distinguished. For substrate affinity measurements of the
recombinant proteins, cinnamic acid was t ested in a
concentration range of 0.1–4 m

M
whereas 2.5–1000 l
M
was used for all other substrates.
The procedure for the indirect evaluation of isoenzyme
activities in crude plant extracts was validated by mixing the
recombinant enzymes (4CL1/4CL2/4CL3 with activity
ratios of 1 : 1 : 1, 10 : 10 : 1, and 1 : 10 : 10, respectively,
based on p-coumaric acid conversion) and subsequent
estimation of isoenzyme activities usin g t he factors given in
Table 1 . The calculated activity measures matched the
predicted values (data not shown).
Protein content was measured according to Bradford [33]
with BSA as standard. P rotein extracts were stored at
)20 °C.
Ó FEBS 2002 4-Coumarate:CoA ligase gene family from Glycine max (Eur. J. Biochem. 269) 1305
Immunoblotting
Protein extracts were separated by SDS/PAGE on 10%
polyacrylamide gels [34]. The proteins were blotted onto
nitrocellulose membranes and blocked with 1% (w/v)
nonfat powdered milk and 1% (w/v) BSA. The blot was
incubated with an antiserum raised against parsley 4CL [35]
at a dilution of 1 : 10000 for 1 h, followed by incubation
with goat anti-(rabbit IgG) Ig conjugated to alkaline
phosphatase (Sigma) and cross-reacting protein b ands were
visualized using 5-bromo-4-chloro-3-indolyl phosphate and
nitro blue tetrazolium as substrates.
Protein purification and analysis
4CL1 was purified from nontreated soybean cell cultures
according to Knobloch and Hahlbrock [1] with some

modifications. The following buffers were used in various
steps of p artial 4CL1 purification: buffer A , 0.2
M
Tris/HCl
pH 8.0, 14 m
M
2-mercaptoethanol, 0.2 m
M
phenylmethane-
sulphonyl fluoride, 30% (v/v) glycerol; buffer B, 0.05
M
Tris/HCl pH 7.9, 0.1 m
M
dithiothreitol, 0.2 m
M
phenyl-
methanesulphonyl fluoride, 30% (v/v) glycerol. All steps
were carried out at 4 °C. Froze n soybean cells (300 g) were
thawed and homogenized with 150 g quartz sand and
150 m L buffer A in a chilled mortar, stirred for 20 min with
0.1 g Dowex 1 · 2 (Serva; equilibrate d w ith buffer A) p er
gram of cells, a nd centrifuged to remove Dowex 1 · 2and
cell debris. 4CL1 activity was precipitated from the super-
natant with (NH
4
)
2
SO
4
(38–72% saturation), dissolved in

buffer B, a nd the extract desalted by chromatography on a
Sephadex G-25M column (Pharmacia). For separating 4CL
isoforms, the protein fraction was applied to a Q Sepharose
Fast Flow column (2.6 · 20 cm) w hich had been equili-
brated with the same buffer. The proteins were eluted with a
linear gradient of 0 –0.4
M
KCl in buffer B and 10 mL
fractions w ere collected. Fractions which showed 4CL1
activity were combined and loaded onto a Cibacron Blue
3G-A column (Pharmacia). After washing the column with
10 mL 0.6
M
KCl in buffer B, proteins were eluted with 2
M
KCl in buffer B . Fractions with 4CL1 activity were pooled
and d esalted b y c hromatography on Sephadex G -25M.
Finally, 4CL1 was separated f rom 4CL2 by anion exchange
chromatography on a R esource Q column (Pharmacia)
using a linear gradient of 0–0.4
M
KCl in buffer B . Protein
fractions with enriched 4CL1 activity were separated on
SDS/PAGE and the protein band corresponding to 4CL1
was used for sequence analysis. Microsequencing of the
N- terminus and two internal oligopeptides obtained after
proteolytic digestion with Lys-C resulted in three pep-
tide sequences: the N-terminal sequence consisted of
APSPQEIIF, s equence S 1 o f ( K)GYLNDPEA, and
sequence S2 of (K)ARLVITQSAYVEK.

DNA and RNA methods
Standard protocols were used for restriction enzyme
digestion, RNA and DNA blots [36]. Total RNA from cell
suspension cultures and s eedlings of G. max L. was isolated
according to [37]. DNA was prepared according t o [38].
Gene-specific hybridization probes have been generated by
either amplification of Gm4CL1 with the oligonucleotide
primers S1 and S2 (Table 2), or by restriction of the cDNAs
releasing a 1.0-kb SalI–KpnI-fragment from Gm4CL2 and a
0.9-kb SacI fragment from pQE-31/Gm4CL3, respectively.
For Southern blot hybridization, the complete open
reading frame of Gm4CL1 cDNA, a HindIII fragment of
pQE-30/Gm4CL2, and a BamHI–HindIII fragment o f
pQE-31/Gm4CL3 were prepared as hybridization probes.
Table 1. Scheme for in direct calculations of 4CL isoenzyme activities.
Isoenzyme
Activity calculated
for substrate
Calculation procedure using
various initial substrates
4CL1 Ferulate 0.6 · 4CL1 activity for
3,4-dimethoxycinnamate
4-Coumarate 1.1 · 4CL1 activity for
3,4-dimethoxycinnamate
4CL2 Ferulate Total activity for ferulate minus
4CL1 activity for ferulate
4-Coumarate 1.4 · 4CL2 activity for ferulate
4CL3/4CL4 4-Coumarate Total activity for 4-coumarate
minus 4CL1 and 4CL2
activities, respectively,

for 4-coumarate
Table 2. Sequences of oligonucleotides. Restrict ion sites contained in the oligonucleotides are underlined.
Designation Sequence
4CL1-GSP1 5¢-GTTGCGTAGGACGAGCAT-3¢
4CL1-GSP2 5¢-CGGATGCCGATTTTGTGGAGG-3¢
4CL1-KpnI5¢-GCT
GGTACCGCACCTTCTCCACAAG-3¢
4CL1-S1 5¢-TCYGGRTCRTTNAGRTADCCTTTCAT-3¢
4CL1-S2 5¢-TBACNCARTCNGCNTAYGTBGARAA-3¢
4CL3-HindIII 5¢-GTTCT
AAGCTTTTAAGGCGTCTGAGTGGC-3¢
4CL13–3¢KpnI5¢-AGTTTCAGGGTCAACAACCCTG-3¢
4CL13-EcoRI 5¢-CTC
GAATTCATGACAACGGTAGCTGCTTCTC-3¢
4CL14-BamHI 5¢-CTC
GGATCCATGGCTGATGATGGAAGCAG-3¢
4CL14-GSP1 5¢-TCAGCGTCACCGTTATCCTC-3¢
4CL14-GSP2 5¢-GTGAGAAATGGAGATGCTGC-3¢
4CL16-GSP1 5¢-TGTTCCGGAGAGCCTCCTC-3¢
4CL16-GSP2 5¢-CAACGGAAGCACGCATAGGAGCAC-3¢
4CL16-SphI5¢-CACC
GCATGCATAACTCTAGCTCCTTCTCTTG-3¢
Seq1 5¢-GTAAAACGACGGCCAGT-3¢
1306 C. Lindermayr et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Hybridization conditions were according to [39]. DNA
fragments were labelled with [a-
32
P]dCTP (Amersham
Pharmacia) using the r andom prime p rocedure (Prime-
a-Gene, Promega). Membranes were washed at moderate

stringency for 20 min twice in 1 · NaCl/Cit, 0.1% (w/v)
SDS at 42 °C o r at high stringency c onditions for 30 min in
0.5 · NaCl/Cit, 0.1% (w/v) SDS at 65 °C, and a utoradio-
graphed between intensifying screens at )80 °C. When
necessary, mem branes were s tripped by incubation in 0.1%
SDS, 5 m
M
EDTA at 95 °C for 30 min.
Cloning and subcloning of genomic DNA
Genomic DNA was isolated from hypocotyls and primary
leaves of 15-day-old G. max L. cv. Harosoy 63 seedlings,
partially digested with MboI, fractionated by size, and
cloned into BamHI-cut kEMBL3. The DNA library was
screened with randomly labelled Gm4CL14 and Gm4CL16
cDNAs t hat h ad be en isolated previously [20]. Positive
plaques were purified by four round s o f s creening, and one
9.5-kb DNA clone (kEMBL/Gm4CL3) was isolated. Res-
triction maps of the genomic clone kEMBL/Gm4CL3 and
subclones w ere c onstructed by single and multiple enzyme
digestions of the clones ligated into pBluescriptIIKS and SK
vectors [40].
cDNA synthesis and selection
RNA from nontreated soybean cell cultures was used for
RT/PCR using the peptide-deduced oligonucleotide primers
4CL1-S1 and 4CL1-S2 (Table 2). RT/PCR was performed
with an increasing annealing t emperature (52.5 °C+
0.1 °CÆcycle
)1
) for 25 cycles, followed by 10 cycles using
55 °C, resulting in a 0.8-kb fragment. This DNA fragment

was used as a gene-specific 4CL1 probe for screening a
cDNA library synthesized from enriched mRNA of
nontreated soybean cell cultures. One Gm4CL1 cDNA
was detected, plaque-purified and i solated a nd was shown
to be almost full length.
Completion of cDNA sequences
5¢-RACE was used to complete the open reading frames of
the partial cDNAs enco ding Gm4CL1 (see above),
Gm4CL2, and Gm4CL4 [20] (Table 3). Amplification was
performed in the presence of the respective nested gene-
specific primers (Table 2) and the universal amplification
primer UAP (Gibco/BRL) after reverse transcription of
soybeancellcultureRNA(1 lg each) using the gene-specific
oligonucleotides Gm 4CL1-GSP1, Gm4CL14-GSP1, and
Gm4CL16-GSP1, respectively, and t ailing with t erminal
transferase and dCTP according to the manufacturer’s
instructions (Gibco/BRL). The following annealing condi-
tions were used during PCR: increasing from 54.5 to 5 8 °C
during 35 cycles with Gm4CL1-GSP2 + UAP, 55 °Cfor
30 cycles using Gm4CL14-GSP2 + UAP after four rounds
of unidirectional a mplification at 58 °C in the presence of
solely the U AP oligonucleotide, and 60 °C with t he primers
Gm4CL16-GSP2 + UAP.
The partial c DNA clone Gm 4CL13, encoding the 4CL3
isozyme, was completed by RT/PCR using the genomic
sequence for the generation of oligonucleotide primers
(Gm4CL13–3¢KpnIandGm4CL13-EcoRI, Table 2). PCR
was performed with an annealing t emperature of 55 °Cfor
30 cycles.
The resulting 5¢ fragments have been cloned and

sequenced on both strands. In all cases, they were shown
to be identical in the overlapping portions when compared
to the respective partial cDNAs.
Construction of
E. coli
expression plasmids
For heterologous expression, either the pTrcHis (Invitro-
gen), or the pQE (Qiagen) vector series were used.
A summary of recombinant plasmids is given i n Table 3.
The introduction of the Gm 4CL1 cDNA into the
expression vector required the elimination of the initiator
codon of the 5¢-RACE product: this was accomplished by
introducing a KpnI restriction site. This modification was
achieved by PCR u sing the 4CL1-Kpn I oligonucleotide and
the vector-binding oligonucleotide Seq1 as primers. The
modified 5 ¢-RACE product was inserted into pZL1 con-
taining the incomplete Gm4CL1 cDNA using KpnIand
SalI. The complete open reading fram e was transferred into
pQE-30 using KpnIandHindIII.
For the construction of pBluescriptKSII/Gm4CL2, the
respective 5¢ fragment was released from the c loning vector
by SalI-restriction and inserted into the single SalIsiteofthe
partial cDNA (pBluescriptKSII/Gm4CL14). Deletion of the
5¢ noncoding region was achieved by PCR using 4CL14-
BamHI and 4CL14-GSP1 a s p rimers and pBluescriptKSII/
Gm4CL2 as template. The amplified product w as digested
with BamHI/SalI and cloned together with the SalI/
Table 3. Summary o f recombinant plasmids.
Plasmid Insert
pZL1/Gm4CL1 Fusion of partial Gm4CL1 recovered from

cDNA library screening and 5¢-end
fragment recovered from 5¢-RACE;
initiator codon was eliminated by
introducing a KpnI restriction site
pQE-30/Gm4CL1 Gm4CL1 cDNA with modified initiator
codon (see pZL1/Gm4CL1)
pBluescriptKSII/
Gm4CL14
Partial Gm4CL2 cDNA [20]
pBluescriptKSII/
Gm4CL2
Fusion of partial Gm4CL2 cDNA and 5¢-end
fragment recovered from 5¢-RACE
pQE-30/Gm4CL2 Full-length Gm4CL2 cDNA; 5¢-noncoding
nucleotides were eliminated by introducing
a BamHI restriction site directly upstream
of the initiator codon
pTZ19R/Gm4CL13 Partial Gm4CL3 cDNA [20]
pTZ19R/Gm4CL3 Fusion of partial Gm4CL3 cDNA and 5¢-end
fragment amplified by PCR
pTrcHisB/Gm4CL3 Full-length Gm4CL3 cDNA
pQE-31/Gm4CL3 Full-length Gm4CL3 cDNA
pTZ19R/Gm4CL16 Partial Gm4CL4 cDNA [20]
pTZ19R/Gm4CL4 Fusion of partial Gm4CL4 cDNA and 5¢-end
fragment recovered from 5¢-RACE;
initiator codon was eliminated
by introducing a SphI restriction site
pQE-30/Gm4CL4 Full-length Gm4CL4 cDNA with modified
initiator codon (see pTZ19R/Gm4CL4)
kEMBL/Gm4CL3 Genomic clone of Gm4CL3 (9.5 kb)

Ó FEBS 2002 4-Coumarate:CoA ligase gene family from Glycine max (Eur. J. Biochem. 269) 1307
HindIII-restricted insert of pBluescriptKSII/Gm4CL14
into pQE-30, previously cut w ith BamHI and Hin dIII.
The resulting construct pQE-30/Gm4CL2 contained an
upstream in-frame extension of 12 codons, including six
histidine codons.
The 4CL3 5 ¢ fragment w as ligated into pTZ19R/
Gm4CL13 using KpnI resulting in the f ull-length clone
pTZ19R/Gm4CL3. The complete open reading frame was
transferred both into the vector pTrcHisB and into pQE-31,
yielding upstream in-frame extensions o f 45 a nd 20 codons,
respectively, including six h istidine codons each. In spite of
different N-terminal extensions, the two expressed 4CL3
proteins showed the same e nzymatic characteristics.
For completion of t he Gm4CL4 cDNA, the initiator
codon of th e 5¢-RACE product was eliminated by PCR
using 4CL16-SphI a nd 4CL16-GSP2 a s primers. The
modified 5¢-RACE fragment was inserted into pTZ19R/
Gm4CL16 using SphIandSalI resulting in pTZ19R/
Gm4CL4. For heterologous expression the complete open
reading frame was amplified by PCR using 4CL16-SphIand
4CL3-HindIII as primers a nd pTZ19R/Gm4CL4 as tem-
plate. The PCR product was inserted into pQE-30 using
SphIandHindIII restriction sites.
Mutagenized and PCR-amplified fragments inserted into
the expression vectors were controlled b y sequencing.
Expression in
E. coli
and isolation of recombinant
proteins

E. coli strain SG13009 harbouring the plasmids pQE-30/
Gm4CL1 or pQE-30/Gm4CL4, as well as the strain M15,
harbouring pQE-30/Gm4CL2 or pQE-31/Gm4CL3, were
grown in Luria–Bertani medium in the presence of
100 lgÆmL
)1
ampicillin and 2 5 lgÆmL
)1
kanamycin. E. coli
JM109, harbouring pQE-30/Gm4CL2 or pTrcHisB/
Gm4CL3 were grown in the presence of ampicillin only.
Cultures were grown until A
600
% 0.5 was reached, induced
with 1.5 m
M
isopropyl-b-
D
-thiogalactopyranoside, and
incubated for 4 h at 37 °C. After centrifugation, the
bacterial cells were resuspended i n an appropriate volume
of buffer [50 m
M
Tris/HCl pH 8.0, 14 m
M
2-mercaptoeth-
anol, and 30% (v/v) glycerol] a nd disrupted by sonication.
After removing cellular debris by centrifugation (20 000 g,
20 min), the crude protein extracts were used for enzyme
activity tests. For 4CL3, extracts were concentrated by the

addition of solid (NH
4
)
2
SO
4
to 75% saturation. The
precipitate was collected b y centrifugation, dissolved i n
buffer (see above), a nd the protein fraction was passed
through a Sephadex G-25M column. The recombinant
proteins were purified by immobiliz ed metal c helate affinity
chromatography using the Ni-NTA Metal affinity matrix
(Qiagen) according to the instructions of the manufacturer.
Adsorbed proteins were eluted from the affinity matrix with
buffer (see above) containing 50 m
M
imidazole.
Analysis of DNA and protein sequences
Double-stranded DNA was s equenced on both strands
using the dideoxy chain-termination method [41] and a
sequenase kit 2.0 (Amersham) or with an ABI Sequencer
using BigDye Terminator chemistry (Botanisches Institut,
LMU Mu
¨
nchen). Computer analysis was carried out
with the PCgene program fro m IntelliGenetics (Geneva),
Chromas from Technelysium (Queensland, Australia), and
the BioEdit Sequence Alignment Editor [ 42].
The following 4CL sequences were used for the protein
sequence alignment (GenBank accession nu mbers given

in parentheses): Arabidopsis thaliana 4CL1 (U18675),
A. thaliana 4CL2 (AF106086), A. thaliana 4CL3
(AF106088), G. max 4CL1 (AF279267), G. max 4CL2
(AF002259), G. max 4CL3 (AF002258), G. max 4CL4
(X69955), Lithospermum erythrorhizon 4CL1 (D49366),
L. erythrorhizon 4CL2 (D49367), Lolium perenne 4CL1
(AF052221), L. perenne 4CL2 (AF052222), L. pe renne
4CL3 (AF0 52223), N icotiana tabacum 4CL (D43773),
N. tabacum 4CL1 (U50845), N. tabacum 4CL2 (U50846),
Oryza sativa 4CL1 (X52623), O. sativa 4CL2 (L43362),
Petroselinum crispum 4CL1 (X13324), P. crispum 4CL2
(X13325), Pinus taeda 4CL1 (U12012), P. taeda 4CL2
(U12013), Populus hybrida 4CL1 (AF008184), P. hybrida
4CL2 (AF008183), Populus tremuloides 4CL1 (AF041049),
P. tremuloides 4CL2 (AF041050), Rubus idaeus
4CL1 (AF239687), R. idaeus 4CL2 (AF239686), R. idaeus
4CL3 (A F239685), Solanum tuberosum 4CL1 (M62755),
S. tube rosum 4CL2 (AF150686), Vanilla planifolia 4CL
(X75542).
The alignment of 4CL amino acid sequences was
generated using
CLUSTAL W
2.0 and corrected by hand.
The resulting data matrix was subsequently analysed u sing
PAUP
version 4.0 [43]. The length of the protein sequences
varied between 535 (P. tremuloides 4CL1) and 636 residues
(L. erythrorhizon 4CL1). For distance and phylogenetic
calculations, overhanging positions were excluded. All
heuristic searches were carried out with the following

settings: RANDOM addition (10 replicates), TBR
branch-swapping, MULPARS, STEEPEST DESCENT,
COLLAPSE and ACCTRAN optimization and character
states specified as unordered and equally weighted. I n the
data matrix all gap characters (–) were scored as missing
data (?). Bootstrap values [44] were calculated from 1000
replicates. The resulting data matrix consisted of 557
characters of which 130 were c onstant, 427 were variable,
and 324 were potentially informative for phylogenetic
analyses. Pair-wise differences varied between 0.02%
(4CL1 and 4CL2 of P. taeda) and 45 .85% (L. erythrorhizon
4CL2 vs. O. sativa 4CL1) with an average pair-wise distance
of 31.2%. For comparison purposes, t he corresponding
nucleotide sequences were aligned and evaluated in the same
manner (data not shown).
RESULTS
The prime objective of t he current investigation was to
extend the molecular survey of 4CL isoenzymes from
soybean by: (a) completing the existing cDNAs [20]; and (b)
by isolating l acking members of the g ene family. Moreover,
we aimed to disclose details of the catalytic capacity of the
isoforms as well as of the d ifferential regulation of t he
isozymes in re lation to specialized branc hes of phenylpro-
panoid metabolism.
Molecular analysis of the 4CL gene family in soybean
Soybean cell cultures have been reported to contain the
4CL1 and 4CL2 isoforms [1]. Of these, 4CL1 is capable
of catalysing the activation of the broadest variety of
1308 C. Lindermayr et al. (Eur. J. Biochem. 269) Ó FEBS 2002
substituted cinnamates including sinapate. This catalytic

property is shared by only the minority of 4CL isoforms
studied to date from any plant. As the cDNA encoding
4CL1 was apparently missing from the pool of partial
cDNA clones isolated earlier [20] (see below), the isolation
of 4CL1 was attempted by purification from soybean cell
cultures as source. The separation of the isoenzymes was
achieved by anion exchange chromatography using
Resource Q a nd verified by using 3,4-dimethoxycinnamate
as substrate which is converted to the CoA ester by 4CL1
only [1]. Microsequencing of the purified 4CL1 established
peptide sequences which facilitated the cloning of the
corresponding cDNA from a cDNA library generated from
untreated soybean cell cultures.
For the isolation of full-length cDNAs encoding the
isozymes 4CL2, 4CL3, and 4 CL4, respectively, t he
5¢-RACE w as used to yield the 5¢ ends of the partial clones
Gm4CL14, Gm4CL13, and Gm4CL16 [20] (for details see
Experimental procedures). In summary, four full-length
cDNAs were obtained, encoding the soybean 4CL isozymes
1, 2, 3, and 4 which displayed divergent levels of similarity to
each other ( Table 4). For example, 4CL3 and 4CL4 s hare a
high identity at the deduced amino acid level (94%),
whereas in a ll othe r cases the i dentity b etween the d educed
4CLisoformsismuchlower(% 60%).
A phylogenetic reconstruction of the k nown plant 4CLs
revealed earlier that two major 4CL classes have evolved
within the angiosperms [18]. The addition of 4CL sequences
deposited i n the databases s ince the earlier rep ort as well as
of those p resented here into the protein alignment and the
subsequent calculation of the most parsimonious phylo-

genetic tree (Fig. 1) confirmed the previous observation of
the e volution of two major 4CL groups. According to the
earlier designation, soybean 4 CL1 and 4CL2 are members
of the class I cluster, whereas 4CL3 and 4 CL4 belong to the
more divergent class II cluster (Fig. 1 , upper and lower
branch of the phylogram, r espectively).
It was s hown previously that 4CL genes can be regulated
at the transcriptional level by both, infection of soybean
seedlings with Phytophthora sojae z oospores and e licitation
of soybean cell cultures [20]. One gene encoding an inducible
isoform of the soybean 4CL has been isolated by screening a
genomic library with a fragment c omprising 700 bp of the
Gm4CL16 cDNA (partial clone of Gm4CL4). The deter-
mination of the complete sequence revealed that the clone
represented the gene corresponding to the Gm 4CL3 cDNA,
which is 93% identical to Gm4CL4. The 4CL3 gene of
soybean was characterized by six exons ranging in s ize from
68 to 1068 bp which are flanked by five introns of 1893, 117,
102, 93, and 170 bp. The exon/intron splice junctions
revealed not only strong similarity to plant junctions in
general [45] but also to both 4CL genes each in parsley [6]
andinpotato[7],the4CL1 gene in rice [46], and the three
4CL genes in Arabidopsis [18]. I t is interesting to note t hat,
for th e increased number of 4CL genes analysed s o f ar, t he
number and the positions of the introns is increasingly
variable. The number of introns in the coding region that
are unrelated with regard to s equence and size range from
three (pine), four (parsley, potato, rice) and five (soybean) to
six (Arabidopsis).
Southern analysis of genomic DNA from cell cultures

was used to verify the number of 4CL genes in soybean.
Hybridization of triplicate blots containing restricted DNA
with gene-specific p robes for 4CL1 and 4CL2, respectively,
or with a probe which was not able to distinguish be tween
the 4CL3 and 4CL4 genes, resulted in the d etection of
distinct sets of fragments (Fig. 2). The hybridization
patterns for 4CL1 and 4CL2 could not be fully explained
by the existence of the respective restriction sites in the
Table 4. Comparison of the soybean 4CL cDNAs and encoded iso-
enzymes. The identity matrix w as calculated in pair-wise alignments
using BioEdit [42] and is giv en in e ach case as p ercentage identity of the
amino acid (up per line) an d t he n ucleotide seq uence (open reading
frame only, l ower line, italic).
Isoenzyme 4CL1 4CL2 4CL3 4CL4
Identity matrix
4CL1 – 63 58 58
65 62 61
4CL2 – – 61
63
60
62
4CL3 – – – 94
93
Amino acid residues 546 547 570 562
Molecular mass (kDa) 59.4 60.2 61.8 61.0
St4CL1
St4CL2
Nt4CL1
Nt4CL
Nt4CL2

Pc4CL1
Pc4CL2
Vp4CL
Le4CL1
Gm4CL2
Ri4CL1
Popt4CL1
Poph4CL1
Poph4CL2
Ri4CL2
At4CL1
At4CL2
Gm4CL1
Gm4CL4
Gm4CL3
Popt4CL2
Ri4CL3
Le4CL2
At4CL3
Lp4CL1
Os4CL2
Lp4CL2
Lp4CL3
Os4CL1
Pt4CL1
Pt4CL2
50 changes
62
2
1

2
1
107
35
23
22
23
16
25
14
22
1
13
19
11
7
13
57
31
31
26
58
62
63
21
26
26
42
39
30

60
39
46
97
51
52
55
32
19
17
45
11
48
57
85
69
53
36
52
58
45
42
60
94
1
100
53
57
78
76

100
87
100
100
5
2
60
100
98
56
70
74
83
61
1
00
100
100
100
99
100
Fig. 1. Heuristic maximum parsimony analysis of 31 4CL protein
sequences depicted as phylogram. The phylogenetic analysis was
calculated using the pine 4CL sequences as the outgroup. T he protein
alignment resulted in one most parsimoniuos tree with a minimal
length of 2155 steps and a consistency index of 0.599 (RI, 0.6 25). The
branch lengths are annotated above the corresponding branches,
bootstrap values for 1000 replicates are indicated below the branch
length only at branches supported by bootstrap analysis.
Ó FEBS 2002 4-Coumarate:CoA ligase gene family from Glycine max (Eur. J. Biochem. 269) 1309

Gm4CL1 and Gm 4CL2 cDNAs. However, the s ingle
fragments detected for 4CL1 by BamHI or EcoRI restric-
tion, respectively, and the double signal for 4CL2 after
restriction with EcoRI, which is co mpatible with the
presence of one EcoRI site in the cDNA sequence, suggested
the existence of single genes encoding each of these isozymes
(Fig. 2 ). The highly similar Gm4CL3 and Gm4CL4 cDNAs
were represented b y a more complex genom ic hybridization
pattern (Fig. 2) which nevertheless could be explained by
the existence of single genes.
Expression pattern of
4CL
genes in soybean seedlings
The spatial distribution of 4CL expression was studied in
soybean seedlings (G. max L. cv. 9007). Expression of
4CL3/4 mRNA at low levels was confined t o roots and
hypocotyls, while 4CL1 and 4CL2 mRNA amounts were
highest in hypocotyls and stems a nd also in young roots
(Fig. 3 ). Only low levels of the latter two mRNAs were
observed in 12-day-old roots. In s hoot tips and leaves, no
mRNA representing any of t he four 4CL isoforms c ould be
detected under the experimental c onditions used.
Differential regulation of
4CL
transcript and enzyme
activity levels
Treatment of soybe an cells with Phytophthora sojae crude
elicitor resulted in differential changes of the activities of the
4CL isoenzymes. At two growth stages of the cell suspen-
sion culture, representing cultures at 1 day after inoculation

(stage I) and at the end of the linear growth phase (stage II),
respectively [29], t he activity of 4CL3/4 strongly increased,
starting from a low basal level and reaching the highest level
at about 10 h following the start of treatment (Fig. 4A;
results f or stage I not shown). Conversely, the activity level
of 4CL1 was strongly redu ced following elicitor treatment
of the cells. After 12 h of treatment, the residual 4CL1
activity represented only % 10% of the level found in
untreated control cells. Only minor changes occurred for the
activity level of 4CL2 when compared with that of untreated
control cells.
The differential expression of the 4CL gene family
members i n soybean ce ll cultures was also a ssessed b y
RNA blot analysis. Northern analyses showed that 4CL1
and 4CL2 mRNA but not 4CL3/4 mRNA could be readily
detected in untreated control cells (Fig. 4B). When chal-
lenged w ith elicitor, differential changes in mRNA levels
occurred that reflected those found for isoenzyme activity
levels (Fig. 4A).
An analysis similar t o that s hown for e licitor-treated cell
cultures was performed with roots of 3-day-old soybean
seedlings following infection with zoo spores of Phytophtho-
ra sojae (Fig. 4C). Under the experimental conditions used,
the levels of 4CL1 and 4CL2 mRNAs were low and
remained unaffected. In c ontrast, 4CL3/4 mRNA levels
were detectable in untreated tissues and increased strongly
after infection (Fig. 4C).
Functional analysis of recombinant 4CL
To elucidate t he biochemical functions of the soybean 4CL
gene family members, the substrate specificity of the

heterologously expressed 4CL isoenzymes was examined
and c ompared w ith that of the previously isolated iso-
enzymes [ 1]. The four cDNAs were expressed in E. coli as
10
kb
B E HB E HB E
4CL3/44CL2
Gm Gm Gm
4CL1
H
8.0
6.0
4.0
3.0
2.5
2.0
1.5
Fig. 2. Southern blot analysis of soybean genomic DNA. DNA samples
(20 lg each) were digested with t he restriction endonucleases BamHI
(B), EcoRI (E), a nd HindIII (H), separated in t riplicate on a 0.6%
agarose gel and transferred t o a nylon membrane. The complete open
reading frame of the Gm4CL1 cDNA, a HindIII-fragment of pQE-30/
Gm4CL2, a nd a BamHI/HindIII-fragment of pQE-31/Gm4CL3 were
used as hybridization probes using high stringency conditions. Posi-
tions of DNA sta ndards are given o n the rig h t.
1.8 kb
1 2 3 4 5 6 7 8
4CL3/4
4CL2
Gm

Gm
Gm
28S rRNA
4CL1
1.8 kb
1.9 kb
2.3 kb
Fig. 3. Spatial expression pattern of the 4CL mRNAs in soybean
seedlings. Total RNA (20 lg each), isolated from different plant tis-
sues, was separated on 1.2% agarose gels, blotted onto n ylon mem-
branes, and hybridized with gene-specific 4CL probes. Blots w ere
washed under high stringency condition s. Hybridization with a 28S
rRNA probe d em onstrated equ al l oading. R NA was isolated from
3- (1) and 12-day (2) -old ro ots, from hypocotyls (3), first (4) and
second (5) internodium, f rom shoot tips (6), and from young (7) and
old (8) leaves detached from 21-day-old plants. The sizes of the
hybridizing RNA species are denoted on t he right.
1310 C. Lindermayr et al. (Eur. J. Biochem. 269) Ó FEBS 2002
inducible fusion proteins containing N-terminal His
6
-tags.
After immobilized metal a ffinity chromatography, t he four
proteins revealed the expected relative molecular masses i n
SDS/polyacrylamide gels. Immunoblot analysis demonstra-
ted that the recombinant proteins interacted with an
antiserum raised against parsley 4CL [35], whereas no
4CL-like protein cross-reacted w ith the antiserum in b acter-
ial e xtracts c ontaining the empty expression vector (Fig. 5).
The recombinant isoenzymes were tested for their relative
abilities t o u se differently substituted cinnamic acids as

substrates (Table 5). The affinities for the cinnamic acid
substrates were determined using Lineweaver–Burk plots.
The recombinant 4CL1 showed simple Michaelis–Menten
kinetics in the presence of 4-coumarate, caffeate, ferulate,
sinapate and 3,4-dimethoxycinnamate, whereas it showed
very low activity towards cinnamate. The recombinant
4CL2 was able to convert cinnamate, 4-coumarate, caffeate,
and f erulate but not sinapate an d 3 ,4-dimethoxycinnamate.
As summarized in Table 5, t he K
m
and r elative V
max
values
for t hese two 4CL isoforms closely resembled those found
previously for partially purified ligase 1 and 2 [1]. Similar
experiments with r ecombinant 4CL3 and 4CL4 demonstra-
ted t hat 4-coumarate and caffeate were most efficiently
converted to the CoA ester, cinnamate and especially
ferulate were converted with very low efficiency, whereas
sinapic and 3,4-dimethoxycinnamic acid were not accepted
as substrates. The K
m
values for the catalytic a ction of the
four heterologously produced soybean 4 CL isoforms for
Fig. 4 . Elicitation of soybean cell cultures and seedlings. (A) Soybean cell cultures of growth s tage II [ 29] were treated with Phy tophthora sojae
b-glucans (80 lg glucose equivalentsÆmL
)1
, filled symbols) or water (open symbols) for the periods indicated. Changes in 4CL isoenzyme activities
were assayed i n crude protein e xtracts a nd d iscriminated by calculating the c ontribution of t he i soenzymes t o t he ove rall 4 CL activity by us ing
isoenzyme-specific substrates. Data points shown are mean values of two independent experiments showing similar results. (B) Differential

expression of individual members of t he 4CL gene family was assayed in soybean cell cultures after elicitor treatment. Soybean cell cultures were
treated as i n ( A) and harvested at t he t imes i ndicated. To tal RNA (20 lg each) fro m elici tor-treated ( E) and untreated (C) c ell c ultures w as separated
on a 1.2% agarose gel a nd blotted o nto a nylon membrane. The b lot was hybridize d with gene-specific 4CL probes corresponding to Gm4CL1,
Gm4CL2, and Gm4CL3/4 and washed at high stringency. (C) Differential expression of 4CL mRNAs in roots of soybean seedlings upon infection
with Phy tophthora so jae was analysed b y Northern b lottin g as described in (B). Roots of soybean (cv. Harosoy 6 3) seedlings were treated with
Phytophthora sojae (race 1) zoospores by dip inoculation (E) a nd harvested a t t he times indicated. C ontrol seedlings were placed in sterile water (C).
Hybridization with soybean 28S rRNA was used to confirm equal l oadin g. The sizes of t he hybridizing RNA species are shown on the right.
Fig. 5. Immunoblot analysis of recombinant soybean 4CL. The four
isoforms were expressed as His
6
-tagged fusion proteins and purified by
immobilized m etal chelate a ffinity chromatography. The purified
proteins were separated by SDS/PAGE and transferred to nitro-
cellulosic filters. For immunodetection, antiserum raised against
parsley 4CL [35] combined with goat a ntirabbit IgG conjugated to
alkaline phosphatase was used. Purified protein extract from b acteria
carrying the empty e xpression vector (pQE-30 ) served as a control. The
relative molecular masses of protein standards are shown on the right.
Ó FEBS 2002 4-Coumarate:CoA ligase gene family from Glycine max (Eur. J. Biochem. 269) 1311
4-coumarate and caffeate were similar t o those r eported for
many purified plant 4CL, whereas differences of the
substrate s pecificity (V
max
/K
m
) b etween members of the
soybean 4CL protein family appeared to be pronounced
and w ere not previously rep orted in several of the other
plant 4CL analysed so far. 4CL1 accepted the broadest
range of hydroxylated and O-methylated cinnamic acids

(highest relative V
max
/K
m
value for ferulate followed by
4-coumarate and sinapate). 4CL2 used a n intermediate
range of substituted cinnamic a cids, while 4CL3 and 4CL4
displayed the highest selectivity towards these acids. All
ligases exhibited low affinity for cinnamate (Table 5). A
major r esult of the comparative substrate studies thus was
the detection of distinct differences in the selectivity of the
soybean 4CL isoforms for the various ring-substituted a nd
unsubstituted cinnamic acids.
DISCUSSION
The r esults of our studies demonstrate t hat 4CL in soybean
is encod ed by a small g ene family consisting of at least four
members. The recombinant proteins expressed from these
genes show pronounced differences in the catalytic efficiency
for metabolically important ring-substituted cinnamic acids.
Expression studies in cell cultures and in seedlings revealed
differential regulation of the f our 4CL genes, supporting
earlier notions on different physiological functions of
membe rs of the 4CL gene family in phenylpropanoid
branch pathways.
One of our goals in the present studies was to generate
full-length 4CL cDNAs for every single member of the gene
family. The nucleotide and a mino acid sequences de duced
from full-size Gm4CL2, Gm4CL3, and Gm4CL4 cDNAs
confirmed the level of identity between the three soybean
4CL genes as d etermined earlier for t he partial cDNAs [ 20].

The present w ork adds a further, a nd presumably l ast,
member to the 4CL family in soybean. A positive detection
in our earlier work [20] w as not possible g iven that RNA
samples from elicitor-treated cells were used. As demon-
strated in this work, these RNA samples were an inadequate
source for 4 CL1 cDNA isolation due to the repression
of 4CL1 mRNA expression in response to elicitor
(Fig. 4 A,B).
A phylogenetic reconstruction (Fig. 1) illustrates the
relationships of 4CL isoforms within the soybean gene
family as well as within the a ngiosperms. As noted earlier,
two classes o f 4CL proteins have ev olved [18]. R emarkably,
there is neither an exclusive bias towards distribution
according to lineage nor according to function. Gene
duplications took place throughout the evolution of these
plant enzymes which partly led to highly divergent struc-
tures (for example Gm4CL3/4 vs. Gm4CL1 or 2; At4CL3 vs.
At4CL1 or 2) which then eventually developed to serve
different environmental needs. Within the class II cluster,
for example, the soybean 4CL3 and 4 are the only i soforms
which are activated strongly in elicited or infected tissues,
whereas the Arab idopsis isoform 3 shows no regulation in
response to pathogen c hallenge but to UV i rradiation [18].
Table 5. Substrate spe cificity of rec ombinant soybean 4CL expressed in E. coli. The K
m
and V
max
values of recombinant 4CL1, 4CL2, 4 CL3 and
4CL4 were determin ed using Lineweaver–Burk plots with a t l east five data points. Each acid was assayed at the lo ng-wave a bsorbance m aximum o f
its CoA ester [32] in the spectrophotometrical test. Relative V

max
values were obtained by setting V
max
of 4-coumarate for each isoform to 100%.
The enzymatic characteristics of the isolated ligases 1 and 2 given in parenthesis were adopted from Knobloch and Hahlbrock [1]. NC., No
conversion.
Isoform Substrate K
m
(l
M
)
Relative V
max
(% of coumarate)
Relative V
max
/K
m
(l
M
)1
)
Gm4CL1 Cinnamate 4400 (1300) 9 (3) 2.0 · 10
)3
4-Coumarate 22 (32) 100 (100) 4.54
Caffeate 33 (40) 40 (56) 1.21
Ferulate 8 (9) 57 (56) 7.13
Sinapate 11 (11) 35 (46) 3.21
3,4-Dimethoxycinnamate 83 (100) 75 (89) 0.91
Gm4CL2 Cinnamate 1700 (4500) 50 (23) 0.03

4-Coumarate 42 (17) 100 (100) 2.38
Caffeate 13 (14) 37 (87) 2.85
Ferulate 140 (130) 71 (96) 0.51
Sinapate NC (NC) – (–) –
3,4-Dimethoxycinnamate NC (NC) – (–) –
Gm4CL3 Cinnamate 1100 45 0.04
4-Coumarate 9 100 11.12
Caffeate 50 74 1.48
Ferulate 3100 25 8.1 · 10
)3
Sinapate NC – –
3,4-Dimethoxycinnamate NC – –
Gm4CL4 Cinnamate 260 20 0.08
4-Coumarate 10 100 10.00
Caffeate 34 50 1.47
Ferulate 1300 30 0.02
Sinapate NC – –
3,4-Dimethoxycinnamate NC – –
1312 C. Lindermayr et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Substrate specificity of all four recombinant soybean 4CL
isoenzymes was a nalysed for a series of cinnamic a cids
bearing phenyl ring substitutions that are typical for
phenylpropanoid compounds of higher plants. A major
conclusion from this part of the results is that, rather
unexpectedly, the substrate specificity of only two recom-
binant 4CL isoforms, Gm4CL1 and Gm4CL2, closely
matched that of the known 4CL isoforms [1], whereas for
Gm4CL3 and 4 there was no known counterpart. A lthough
structural constraints in fusion prote ins g enerated from
cDNAs could affect substrate conversion, such an effect

appears to be unlikely for the observed catalytic properties
of recombinant 4CL3 o r 4CL4. I t appears more likely that
4CL3 and 4 had not been identified in earlier work because
proteins had been extracted from unstressed tissues [1] o r a
differentiation b etween 4CL2 and 4 CL3 or 4 in unfraction-
ated protein extracts was not possible due to the lack of
knowledge of their catalytic properties (Table 5) [20].
The analysis of the s ubstrate specificity of t he recombin-
ant soybean 4CL expressed in E. coli (Table 5) revealed
pronounced differences in the ability of the isoenz ymes to
utilize differently r ing-substituted cinnamic acids. The four
members of soybean 4CL thus represent enzymes with
broad (4CL1), intermediate (4CL2), and more restricted
substrate specificity ( 4CL3 and 4CL4). Soybean 4CL1 thus
far is the only plant isoenzyme capable of activating
sinapate, a presumed p recursor f or the syringyl monolignol
formation, for whic h a c DNA is available. Alternatively,
woody angiosperms have been described to obviate the need
for the activation o f highly ring-substituted cinnamic acids
as precursors for monolignol biosynthesis by using ring
substitution-specific methylations acting on already activa-
ted m etabolites [12]. Loss-o f-function experiments i n a lfalfa
(Medicago sativa) likewise indicated no necessity for CoA-
ligase isozymes converting already highly substituted cin-
namic acids [14]. The existence of the soybean 4CL1
isoform, using ferulate and sinapate with very high
efficiency, thus indicates an even higher flexibility in the
metabolic grid responsible for the distribution of phenyl-
propanoids as presently thought [47].
The molecular c haracterization of 4CL isoforms expres-

sing pronounced differences in the substrate specificity may
facilitate studies on the a ctive site of this class of enz ymes to
identify amino acids that are of functional i mportance. This
may include amino acid motifs such as a putative AMP-
binding domain [48], but also am ino a cids that are
responsible for a broad or narrow specificity towards ring-
substituted cinnamates. The central position of 4CL in
phenylpropanoid branch pathways therefore makes t his
enzyme a potentially valuable target for pathway or product
engineering in higher plants. Attempts towards this goal
have recently been reported for 4CL2 from Arabido psis
thaliana [49,50].
Another particularly striking observation is the differen-
tial expression of the four 4CL isoforms. Based o n enzyme
activity measurements, 4CL1 and 4CL2 are both e xpressed
in the unstresse d cell culture whereas based on RNA blot
analyses and activity measurements 4CL3 and 4CL4 appear
to be the major elicitor-induced forms being not expressed
in untreated cells. As the cloned cDNAs for 4CL3 and 4CL4
cross-hybridized under the conditions used, it is not possible
to analyse separately the t ranscript levels of Gm4CL3 and
Gm4CL4. Even though some uncertainty remains about the
relative proporti on of the e xpressed t ranscript levels corres-
ponding to the two closely related genes, 4CL3 or the closely
related 4CL4 protein very probably represent the highly
elicitor-induced enzyme. By contrast, the expression of
4CL1 in the soybean cell culture is r educed by elicitor
treatment, a behaviour which is reported only rarely for
enzymes c ommitted to the biosynthesis of plant p rotective
compounds. A consequence of this differential elicitor

responsiveness c ould b e that the overall product profile of
CoA esters of cinnamic acids in soybean c ells is shifted after
elicitation due to the large differences in substrate preference
of the 4 CL isoform 1 vs. 3 or 4. A similar c onsequence
might a pply to the soybean seedling after infection. Again
transcript levels of predominantly isoforms 3 or 4 are
enhanced close to the infection sites with a time-course
comparable to that observed for o ther enzymes of phenyl-
propanoid metabolism [51].
A genomic clone from soybean containing a complete
copy of one of the genes encoding an inducible 4CL
isoenzyme, Gm4CL3, was isolated. Although promoter
analyses for this gene have not yet been carried out, the
presence of a putative T ATA box and other boxes (A, E, L,
P, data not shown), which are conserved among several
plant g enes in phenylpropanoid m etabolism ( phenylalan ine
ammonia-lyase, 4CL, caffeoyl-CoA O-methyltransferase)
[19,52–54], indicate common principles of gene regulation
under various metabolic conditions.
Pathogen attack or elicitor treatment in soybean affects
various metabolic activities, i ncluding different branches of
phenylpropanoid metabolism. Phenylpropanoid responses
are temporally and spatially coord inated [26,28,55]. They
lead t o the massive deposition o f cell wall phenolics, release
of isoflavones from conjugates, and the production of
the soybean phytoalexins, glyceollins. All together, these
Fig. 6 . Scheme illustrating the central position of hydroxycinnamate
CoA esters and 4CL isoforms in the biosynthesis of various phenyl-
propanoid metabolites in soybean under different developmental and
environmental con ditions.

Ó FEBS 2002 4-Coumarate:CoA ligase gene family from Glycine max (Eur. J. Biochem. 269) 1313
responses are thought to contribute to the toxic environment
of both cell layers (immediately) proximal to the infection
site and tissues distal to the proximal cells. Evidence has
been presented that the nature, timing, and spatial aspects of
the proximal a nd distal cell responses of soybean t o g lucan
elicitor are similar to those occurring in incompatible
infected tissues [55]. As various of the phenylpropanoid
defence responses depend on the action of 4CL, it is likely
that the differential regulation of 4CL isoenzymes, as
observed i n t he present and in previous studies [20], reflects
the metabolic demands for CoA thioesters of substituted
cinnamic acids in the different phenylpropanoid branches of
soybean following infection. 4CL isoenzymes may therefore
influence to a certain degree the substitution pattern of
subsequent phenylpropanoid branches that require suitably
ring-substituted cinnamoyl CoA esters as substrates
(Fig. 6 ). However, phenyl ring modification involving
hydroxylation and O-methylation can basically occur by
different pathways, namely by modifications at the free acid
level, by s ubstitutions at the level of conjugated intermedi-
ates, such as CoA esters, and at the level of the aldehyde and
alcohol intermediates of monolignol synthesis [ 21,47,56].
The metabolic interconversions of cinnamic acids could add
to the complexity of the final phenylpropanoid products,
their c ellular l o calization, and the dynamics of their
synthesis. In any case, the coordinated r egulation of 4 CL3
or 4 with a ll other known enzymes of phytoalexin biosyn-
thesis in soybean [24] indic ate that at least these i soenzymes
are involved in defence-related pathways, whereas 4CL1

and 2 may have different functions in phenylpropanoid
metabolism of this plant.
ACKNOWLEDGEMENTS
We thank K. Hahlbrock (Ko
¨
ln, Germany) for providing Petroselinum
crispum 4CL antiserum and A. Mitho
¨
fer for critically reading the
manuscript and for valuable discussions. T h is work w as supported b y
the Deutsche Forschungsgemeinschaft (grant E b62/11-3), the Fonds
der C hemischen Industrie, a nd by a fellowship o f the state of Bavaria
(to C. L .).
REFERENCES
1. Knobloch, K .H. & Hahlbrock, K . (1975) Isoenzymes o f
p-coumarate:CoA ligase f ro m c ell s uspension cultures of Glycine
max. Eur. J. Biochem. 52 , 311–320.
2. Ranjeva, R., Boudet, A. & Faggiox, R. (1976) Phe nolic me tabo-
lism in petunia tissues. IV. Properties of p-coumarate:coenzyme A
ligase isoenzymes. Biochimie 58, 1255–1262.
3. Wallis, P.J. & Rhodes, M.J.C. (1977) Multiple forms of hydro-
xycinnamate: CoA ligase in etiolated pea seedling. Phytoc hemistr y
16, 1891–1894.
4. Knogge, W., Beulen, C. & Weissenbo
¨
ck, G. (1981) Distribution of
phenylalanine ammonia-lyase und 4-coumarate:CoA ligase in oat
primary leaves. Z. Naturforsch. 36 c, 389–395.
5. Grand, C., Boudet, A. & Boudet, A.M. (1983) Isoenzymes of
hydroxycinnamate:CoA ligase from poplar s tems, properties a nd

tissue distribution. Plant a 158, 2 25–229.
6. Lozoya, E., Hoffmann, H., Douglas, C.J., S chulz, W., Scheel, D .
& Hahlbrock, K . (1988) Primary structures and catalytic proper-
ties of isoenzymes encoded by two 4-coumarate:CoA ligase genes
in parsley. Eur. J. Biochem. 176, 661–667.
7. Becker-Andre
´
, M., Schulze-Lefert, P. & Hahlbrock, K. (1991)
Structural comparison, modes of expression, and putative
cis-acting elements o f the two 4 -coumarate:CoA ligase genes in
potato. J. Biol. Chem. 266, 8 551–8559.
8. Voo, K.S., Whetten, R.W., O’Malley, D.M. & Sederoff, R.R.
(1995) 4-coumarate:coenzyme A ligas e f rom l oblolly pine xylem.
Plant Physiol. 108, 8 5–97.
9. Lee, D. & Douglas, C.J. (1996) Two divergent members of a
tobacco 4-coumarate:coenzyme A ligase (4CL) gene family. Plant
Physiol. 112 , 193–205.
10. Brodelius, P.E. & Xue, Z T. (1997) Isolation and characterization
of a cDNA from cell suspension cultures of Vanilla planifolia
encoding 4-coumara te:Coenzyme A ligase. Plant Physiol.
Biochem. 35, 497–506.
11. Humphreys, J.M., Hemm, M.R. & Chapple, C. ( 1999) New r outes
for lignin b iosynth esis defined by biochemical characterization of
recombinant ferulate 5-hydroxylase, a m ultifunctional cytochrome
P450-dependent m onooxygenase. Proc. Natl A cad. Sci. USA 96,
10045–10050.
12. Li, L., Popko, J.L., Umezawa, T. & Chiang, V.L. (2000)
5-Hydroxyconiferyl aldehyde modulates enzymatic methylation
for syringyl monolignol formation, a new view o f monolignol
biosynthesis in angiosperms. J. Biol. Chem. 275, 653 7–6545.

13. Li, L ., Cheng, X.F. , Leshkevich, J., Umezawa, T ., Harding, S.A.
& Chiang, V.L. (2001) The last step of syringyl m onolignol bio -
synthesis in angiosperms is regulated by a novel gene encoding
sinapyl a lcohol de hydrogenase. Plant Cell 13, 1567–1585.
14. Guo, D., Chen, F., Inoue, K., Blount, J.W. & Dixon, R.A. (2001)
Downregulation of c affeic ac id 3-O-methyltran sferase and c a ffeoyl
CoA 3-O-methyltransferase in transgenic alfalfa: i mpacts o n lignin
structure and implications for the biosynthesis of G and S lignin.
Plant Cell 13 , 73–88.
15. Douglas, C.J., Hoffmann, H., Schulz, W. & Hahlbrock, K. (1987)
Structure and elicitor or U. V light-stimulated expression of
two 4 -coumarate:CoA l igase g enes in parsley. EMB O J. 6,
1189–1195.
16. Zhang, X.H. & Chiang, V.L. (1997) Molecul ar cloning of 4-cou-
marate:coenzyme A ligase in loblolly p ine and the roles of this
enzyme in the biosynthesis of lignin in compression wood. Plant
Physiol. 113 , 65–74.
17. Allina, S.M., Pri-Hadash, A., Theilmann, D.A., Ellis, B.E. &
Douglas, C.J. (1998) 4-Coumarate:coenzyme A ligase in hybrid
poplar. Properties of native enzymes, cDNA cloning, and analysis
of recombinant e nzym es. Plant Physiol. 116, 7 43–754.
18. Ehlting, J., Bu
¨
ttner, D., Wang, Q., Dougl as, C.J., Somssich, I .E. &
Kombrink, E. ( 1999) Three 4-coumarate:coenzyme A ligases in
Arabidopsis thaliana represent t wo evolutionary classes in
angiosperm s. Plant J. 19, 9–20.
19. Hu, W J., Kawaoka, A., Tsai, C J., Lung, J., Osakabe, K.,
Ebinuma, H. & Chiang, V.L. ( 1998) Compartmentalized expres-
sion of two structurally and functionally distinct 4-coumarate:

CoA ligase genes in aspen (Populus tremuloides). Proc. Natl Acad.
Sci. USA 95 , 5407–5412.
20. Uhlmann, A. & Ebel, J. (19 93) Molecular cloning and e xpression
of 4-coumarate:coenzyme A ligase, an enzyme involved in the
resistance response of soybean (Glycine max L.) against pathogen
attack. Plant Physiol. 102, 1 147–1156.
21. Schoch, G ., Goepfert, S., Morant, M., Hehn, A., Meyer, D.,
Ullmann, P. & Werck-Reichhart, D. (2001) CYP98A3 from
Arabidopsis thaliana is a 3¢-hydroxylase of phenolic esters, a
missing link in the phenylpropanoid pathway. J. Biol. Chem. 276,
36566–36574.
22. Pakusch, A. & Matern, U. (1991) Kinetic characterization of
caffeoyl-coenzyme A-specific 3-O-methyltransferase from elicited
parsley cell s uspensions. Plant Physiol. 96, 327–330.
23. Ye, Z.H., Kneusel, R.E., Matern, U. & Varner, J.E. (1994) An
alternative methylation pathway in lignin biosynthesis in Z innia.
Plant Cell 6 , 1427–1439.
1314 C. Lindermayr et al. (Eur. J. Biochem. 269) Ó FEBS 2002
24. Ebel, J. & Grisebach, H. (1988) Defense strategies of soybean
against the fungus Phytophthora megasperma f. sp. glycinea:a
molecular analysis. Trends Bioc hem. Sci. 13, 2 3–37.
25. Graham, T.L. & G raham, M.Y. ( 1991) Cellular c oordination of
molecular responses in plant d efense. Mol. Plant-Microbe Interact.
4, 415–422.
26. Morris, P.F., Sa vard, M.E. & Ward, E.W. (1991) Identification
and accumulation of isoflavonoids and isoflavone glucosides in
soybean leaves and hypocotyls in resistance responses to Phyto-
phthora megasperma f. sp. glycinea. Physiol. Mol. Plant Pathol. 39,
229–244.
27. Graham, M.Y. & Graham, T.L. (1991) Rapid accumulation of

anionic peroxidases and phenolic polymers in soybean cotyledon
tissues following t reatment with Phyto phthora megasperma f. sp.
glycinea wall glucan. Plant Physiol. 97 , 1445–1455.
28. Hahn, M.G., Bonhoff, A. & Grisebach, H. (1985) Quantitative
localization of the phytoalexin glyceollin I in relation to fungal
hyphae in soybean roots infected with Phytophthora meg asperma
f. sp. gl ycinea. Plant Physiol. 77 , 591–601.
29. Hille,A.,Purwin,C.&Ebel,J.(1982)Inductionofenzymesof
phytoalexin synthesis in cultured soybean cells by an elicitor from
Phytophthora megasperma f. sp. glycinea. Plant Cell Reports 1,
123–127.
30. Sharp, J.K., Valent, B. & Albersheim, P . (1984) Purification and
partial characterization of a b-glucan fragment that elicits
phytoalexin a ccumulation in s oybean. J. Biol . Chem. 259, 11312–
11320.
31. Schmidt, W.E. (1986) Spezifische Bindung eines b-Glucanelicitors
aus Phy tophthora megasperma f. sp. glyc inea. Plasmamembranen
aus Sojabohne ( Glycine max ). PhD Thesi s. University of Freiburg,
Freiburg, Ger m any.
32. Gross,G.G.&Zenk,M.H.(1966)DarstellungundEigenschaften
von Coenzym A-Thiolestern substituierter Zimtsa
¨
uren. Z.
Naturforsch. 21b , 683–690.
33. Bradford, M.M. (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing t he
principle of protein-dye binding. Anal. Biochem. 282, 152–160.
34. Laemmli, U .K. (1970) Cleavage of structural proteins during the
assembly of the head o f bacteriophage T4. Nature 227 , 680–685.
35. Ragg, H., Kuhn, D .N. & Hahlbro ck, K. (1981) Coordinated

regulation of 4 -coumarate:CoA ligase and phenylalanine
ammonia-lyase mRNAs in cultured plant cells. J. Biol. C hem. 256,
10061–10065.
36. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular
Cloning: a Laboratory Manual, 2nd edn. Cold Spring. Harbor
Laboratory Press, New Y ork.
37. Chang, S., Pur year, J. & C airney, J. (1993) A s imple and efficient
method for isolating RNA from pine trees. Plant Mol. Biol.
Reporter 11, 113 –116.
38. Draper, J., Scott, R., Armitage, P. & Walden, R. (1988) Plant
Genetic T ransformation and Ge ne Expression. B lackwell Scientific
Publications, Oxford.
39. Fliegmann, J. & Sandermann, H. Jr (1997) Maize glutathione-
dependent formaldehyd e dehydrogenase cDNA: a novel plant
gene of detoxification. Plant Mol. Biol. 34, 843–854.
40. Short, J.M., Fernandez, J.M., Sorge, J.A. & Huse, W.D. (1988 )
k Zap: a b acteriop hage k expression vecto r with in vivo excision
properties. Nuclei c Acids Res. 16 , 7583–7600.
41. Sanger, F., Nicklen, S. & Coulson, A.R. (1977) DNA sequencing
with c hain-terminating inhibitors. Proc. Natl Acad. Sci. USA 74 ,
5463–5467.
42. Hall, T.A. (1999) BioEdit. A User-Friendly Biological Sequence
Alignment and Analysis Program for Windows 95/98/NT. Nucleic
Acids Symposium S er. 41, 95–98.
43. Swofford, D.L. (1998) Paup: Phylogenetic Analysis Using
Parsimony, V ersion 4.0b1. Co mputer program distributed by the
Illinois Natural History Surve y, Champaign, IL, USA.
44. Felsenstein, J. (1985) Confidence limits on phylogenies: an
approach usi ng the bootstrap. Evolution 39, 783–791.
45. Brown, J.W.S. (1986) A catalogue of splice junctions and putative

branch point sequences from plant introns. Nucleic Acids Res. 14,
9549–9559.
46. Zhao, Y., Kung, S.D. & Dube, S .K. (1990) Nucleotide seq uence of
rice 4-coumarate:CoA l igase gene, 4CL-1. N ucleic A cids Res. 18,
6144.
47. Dixon, R.A., Chen, F., Guo, D. & Parvathi, K. (2001) The
biosynthesis of monolignols: a Ômetabolic gridÕ,orindepend-
ent pathways to guaiacyl and syringyl units? Phytochemistry 57,
1069–1084.
48. Bairoch, A. (1991) Putative AMP-binding Domain Signature.
PROSITE: a Dictionary of Sites and Patterns in Proteins. Release
8.00.
49. Stuible, H P., Bu
¨
ttner, D., Ehlting, J., Hahlbrock, K. &
Kombrink, E. (2000) Mutational analysis of 4-coumarate:CoA
ligase identifies functionally important amino acids and verifies its
close relationship to other adenylate-forming enzymes. FEBS Lett.
467, 117–122.
50. Stuible, H P. & Kombrink, E. (2001) Identification of the
substrate specificity-conferring amino acid r esidues o f 4 -coumar-
ate:coenzyme A ligase allows the rational design of mutant
enzymes with new catalytic properties. J. Biol. Chem. 276, 26893–
26897.
51. Habereder, H., Schro
¨
der, G. & Ebel, J. (1989) Rapid induction of
phenylalanine ammonia-lyase and chalcone synthase mRNAs
during fungus infection of soybean (Glycine max L.) r oots or
elicitor treatment of soybean cell cultures at the onset of phyto-

alexin synthesis. Planta 177 , 58–65.
52. Lois, R., Dietrich, A., Hahlbrock, K. & Schulz, W. (1989) A
phenylalanine ammonia-lyase g ene from parsley: s tructure, r egu-
lation and identification of elicitor and light responsive cis-acting
elements. EMBO J. 8, 1641–1648.
53. Logemann, E., Parniske, M. & Hahlbrock, K. (1995) Modes of
expression an d common s tructural features o f t he complete ph e-
nylalanine ammonia-lyase gene family in parsley. Proc. Natl Acad.
Sci. USA 92, 5905–5909.
54. Grimmig, B. & Matern, U. ( 1997) Structure of the parsley
caffeoyl-CoA O-methyltransferase gene, harbouring a novel
elicitor responsive cis -acting element. Plant Mol. Biol. 33, 323–
341.
55. Graham, M.Y. & G raham, T.L. (1994) Wound-associated c om-
petency factors are required for the proximal cell responses of
soybeantothePhytophthora sojae wall glucan elicitor. Plant
Physiol. 105, 571–578.
56. Heller, W. & Forkmann, G. (1994) Biosynthesis of flavonoids. In:
The Flavonoids: Advances in Research Since 1986 (Harborne, J.B.,
ed.), pp. 499–535. Chapman & Hall Publishers, London.
Ó FEBS 2002 4-Coumarate:CoA ligase gene family from Glycine max (Eur. J. Biochem. 269) 1315