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

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

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-

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

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

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.

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

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

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)

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

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

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-

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

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

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

(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

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

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

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

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

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

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

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