The Plant Journal (1999) 19(1), 9±20
Three 4-coumarate:coenzyme A ligases in Arabidopsis
thaliana represent two evolutionarily divergent classes in
angiosperms
JuÈrgen Ehlting1, Daniela BuÈttner1, Qing Wang2,
Carl J. Douglas2, Imre E. Somssich1 and
Erich Kombrink1,*
1
Max-Planck-Institut fuÈr ZuÈchtungsforschung, Abteilung
Biochemie, Carl-von-Linne Weg 10, D-50829 KoÈln,
Germany, and
2
Department of Botany, University of British Columbia,
Vancouver, British Columbia V6T 1Z4, Canada
Summary
The enzyme 4-coumarate:CoA ligase (4CL) plays a key
role in channelling carbon ¯ow into diverse branch
pathways of phenylpropanoid metabolism which serve
important functions in plant growth and adaptation to
environmental perturbations. Here we report on the
cloning of the 4CL gene family from Arabidopsis thaliana
and demonstrate that its three members, At4CL1, At4CL2
and At4CL3, encode isozymes with distinct substrate
preference and speci®cities. Expression studies revealed
a differential behaviour of the three genes in various
plant organs and upon external stimuli such as wounding
and UV irradiation or upon challenge with the fungus,
Peronospora parasitica. Phylogenetic comparisons
indicate that, in angiosperms, 4CL can be classi®ed into
two major clusters, class I and class II, with the At4CL1
and At4CL2 isoforms belonging to class I and At4CL3 to
class II. Based on their enzymatic properties, expression
characteristics and evolutionary relationships, At4CL3 is
likely to participate in the biosynthetic pathway leading
to ¯avonoids whereas At4CL1 and At4CL2 are probably
involved in lignin formation and in the production of
additional phenolic compounds other than ¯avonoids.
Introduction
The general phenylpropanoid pathway channels carbon
¯ow from primary metabolism to different branch path-
ways of secondary phenolic metabolism via the sequential
action of the enzymes phenylalanine ammonia-lyase (PAL),
cinnamate 4-hydroxylase (C4H) and 4-coumarate:CoA
ligase (4CL). The latter enzyme, 4CL (EC 6.2.1.12), converts
4-coumaric acid and other substituted cinnamic acids such
Received 18 January 1999; revised 26 April 1999; accepted 3 May 1999.
*For correspondence (fax +49 2215062 313;
e-mail kombrink@mpiz-koeln.mpg.de).
ã 1999 Blackwell Science Ltd
as caffeic acid and ferulic acid into the corresponding CoA
thiol esters which are used for the biosynthesis of
numerous phenylpropanoid-derived compounds including
¯avonoids, iso¯avonoids, lignin, suberins, coumarins and
wall-bound phenolics (Dixon and Paiva, 1995; Douglas,
1996). It has been proposed that 4CL isoforms differing in
their substrate speci®cities may direct the ¯ux from general
phenylpropanoid metabolismo into the different end
product-speci®c pathways by supplying appropriate mix-
tures of substrates for subsequent reactions and/or speci®c
metabolic needs. In support of such functions, native 4CL
isoforms with distinct capacities to convert different
substituted 4-hydroxycinnamates have been reported in
soybean, petunia and pea (Allina et al., 1998, and references
therein; Hahlbrock and Grisebach, 1979).
4CL genes have been cloned from numerous plant
species, where they exist in small gene families. In some
plants, e.g. parsley, potato and loblolly pine, the cloned
genes encode identical or nearly identical proteins (Becker-
Andre et al., 1991; Douglas et al., 1987; Zhang and Chiang,
1997), whereas in other plants, e.g. tobacco, soybean,
hybrid poplar (Populus trichocarpa 3 P. deloides) and
aspen (Populus tremulopides), structurally divergent forms
have been isolated (Allina et al., 1998; Hu et al., 1998; Lee
and Douglas, 1996; Uhlmann and Ebel, 1993). However,
only in few cases has the existence of functionally different
4CL proteins been correlated with the differential expres-
sion of divergent 4CL gene family members (Allina et al.,
1998; Hu et al., 1998). In aspen, Pt4CL1 has been associated
with lignin biosynthesis since it has activity towards
substituted hydroxycinnamic acids including 5-hydroxy-
cinnamate, and the corresponding gene is expressed in
lignifying xylem (Hu et al., 1998). Aspen Pt4CL2 is consid-
ered to be involved in the biosynthesis of other phenolics
such as ¯avonoids, since it has highest activity towards
coumarate, lacks activity towards 5-hydroxyferulate, and
the corresponding gene is preferentially expressed in the
epidermis of leaves and stems (Hu et al., 1998). Likewise,
the two soybean 4CL genes are differentially regulated.
Based on its expression pattern, the pathogen-inducible
GM4CL16 is speculated to encode a ¯avonoid-speci®c
isoform involved in the biosynthesis of phytoalexins,
whereas the biochemical role of the second, non-inducible
isoform is unclear (Uhlmann and Ebel, 1993). Thus, despite
its central position in phenylpropanoid metabolism, the
precise function of 4CL isoforms in directing metabolic ¯ux
to speci®c phenolic compounds is largely unknown.
9
10 JuÈrgen Ehlting et al.
Con¯icting evidence exists concerning the ability of 4CL
to activate sinapate in preparation for reduction to sinapyl
alcohol, a lignin monomer incorporated into the syringyl
lignin of angiosperms. Isoforms from soybean, petunia
and pea have been reported to convert sinapate to sinapyl-
CoA, whereas native or recombinant enzymes from most
other plants do not (Allina et al., 1998, and references
therein). Antisense suppression of 4CL activity in Arabi-
dopsis and tobacco has also led to con¯icting results
(Kajita et al., 1997; Lee et al., 1997).
Phenylpropanoid metabolism is regulated primarily via
transcriptional control of the corresponding genes (Dixon
and Paiva, 1995). The genes PAL, C4H and 4CL have been
demonstrated to be coordinately expressed in an organ-,
cell-type-, and stimulus-speci®c manner (Koopmann et al.,
1999; Logemann et al., 1995; Reinold and Hahlbrock, 1997).
4CL expression is induced upon environmental stresses
such as wounding or UV irradiation, conditions under
which phenylpropanoid derivatives play an important
protective role (Dixon and Paiva, 1995; Lee et al., 1995).
Transcriptional activation of 4CL has also been shown after
elicitor treatment of cultured cells from different plants
including soybean, parsley and Arabidopsis (Douglas
et al., 1987; Trezzini et al., 1993; Uhlmann and Ebel, 1993),
and it has been demonstrated in soybean and parsley
following inoculation with Phytophthora sojae and in
potato infected with Phytophthora infestans (Becker-AndreÂ
et al., 1991; Schmelzer et al., 1989; Uhlmann and Ebel,
1993). In Arabidopsis, transient accumulation of 4CL
mRNA was detected upon in®ltration of leaves with an
avirulent strain of the bacterial pathogen, Pseudomonas
syringae pv maculicola (Lee et al., 1995).
Previously, 4CL was reported to be encoded by a single
gene in Arabidopsis and the corresponding cDNA was
characterized (Lee et al., 1995). In this study, we report on
the isolation of two additional, divergent Arabidopsis
cDNAs as well as the three genes encoding 4CL. Differ-
ences in enzymatic properties, endogenous expression
patterns and transcript accumulation following infection
with the fungus, P. parasitica, wounding or UV irradiation
strongly suggest different physiological functions of the
three isozymes in Arabidopsis. Based on phylogenetic
reconstruction, two classes of 4CL isoforms were identi®ed
among angiosperms and the functional differences of
these evolutionary divergent classes are discussed.
Results
Isolation and characterization of the 4CL genes and
cDNAs
A genomic 4CL clone was isolated from an Arabidopsis
Col-0 genomic library, using the published Arabidopsis
4CL cDNA as a probe (Lee et al., 1995). This gene contained
1.6 kb of putative promoter sequence, an open reading
frame interrupted by three introns, and 3.2 kb of 3¢-non-
coding sequence. Although 4CL was presumed to be a
single-copy gene in Arabidopsis (Lee et al., 1995), the open
reading frame of the genomic clone showed only 80%
identity to the cDNA. We designated the cDNA At4CL1 and
the isolated gene At4CL2 (Figure 1). To isolate the corre-
sponding At4CL2 cDNA, an Arabidopsis cDNA library was
screened with a subclone of At4CL2 containing the ®rst
exon. The resulting cDNA contained an open reading
frame of 1671 bp, 51 bp 5¢-untranslated region, 211 bp 3¢-
non-coding region, and a poly(A) tail. The sequence of the
open reading frame was identical to that of At4CL2.
At4CL1 was PCR-ampli®ed from genomic DNA using
gene-speci®c primers derived from the 5¢ and 3¢ untrans-
lated regions of the cDNA. The product obtained contained
an open reading frame identical to that of the At4CL1
cDNA, interrupted by three introns (Figure 1). The promo-
ter sequence of At4CL1 was PCR-ampli®ed from genomic
DNA using the RAGE procedure (Cormack and Somssich,
1997). The PCR product contained 474 bp of the coding
region and 1 kb of 5¢ upstream promoter sequence. The
sequence of the coding region was in complete agreement
with that of the At4CL1 cDNA and the authenticity of the
At4CL1 promoter region was veri®ed by sequence com-
parison to an independent PCR ampli®cation product.
A third divergent gene, designated At4CL3, was identi-
®ed upon sequence analysis of a genomic clone previously
isolated by cross-hybridization to a parsley 4CL cDNA
(Trezzini et al., 1993). This truncated clone contained the
major part of the 4CL coding region interrupted by six
introns of different sizes and 0.6 kb of 3¢-non-coding
sequence (Figure 1). The coding region of At4CL3 showed
only 65% and 64% identity to At4CL1 and At4CL2,
respectively. The missing 5¢ region of At4CL3 and 645 bp
of putative promoter sequence was PCR-ampli®ed using
the RAGE procedure as described above. The correspond-
ing At4CL3 cDNA was isolated by screening a cDNA library
with the genomic At4CL3 insert. Sequence analysis of the
At4CL3 cDNA showed that it contains the complete coding
region of 1686 bp identical to that of At4CL3, ¯anked by
46 bp of 5¢ and 147 bp of 3¢ untranslated sequences.
Sequence comparisons of At4CL1, At4CL2 and At4CL3
indicated that the positions of three introns found in these
genes are conserved but that the introns differ both in
length and sequence. At4CL3 contains three additional
introns, one interrupting the region corresponding to the
®rst exon of At4CL1 and At4CL2, and two in the region of
their second exons (Figure 1). The 5¢ untranslated and
putative promoter regions show no overall sequence
similarities. Putative TATA boxes are located 124, 103
and 116 bp upstream of the ATG translation initiation sites
of At4CL1, At4CL2 and At4CL3, respectively (Figure 1).
Three sequence motifs, box P, box A and box L, which are
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 9±20
 Arabidopsis 4-coumarate:CoA ligase genes 11

Figure 1. Structural features of the three 4CL

genes from Arabidopsis.
(a) Schematic representation of the gene
structures of At4CL1, At4CL2 and At4CL3.
Exons are represented as dark grey boxes,
open reading frames corresponding to each
other are indicated by the light grey
shading.
(b) Relative positions of the putative CAAT,
TATA, P-, A- and L-boxes within the ®rst
250 bp of the three At4CL promoters.
(c) Comparison of the consensus sequences
of P-, A- and L-boxes (Logemann et al.,
1995) with the corresponding motifs found
within the At4CL promoters.

conserved among several plant PAL, C4H and 4CL genes,
including those in Arabidopsis (Logemann et al., 1995), are
also present in the At4CL3 gene promoter, whereas the
promoters of At4CL1 and At4CL2 contain only boxes P and
L (Figure 1).
Genomic DNA blot analyses
The limited sequence identity amongst the three genes
may explain why 4CL was previously assumed to be a
single-copy gene in Arabidopsis (Lee et al., 1995; Trezzini
et al., 1993). Indeed, unique sets of restriction fragments
were observed on genomic DNA blots using distinct
probes for each gene and high-stringency ®lter washing
conditions (Figure 2). When restriction enzymes were used
that do not cut within the probes, single hybridization
signals were observed in each case; with other enzymes,
all hybridization signals could be explained on the basis of
the available genomic sequences. Even under low-strin-
gency conditions, no clear cross-hybridization was
observed to additional fragments or between the Arabi-
dopsis 4CL genes (data not shown).
Enzymatic activities of the recombinant At4CL proteins
To examine the biochemical properties of the Arabidopsis
4CLs, the three cDNAs were expressed in E. coli in two
forms, as fusion proteins both containing and lacking N-
terminal His6-tags. Figure 3 shows that the recombinant
proteins had apparent molecular masses of approximately
66 kDa and reacted with an antibody raised against parsley
4CL. At4CL2 migrated to a similar position in the denatur-
ing polyacrylamide gel as did the 4CL present in protein
extracts of bolting stems. The slower migration of the
recombinant At4CL1 and At4CL3 is due to both having
longer N-terminal extensions (see Experimental proce-
dures).
Extracts from bacteria expressing the three recombinant
proteins lacking the His6-tag were tested for their abilities
to utilize different substituted cinnamic acids as substrates.

ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 9±20

12 JuÈrgen Ehlting et al.
All recombinant proteins converted 4-coumarate to the
CoA-ester and hence are bona ®de 4CLs. The Km values of
At4CL1 and At4CL3 for coumarate are similar to those
reported for many puri®ed plant 4CLs, whereas that of
At4CL2 is considerably higher, resulting in a 10-fold lower
speci®city (Vmax/Km) for this substrate (Table 1). The
Figure 2. Genomic DNA blot analysis of At4CL genes.
Arabidopsis DNA (10 mg lane±1) was digested to completion with the
indicated restriction enzymes, separated on agarose gels and blotted
onto a nylon ®lter. The ®lter was ®rst hybridized with the At4CL1 cDNA,
and after washing off the probe, subsequently rehybridized with a
genomic 1.5 kb HindIII fragment of At4CL2 containing the ®rst exon, and
®nally with a genomic 4.5 kb EcoRI/XbaI fragment of At4CL3.
apparent Km, relative Vmax and relative Vmax/Km values
determined for the different substrates tested are listed in
Table 1. It should be noted that cinnamate was also
converted with low ef®ciency by all three enzymes,
whereas sinapate was not converted at all.
No differences in 4CL activities were detected when the
proteins were expressed with or without an N-terminal
His6-tag (data not shown). In extracts of bacterial strains
harbouring the empty expression vector (pQE), no 4CL
protein (Figure 3) or 4CL activity (not shown) was detect-
able.
Quanti®cation of low-abundance mRNAs by RT±PCR
Since no cross-hybridization between the three 4CL genes
was observed by DNA blot analyses, we used the same
probes and hybridization conditions to study expression of
At4CL1, At4CL2 and At4CL3 by RNA blot analysis. How-
ever, At4CL3 mRNA was not detectable, presumably due
to its low abundance. To determine the relative mRNA
amounts of At4CL3 and compare its pattern of expression
to that of At4CL1 and At4CL2, we developed a rapid and
simple RT±PCR method for quanti®cation of low-abun-
dance mRNA. Using At4CL3-speci®c primers (one span-
ning an exon±intron junction to avoid ampli®cation of
genomic DNA), a PCR product of 340 bp was generated
and quanti®ed by staining with the ¯uorescent dye Sybr-
Green I after separation on polyacrylamide gels. Within the
range of 10±180 ng of total cotyledon RNA used as
template, a linear increase in the amount of PCR product
and ¯uorescence signal output was observed. A similar
relationship was obtained using At4CL3 cDNA as template
for PCR, whereas no ampli®cation was detectable from
At4CL1 or At4CL2 cDNA templates under the same
conditions. Up to a relative ¯uorescence of 25 000 units,
no saturation of the signal intensity was observed. Thus,
for further analyses, 100 ng of total RNA was used for RT±
PCR with relative ¯uorescence values not exceeding
Figure 3. Immunoblot analysis of the
recombinant Arabidopsis 4CLs.
Three 4CLs from Arabidopsis were
expressed as fusion proteins with N-
terminal His6-tag in E. coli and puri®ed
from bacterial extracts using Ni-NTA
agarose columns. The puri®ed, recombin-
ant proteins (0.5 mg lane±1), total protein
extracts from Arabidopsis bolting stems
and from elicitor-treated parsley cells
(5 mg lane±1) were separated by SDS±PAGE,
transferred to nitrocellulose membranes
and probed with antiserum raised against
puri®ed 4CL from parsley at a dilution of
1:5000 for 1 h. Protein extracts (0.5 mg
lane±1) from bacteria containing the empty
expression vector (pQE) served as control
lane.
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 9±20
 Arabidopsis 4-coumarate:CoA ligase genes 13

Table 1. Kinetic properties of recombinant Arabidopsis 4CLs

Km Relative Vmax Relative Vmax /Km

(mM) (% of coumarate) (1/mM)

At4CL1 Cinnamate 6320 103 0.02

4-coumarate 38 100 2.6
Caffeate 11 27 2.5
Ferulate 199 53 0.26
Sinapate n.c. ± ±
At4CL2 Cinnamate 6630 21 0.003
4-coumarate 252 100 0.39
Caffeate 20 74 3.7
Ferulate n.c. ± ±
Sinapate n.c. ± ±
At4CL3 Cinnamate 2070 164 0.08
4-coumarate 23 100 4.4
Caffeate 374 129 0.35
Ferulate 166 86 0.52
Sinapate n.c. ± ±

Km and Vmax were determined by linear regression of v against v/s (Eadie±Hofstee plot) with at least six data points. Absolute Vmax
values with 4-coumarate as substrate were 1.13, 0.28 and 0.19 pkat ml±1 protein extract for At4CL1, At4CL2 and At4CL3, respectively.
Relative Vmax values were obtained by setting Vmax for 4-coumarate for each enzyme to 100%. The data shown are representative for
three independent measurements. n.c., no conversion.

25 000 units. Using gene-speci®c primers, the relative
abundance of At4CL1 and At4CL2 mRNAs was also
determined by this method and resulted in expression
patterns virtually identical to the RNA blots shown below.
Expression patterns of the At4CL genes
The differential expression of the Arabidopsis 4CL gene
family was assessed by RNA blot analysis or quantitative
RT±PCR, as outlined above. Since the general phenylpro-
panoid pathway is pathogen-activated in all plants ana-
lysed to date, we inoculated Arabidopsis with the fungus
Peronospora parasitica, the causal agent of the downy
mildew disease, and studied expression of the At4CL
genes in comparison to other inducible Arabidopsis
phenylpropanoid and defence-related genes (Trezzini
et al., 1993). When challenged with P. parasitica pathovar
Noks1, an incompatible interaction is established on
Arabidopsis ecotype Ler-1 cotyledons which is character-
ized by formation of necrotic cavities without sporulation
of the fungus (Holub et al., 1994). Figure 4 shows that
At4CL1 and At4CL2 mRNA accumulation was strongly
induced, starting 12 h and 24 h after inoculation with
fungal spores, respectively. In contrast, the amount of
At4CL3 mRNA was not affected by infection (Figure 4b). As
expected, strong accumulation of AtPAL and AtC4H
mRNAs was also induced by infection, with patterns
similar to those of At4CL1 and At4CL2, respectively.
Expression of the peroxidase gene, AtPOX (Trezzini et al.,
1993), reached a maximum 4 days or later after inoculation
with fungal spores (Figure 4a).
Abiotic stresses such as wounding or irradiation with UV
light also activate phenylpropanoid gene expression
(Dixon and Paiva, 1995). At4CL1 mRNA accumulated
rapidly and transiently in wounded Arabidopsis leaves
(Figure 5a), consistent with previous reports (Lee et al.,
1995). The wound-induced expression pattern of At4CL2
was similar to that of At4CL1; however, the maximum
amount of At4CL2 mRNA was considerably lower than that
of At4CL1 or the wound-inducible allene oxide synthase
gene, AtAOS (Laudert et al., 1996), which served as a
positive control for wounding ef®ciency. At4CL3 mRNA
levels were not affected by wounding (Figure 5a).
When dark-adapted Arabidopsis plants were illuminated
with UV-containing white light, both At4CL1 and At4CL2
mRNAs accumulated rapidly and transiently, reaching
maximum levels 6 h after the onset of irradiation (Figur-
e 5b). Quantitative RT±PCR revealed that At4CL3 mRNA
accumulated more strongly after UV irradiation, but in
contrast to At4CL1 and At4CL2 remained at this elevated
level from 6 h to at least 24 h (Figure 5b). However, even
these elevated At4CL3 mRNA levels remained undetect-
able on RNA blots containing 20 mg of total RNA (data not
shown).
Analysis of the organ-speci®c expression pattern re-
vealed that mRNA levels of At4CL1 were highest in
seedling roots and in bolting stems of mature plants,
siliques contained intermediate levels, whereas mature
leaves and ¯owers contained only low levels (Figure 5c).
At4CL2 mRNA amounts were also high in seedling roots,
whereas low amounts were present in all other organs.
Both At4CL1 and At4CL2 were also expressed in 2- and 3-

ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 9±20

14 JuÈrgen Ehlting et al.
day-old seedlings, with higher levels of both transcripts in
3-day-old seedlings as previously reported (Lee et al., 1995)
(data not shown). A different expression pattern was
observed for At4CL3. The highest relative mRNA amount
was detected in ¯owers, intermediate mRNA amounts
were present in mature leaves and siliques, and low mRNA
Figure 4. Induction of 4CL and other defence-related mRNAs in
Arabidopsis upon infection with P. parasitica.
(a) Arabidopsis Ler-1 seedlings were spray-inoculated with P. parasitica
pv Noks1 and harvested at the times indicated. Control plants were
sprayed with water. Total extractable RNA (20 mg lane±1) was separated
on agarose gels, blotted and hybridized with DNA probes speci®c for
At4CL1, At4CL2, C4H, PAL, peroxidase (POX). (b) At4CL3 mRNA was
ampli®ed from 100 ng total RNA by quantitative RT±PCR as described in
Experimental procedures. The products were separated on poly-
acrylamide gels, stained with SybrGreen I and visualized with a
¯uorescence imager. A false colour image is shown.
amounts were observed in seedling roots and bolting
stems of mature plants (Figure 5c).
Phylogenetic comparison of 4CL genes
Although clearly divergent, the deduced amino acid
sequences of the isolated At4CL genes show signi®cant
similarity throughout their complete lengths, with the
predicted At4CL1 and At4CL2 amino acid sequences being
more similar to each other (86% identity) than to the
predicted At4CL3 sequence (71% and 73% identity, respec-
tively). Amino acid sequence similarity is most pro-
nounced in the central and C-terminal parts of the
proteins. Beginning at position G407 of At4CL1, the amino
acid motif GEICIRG is conserved at the same relative
positions in all plant 4CLs analysed to date, including
At4CL2 and At4CL3. This motif has been proposed to be
associated with stability and catalytic activity of 4CL and
related enzymes (Becker-Andre et al., 1991). In addition, all
At4CLs contain a motif within the putative AMP binding
domain that is highly conserved in all known 4CLs as well
as in other enzymes that share a similar reaction mechan-
ism (Uhlmann and Ebel, 1993).
A phylogenetic reconstruction based on the different
plant 4CL amino acid sequences available from databases
is shown in Figure 6, and revealed that within the
angiosperms, two major 4CL groups have evolved, which
we designated class I and class II. At4CL1 and At4CL2 are
most closely related to each other within the class I cluster,
whereas At4CL3 is in the divergent class II cluster.
Discussion
Although 4CL was previously assumed to be encoded by a
single-copy gene in Arabidopsis, we have shown in this
paper that it actually comprises at least three genes. Based
Figure 5. Wound-, UV-light- and organ-
speci®c accumulation of At4CL mRNAs.
(a) For wounding, Arabidopsis leaves were
cut into 3±5 mm strips and incubated at
high humidity for the time periods
indicated. Time point 0 h represents leaves
of untreated plants. Total extractable RNA
(20 mg lane±1) was separated on agarose
gels, blotted and hybridized with DNA
probes speci®c for At4CL1, At4CL2 and a
wound-inducible control gene, AtAOS,
encoding allene oxide synthase. At4CL3
mRNA quanti®cation by RT±PCR was
performed as described in Figure 4.
(b) For UV irradiation, dark-adapted
Arabidopsis seedlings were exposed to UV-
containing white light for the times
indicated and used for RNA extraction and
analysis as above.
(c) Organs were separated and used for
RNA extraction and analysis as above.
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 9±20

on the enzymatic activities of the recombinant proteins, all
three genes encode bona ®de 4CLs. However, the three
isoforms show clear differences in their substrate speci®-
cities. Detailed expression studies also demonstrated
differential regulation of the three At4CL genes, indicative
of different physiological functions. Because we failed to
isolate clones containing additional 4CL genes during
extensive screenings of both genomic and cDNA libraries,
we believe that these may represent the full complement
of 4CL genes in Arabidopsis. Search for `4CL-like'
sequences in the Arabidopsis databases and analysis of
two such sequences (gene F13C5.180 and EST 144C1t7)
suggested that they are unlikely to encode authentic 4CLs
since the degree of amino acid identity relative to bona
®de 4CLs is low (38±44%) and the amino acid motif
GEICIRG within the putative catalytic domain is poorly
conserved (T. Otke and C. Douglas, unpublished data).
Phylogenetic reconstruction with available amino acid
sequences revealed that 4CL forms two major clusters
among the angiosperms (Figure 6). Within the class I
cluster, the Arabidopsis 4CL1 and 4CL2 isoforms are most
closely related to each other, as are the isoforms from
hybrid poplar, tobacco, parsley and potato. This indicates
that class I isoforms have evolved relatively recently after
independent gene duplications within the respective plant
lineages. In contrast, the At4CL3 isoform groups within the
class II cluster of angiosperm 4CL sequences. The greater
Figure 6. Phylogenetic reconstruction based on 4CL amino acid
sequences.
The most parsimonious tree was found using the TBR heuristic branch
swapping algorithm with the PAUP 3.1.1 program. The number of steps
between nodes is shown above the branch length, and bootstrap values
(500 replications) are shown below the branch length. The tree has a
consistency index of 0.793. The allelic pine 4CL1 and 4CL2 sequences
were used as the outgroup to root the tree.
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 9±20
Arabidopsis 4-coumarate:CoA ligase genes 15
evolutionary distance between At4CL3 and the other two
Arabidopsis genes is also obvious at the structural level,
with At4CL3 containing three additional introns that are
not present in At4CL1 and At4CL2. Genes encoding
divergent 4CL isoforms represented in both clusters have
also been cloned from soybean, aspen and Lithospermum,
indicating an early gene duplication event during angio-
sperm evolution. Clear differences in expression patterns
and/or enzymatic properties have been described that
distinguish class I and class II 4CL isoforms in Arabidopsis
(this study), soybean (Uhlmann and Ebel, 1993) and aspen
(Hu et al., 1998). In contrast, the analysis of class I isoforms
has revealed few if any enzymatic differences in several
plants (Allina et al., 1998, and references therein). How-
ever, in Arabidopsis, the class I isoforms do show
divergence in expression patterns and enzymatic proper-
ties. Therefore, an early gene duplication most likely has
lead to the evolution of functionally divergent 4CL iso-
forms of classes I and II, whereas the relatively recent
independent gene duplication events within the class I
cluster have not yet resulted in functionally distinct 4CLs in
all cases.
Although still based on limited data, we hypothesize that
a primary function of class II 4CL isoforms is to channel
activated 4-coumarate to chalcone synthase and subse-
quently to different branch pathways of ¯avonoid second-
ary metabolism leading to ¯ower pigments and UV
protective ¯avonols and anthocyanins (Figure 7). For
Arabidopsis, this proposal is based on our observations
that: (a) At4CL3 is expressed to relatively high levels in
¯owers, but not in ligni®ed organs such as bolting stems
and roots; (b) At4CL3 expression is strongly induced upon
UV irradiation whereas fungal infection and wounding
have no effects, and (c) At4CL3 has a high speci®city for 4-
coumarate, relative to caffeate and ferulate (the more
immediate precursors for lignin biosynthesis). A similar
function in the biosynthesis of non-lignin-related phenyl-
propanoids has been postulated for the related class II 4CL
from aspen (Pt4CL2), which is preferentially expressed in
immature organs but not in lignifying secondary xylem,
and whose enzymatic properties are consistent with it
being involved in ¯avonoid biosynthesis (Hu et al., 1998).
In addition, the class II 4CL isoform from soybean (4CL16)
is speci®cally induced in infected plants in coordination
with other enzymes of phenylpropanoid metabolism, such
as PAL and chalcone synthase, and is thus assumed to
participate in the biosynthesis of anti-microbial phytoalex-
ins derived from the ¯avonoid pathway (Uhlmann and
Ebel, 1993). Unlike soybean and other legumes, the
phytoalexins in Arabidopsis (Tsuji et al., 1992) are not
¯avonoids, perhaps explaining the lack of At4CL3 induc-
tion upon infection with P. parasitica.
The apparent low overall expression levels of At4CL3 in
all organs and upon all treatments tested may not
16 JuÈrgen Ehlting et al.
necessarily re¯ect the true cellular situation. At present it
cannot be excluded that At4CL3 mRNA accumulation is
highly restricted to a few speci®c cell types, e.g. epidermal
cells, as suggested for Pt4CL2 in aspen (Hu et al., 1998) Use
of At4CL3 promoter±reporter gene constructs for trans-
genic studies, and in situ localization of At4CL3 mRNA, will
certainly help to clarify this point.
All 4CL isoforms known so far from parsley, tobacco,
potato and hybrid poplar group within the class I cluster.
One possibility is that divergent class II isoforms exist in
these plants, but remain to be isolated. Indeed, a PCR-
based approach has revealed the existence in hybrid
poplar of an orthologue of the aspen gene Pt4CL2 (D.
Cukovic and C. Douglas, unpublished data). It is also
conceivable that class II isoforms have been lost during
evolution and have been functionally replaced by class I
isoforms in some plant species.
The two Arabidopsis class I 4CLs described here display
signi®cant differences in their enzymatic properties and in
their organ-speci®c expression patterns. At4CL1 shows a
high speci®city for 4-coumarate and caffeate in compar-
ison to ferulate. At4CL1 is also the only gene family
member strongly expressed in the ligni®ed bolting stems
of adult plants (Figure 5c), where in situ hybridization
showed that its expression is speci®c to the xylem and
sclerenchyma (Q. Wang and C. Douglas, unpublished
data). It is also expressed at the onset of lignin deposition
in seedling roots and cotyledons (data not shown) (Lee
et al., 1995). Together, these data strongly support a
primary role for At4CL1 in the biosynthesis of p-hydro-
Figure 7. Proposed roles of Arabidopsis 4CL isoforms in biosynthetic
pathways of phenylpropanoids.
For 4CL, the arrow size re¯ects relative substrate speci®cities and
conversion rates (Vmax/Km) by the indicated isoforms, as determined in
this paper. PAL, phenylalanine ammonia-lyase; C4H, cinnamate-4-
hydroxylase; C3H, coumarate-3-hydroxylase; COMT, caffeic acid O-
methyltransferase; F5H, ferulate-5-hydroxylase; CHS, chalcone synthase;
CCo3H, coumaroyl-CoA-3-hydroxylase; CCoOMT, caffeoyl-CoA 3-O-
methyltransferase; n.d., not determined; n.c., no conversion.
xyphenyl and guaiacyl lignin subunits, the latter requiring
methylation of caffeoyl-CoA to feruloyl-CoA (Figure 7). A
similar role in lignin biosynthesis has also been postulated
for the class I Pt4CL1 isoform from aspen (Hu et al., 1998),
for genes encoding other class I 4CL isoforms such as
tobacco 4CL1 and 4CL2, which are highly expressed in
lignifying stems (Lee and Douglas, 1996), and for the
parsley 4CL1 gene, whose promoter directs strong expres-
sion to lignifying tissue (Hauffe et al., 1991).
The enzymatic properties of At4CL2 are unusual in so far
as caffeate is converted with higher ef®ciency than 4-
coumarate, and ferulate is not converted at all. But the
existence of an O-methylation pathway that operates on
caffeoyl-CoA to produce feruloyl-CoA (Ye et al., 1994)
suggests that At4CL2 could participate in the biosynthesis
of lignin and/or other caffeoyl-CoA derived phenolic
compounds, especially in seedling roots where At4CL2 is
expressed along with At4CL1. Since the existence of
caffeoyl-derived phenolics such as chlorogenic acid is
unknown in Arabidopsis, this possibility remains to be
experimentally addressed.
The lack of any detectable conversion of sinapate by any
of the Arabidopsis 4CL isoforms con®rms and extends
similar observations made with a number of other plants
and supports the hypothesis that the biosynthesis of
sinapyl alcohol and syringyl lignin may occur via a 4CL-
independent, as yet uncharacterized, pathway (Allina et al.,
1998; Lee et al., 1997). Independent gene duplications
within the class I cluster of different angiosperm plant
lineages may re¯ect the on-going evolution of divergent
and functionally specialized 4CL isoforms, such as At4CL1
and At4CL2 in Arabidopsis, as opposed to functionally
redundant 4CL isoforms, present in plants such as parsley,
potato and tobacco.
The fact that not only At4CL3, but also At4CL1 and
At4CL2, are induced after irradiation with UV-containing
white light, and that all three isoforms support the
biosynthesis of 4-coumaroyl-CoA, may argue against a
strict functional separation between class I and class II
isoforms. However, the light stress response of class I
isoforms may be required to support the activity of the
class II isoforms by increasing the supply of 4-coumaroyl-
CoA esters for the ¯avonoid biosynthetic pathway. Alter-
natively, other phenylpropanoid compounds in addition to
¯avonoids may be required to protect the plant from
damage by UV irradiation, and the differences in the extent
and timing of induction of At4CL3, as opposed to that of
At4CL1 and At4CL2, may re¯ect their distinct roles in
protection. Furthermore, it is likely that treatment with UV
light initiates developmental processes requiring the
synthesis of other phenylpropanoid compounds derived
from pathways controlled by At4CL1 and At4CL2.
The expression patterns of At4CL1 and At4CL2 in
response to UV irradiation, wounding or infection with
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 9±20

the fungus P. parasitica, showed no signi®cant differences,
except that the amounts of At4CL1 mRNA were, in general,
5±10 times higher than those of At4CL2 (data not shown).
These and earlier results, demonstrating rapid accumula-
tion of At4CL1 mRNA in leaves in®ltrated with an avirulent
strain of the bacterial pathogen P. syringae (Lee et al., 1995),
indicate that both genes may have important functions in
pathogen defence. These may include the biosynthesis of
lignin and other cell-wall-bound phenolic compounds at
the site of pathogen ingress, aimed at restricting the growth
of the micro-organisms. Cell-wall thickening and the
appearance of auto¯orescent, phenolic material at infection
sites are consistent with such a function and have been
observed in Arabidopsis (data not shown) and numerous
other plants (Kombrink and Somssich, 1995). In addition,
cell wall reinforcement and/or sealing of wounded tissue
with phenolics may help to limit subsequent, secondary
infections caused by opportunistic micro-organisms. A
defensive role for 4CL in Arabidopsis is further supported
by results obtained using Arabidopsis plants transgenic for
a parsley class I 4CL promoter±GUS fusion. In these plants,
a strong and rapid activation of the GUS reporter gene is
induced by Peronospora infection and locally restricted to
the vicinity of fungal infection sites (data not shown).
The interaction between Arabidopsis and P. parasitica is
an intensively studied plant pathosystem (Kunkel, 1996)
and a large number of genetically well-de®ned interaction
types have been described (Holub et al., 1994). Several
signal transduction mutants in defence response pathways
showing altered susceptibility to P. parasitica have been
isolated (Dong, 1998), along with a major resistance gene,
RPP5, conferring resistance against P. parasitica pv Noko1
(Parker et al., 1997). Little is known about the molecular and
biochemical mechanisms leading to the resistant pheno-
type and to the transcriptional activation of defence-related
genes at sites of fungal ingress. In this respect, further
studies of At4CL1 and At4CL2 should prove extremely
valuable, particularly for mutagenesis screens aimed at
identifying regulators of this locally activated plant defence
reaction. Finally, the isolation and analysis of knock-out
mutants in the three divergent 4CL genes should help to
elucidate the speci®c metabolic functions of the corre-
sponding enzymes.
Experimental procedures
Plant material and stress treatments
For organ isolation, Arabidopsis thaliana Col-0 plants were grown
under short-day conditions (8 h light, 16 h dark, 20°C) in phyto-
chambers or under long-day conditions (16 h light, 8 h dark) in the
greenhouse. Healthy organs were isolated and immediately
frozen in liquid nitrogen.
Peronospora parasitica pathovar Noks1 was maintained by
weekly inoculation of susceptible Arabidopsis ecotype Col-5 as
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 9±20
Arabidopsis 4-coumarate:CoA ligase genes 17
previously described (Dangl et al., 1992). For infection studies, 10-
day-old Arabidopsis (Ler-1) seedlings grown in soil or on AM
plates (0.5 3 MS salts supplemented with vitamins (Sigma,
Deisenhofen), 1% sucrose, 0.6% agar) were spray-inoculated with
conidia suspensions (50 per ml), maintained in humidity chambers
under short-day conditions (except that the temperature in the
dark was 16°C). For each sample, approximately 5000 seedlings
were infected and harvested by freezing the whole pot or agar
plate in liquid nitrogen. Aerial parts of the seedlings (cotyledons
and hypocotyl) were scratched off the frozen surface and stored at
±80°C. Control plants were sprayed with water.
For wounding experiments, Arabidopsis Col-0 plants were
grown under short-day conditions for 3 weeks in phytochambers.
Healthy looking, fully expanded leaves were detached, cut into 3±
5 mm strips, placed on MS-soaked Whatman paper in sealed Petri
dishes, and incubated in the light. Samples were frozen in liquid
nitrogen at the time points indicated. Control leaves were
detached and incubated without further wounding.
For UV-light treatment, Arabidopsis Col-0 plants were grown
under short-day conditions for 12 weeks. Plants were dark-
adapted for 36 h and subsequently irradiated with UV-containing
white light (Hartmann et al., 1998). Plants were harvested at
different times after onset of irradiation and stored at ±80°C.
Isolation of At4CL genomic and cDNA clones
Genomic regions of At4CL1 were PCR-ampli®ed from 100 ng
genomic Col-0 DNA with gene-speci®c primers deduced from the
Arabidopsis 4CL1 cDNA (Lee et al., 1995) (forward primer B6F: 5¢-
AATGGCGCCACAAGAACAAGCAGTTTCTCA-3¢, position 5±34 in
the cDNA; reverse primer B8R: 5¢-CACATTGAAATCATCTTTGC-
TTTTTGGTTG-3¢, position 1731±1702). PCR was performed with
pwo polymerase (Boehringer, Mannheim) with an annealing
temperature of 60°C for 30 cycles. PCR products of two
independent ampli®cations were blunt-end-cloned into EcoRV-
digested pBluescriptSK vector. Promoter regions of At4CL1 were
isolated using the RAGE procedure (Cormack and Somssich,
1997). Brie¯y, genomic Col-0 DNA was digested with HindIII and
subsequently polyadenylated with terminal deoxynucleotide
transferase. The ®rst PCR ampli®cation was done with a gene-
speci®c upstream directed primer, B4R2 (5¢-AGTCGATGACTTCT-
GATGCCTCGGTGGTC-3¢, position 591±562 in the Arabidopsis
4CL1 cDNA) and a universal-T17 primer using Taq+ polymerase
(30 cycles at 94°C for 15 sec, 60°C for 30 sec, and 72°C for 3 min).
Re-ampli®cation was performed under the same conditions with a
nested gene-speci®c primer, B7R (5¢-ATGACTACTCCGTCGTCG-
TTTTGAAGTGGT-3¢, position 490±461) and the universal primer.
The resulting 1686 bp PCR product was blunt-end-cloned into the
pBluescriptSK vector. To verify the DNA sequence of the RAGE
product, the isolated promoter region was ampli®ed from
genomic DNA using the pwo polymerase with two speci®c
primers deduced from the RAGE product, B11F (5¢-ATTAACTG-
CAGACACTTTTAGCCCATAACTTTC-3¢) and B11R (5¢-CTTGTGGC-
GCCATGGTAAATAGTAAATATTGTG-3¢), and the resulting PCR
product was cloned and sequenced.
At4CL2 was isolated by screening a lEMBL4 genomic library
using the Arabidopsis 4CL1 cDNA insert (Lee et al., 1995) as a
probe under high-stringency hybridization and washing (0.23
SSC, 0.1% SDS, 65°C) conditions. Overlapping restriction frag-
ments were subcloned into the pBluescriptSK vector.
Sequence analysis of a previously isolated genomic 4CL clone
(Trezzini et al., 1993) revealed that it contained the major part of
the At4CL3 coding region. Clones containing the 5¢ end and the
promoter region of At4CL3 were isolated by the RAGE procedure
18 JuÈrgen Ehlting et al.
as described above using XhoI-digested genomic DNA. The ®rst
PCR was performed with the universal-T17 primer and the gene
speci®c primer R3 (5¢-GATTATGAGTTTGGCTCCGGAAGATT-
TAAGC-3¢), the second PCR with the universal primer and a
nested gene-speci®c primer R2 (5¢-CGGTAGTGATCAGCGT-
GAGGTTTTCTCC-3¢). The isolated region was subsequently
ampli®ed from genomic DNA using speci®c primers deduced
from the RAGE product and cloned into the pBluescriptSK vector.
cDNA clones for At4CL2 and At4CL3 were isolated from a lZAP
cDNA library (Korfhage et al., 1994). Approximately 160 000
plaques were screened with a 1.6 kb HindIII genomic subclone
spanning the ®rst exon of At4CL2 or with the 4.5 kb EcoRI/XhoI
genomic subclone of At4CL3 (Trezzini et al., 1993). For At4CL2 and
At4CL3, 16 and two positive clones, respectively, were identi®ed
in the initial screening round. Two full-length cDNA clones
corresponding to both genes were puri®ed and subsequently
subcloned into the pBluescriptSK vector.
DNA sequences of all isolated clones were determined by the
ADIS service unit (Max-Planck-Institut fuÈr ZuÈchtungsforschung,
KoÈln, Germany) on ABI PRISM 377 DNA sequencers (PE Applied
Biosystems, Foster City, California, USA) using BigDye Termina-
tor chemistry.
Nucleic acid methods
Standard molecular biology procedures were performed accord-
ing to Sambrook et al. (1989). Genomic DNA was isolated starting
from 1 g of Arabidopsis Col-0 leaves using the `plant DNA
isolation kit' (Boehringer, Mannheim, Germany) according to
the manufacturer's protocol. For DNA blot analyses, 10 mg of total
genomic DNA per lane was digested with appropriate restriction
enzymes, separated on 0.8% agarose gels, and blotted onto nylon
membranes (Amersham Pharmacia Biotech, Freiburg, Germany).
Probes were 32P-labelled using the `random primed labelling kit'
(Boehringer). After hybridization, blots were washed under
moderate (23 SSC, 0.5% SDS, 65°C) or high-stringency conditions
(0.23 SSC, 0.1% SDS. 65°C). Total RNA was isolated with the `total
RNA Maxi Kit' (Qiagen, Hilden) according to the manufacturer's
protocol. For RNA blot analyses, 20 mg of total RNA per lane were
separated on 1.2% agarose gels containing formaldehyde and
blotted onto nylon membranes. Filters were washed under high-
stringency conditions (0.23 SSC, 0.1% SDS, 65°C) after hybridiza-
tion. Equal loading was monitored by ethidium bromide staining
of the gels.
The Arabidopsis C4H probe was generated by PCR using
speci®c primers (forward: 5¢-GCCGACGATTTTCTCACCGG-3¢ and
reverse: 5¢-TCGTAGAACGAACCATTTAAAGAC-3¢) deduced from
the published C4H sequence (Bell-Lelong et al., 1997). Synthetic
oligonucleotides were from MWG (Ebersbach, Germany). A
previously isolated Arabidopsis genomic PAL clone (Trezzini
et al., 1993) was used as probe.
Quantitative RT±PCR
The concentration of total RNA was determined after dilution to
approximately 50 ng ml±1 using the ¯uorescence RNA stain Ribo-
Green (Molecular Probes, Eugene, Oregon, USA) in order to
ensure appropriate concentrations of template RNA, and subse-
quently diluted to 10 ng ml±1. For quantitative RT±PCR, 100 ng of
total RNA was used as template. RT±PCR was performed using
the `Titan-one-tube-RT±PCR system' (Boehringer) according to the
manufacturer's protocol with the following temperature pro®le:
50°C for 30 min; 94°C for 2 min; 25 cycles with 94°C for 15 sec, 64°C
for 30 sec and 68°C for 30 sec; and 68°C for 10 min. The gene-
speci®c primers D3F (5¢-CCCTTACTATTTGAATTTACATTAC-3¢)
and D1R (5¢-CTTAGTGTCCAACATCTATTGCGTCAAAGGATC-3¢)
amplify a 340 bp product of the At4CL3 mRNA. D3F was deduced
from the cDNA such to span an exon±intron junction and
therefore does not bind to genomic DNA templates. RT±PCR
products obtained after 25 cycles were separated on polyacryla-
mide gels and stained with the ¯uorescence DNA dye SybrGreen I
(Molecular Probes). PCR products were visualized by scanning the
gel with a ¯uorescence imager (STORM860, Molecular Dynamics,
Sunnyvale, California, USA) and quanti®ed using ImageQuant
software.
Expression of At4CL proteins in E. coli
All three At4CL cDNAs were expressed in E. coli (M15) using the
QIAexpressionist expression vectors of the pQE50 (including a
His6-tag) and pQE30 (without His6-tag) series (Qiagen). The
complete At4CL1 cDNA was cloned in-frame into the pQE31 and
pQE51 vectors. This results in proteins containing 26 additional N-
terminal amino acids derived from the 5¢ untranslated region and
part of the polylinker of the pQE vectors. The At4CL2 cDNA was
cloned into the pQE30 and pQE50 vectors. However, since the
original At4CL2 5¢ untranslated region contains two in-frame stop
codons, it was deleted using PCR. This resulted in an expressed
protein with ®ve additional vector-derived N-terminal amino
acids. The 5¢ untranslated region of At4CL3 was deleted using a
PstI site at position +6 in the coding region and the PstI site in the
polylinker of the pBluescript vector and was subsequently cloned
into both the pQE32 and pQE52 expression vectors. The resulting
proteins therefore lack three of the original N-terminal amino
acids but contain six additional amino acids derived from the
expression vector.
Proteins were extracted from exponentially growing bacteria (in
100 ml LB medium) 3 h after induction of 4CL expression with
2 mM IPTG (A600 = 0.7). Bacterial pellets were resuspended in 10 ml
buffer (50 mM Na-phosphate, pH 7.8; 300 mM NaCl; 1 mg ml±1
lysozyme), incubated on ice for 30 min and the bacterial cells
were disrupted by soni®cation. Following centrifugation
(20 000 g), the crude protein extracts were supplemented with b-
mercaptoethanol (2 mM) and immediately used for enzyme
activity tests. His6-tagged 4CL proteins were puri®ed using Ni-
NTA spin columns (Qiagen) according to the manufacturer's
protocol. Puri®ed proteins were separated by SDS±PAGE, blotted
onto nitrocellulose membranes, and detected using an antiserum
raised against the parsley 4CL (Ragg et al., 1981).
Enzyme assays
4CL activity was measured at room temperature using a spectro-
photometric assay to detect the formation of CoA esters of
various cinnamic acid derivatives (Knobloch and Hahlbrock,
1977). The change in absorbance was monitored at wavelengths
of 311, 333, 369, 345 and 352 nm according to the absorption
maxima for cinnamoyl-CoA, 4-coumaroyl-CoA, caffeoyl-CoA,
feruloyl-CoA and sinapoyl-CoA, respectively (StoÈckigt and Zenk,
1975). Phenolic substrates (Sigma, Deisenhofen, Germany) were
used at concentrations ranging from 0.1 to 4 mM (cinnamate) and
from 0.005 to 0.8 mM (all other substrates). The kinetic constants,
Km and Vmax, for the phenolic substrates were determined at ®xed
concentrations of all other substrates by linear regression of v
against v/s (Eadie±Hofstee plot). Similar values were obtained
from Hanes plots (s/v against s). Each plot contained at least six
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 9±20

points. For comparison of activities in different bacterial extracts,
the apparent Vmax values were normalized by setting Vmax for 4-
coumarate to 100%.
Sequence comparisons
An alignment of published 4CL amino acid sequences was
generated using the PILEUP program within the GCG program
package, version 8.1 (Devereux et al., 1984) with manual optimiza-
tion. Based on this alignment, a maximum parsimony analysis
was performed (Fitch, 1977) using the PAUP 3.1.1 program
(Smithsonian Institution, Washington DC, USA). The most
parsimonious tree was found using the heuristic search option
with the `tree bisection reconnection' (TBR) branch swapping
algorithm (Swoffort and Olsen, 1990). For statistical analysis, 500
bootstrap replications (Felsenstein, 1985) were analysed. The
following plant 4CL sequences were used (GenBank accession
numbers given in brackets): Arabidopsis thaliana 4CL1 (U18675),
soybean 4CL14 (X69954), soybean 4CL16 (X69955), Lithosper-
mum erythrorhizon 4CL1 (D49366), Lithospermum erythrorhizon
4CL2 (D49367), tobacco 4CL (D43773), tobacco 4CL1 (U50845),
tobacco 4CL2 (U50846), rice 4CL1 (X52623), rice 4CL2 (L43362),
parsley 4CL1 (X13324), parsley 4CL2 (X13325), loblolly pine 4CL1
(U12012), loblolly pine 4CL2 (U12013), hybrid poplar 4CL1
(AF008184), hybrid poplar 4CL2 (AF008183), aspen 4CL1
(AF041049), aspen 4CL2 (AF041050), potato 4CL1 (M62755),
potato 4CL2 (AF150686) and vanilla 4CL (X75542).
Acknowledgements
We thank Dr W. Knogge and Professor K. Hahlbrock for critical
comments on the manuscript, and Professor K. Hahlbrock also for
his continuous interest and support of this project. We also thank
Dr E. Weiler (Ruhr-UniversitaÈt, Bochum, Germany) for providing
the allene oxide synthase cDNA, Dr Eric B. Holub (Horticulture
Research International, East Malling, UK) for providing the P.
parasitica isolate Noks1, Dr T. Debener (Bundesanstalt fuÈr
ZuÈchtungsforschung, Ahrensburg, Germany) for help in estab-
lishing the pathosystem in our laboratory, and Dr H. Kranz for the
supply of RNA of UV-treated plants. The excellent technical
assistance of Eva SchloÈsser is also acknowledged. This work was
supported in part by the Deutsche Forschungsgemeinschaft (Ko
1192/4-1) and by a research grant from the National Sciences and
Engineering Research Council of Canada to C.J.D.
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