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4-Coumarate:CoA ligase (4CL) is involved in the formation of coenzyme A thioesters of hydroxycinnamic acids that are central substrates for subsequent ...
Eur. J. Biochem. 269, 1304–1315 (2002) Ó FEBS 2002

Divergent members of a soybean (Glycine max L.) 4-coumarate:coenzyme A ligase gene family Primary structures, catalytic properties, and differential expression Christian Lindermayr1, Britta Mo¨llers1, Judith Fliegmann1, Annette Uhlmann1, Friedrich Lottspeich2, Harald Meimberg3 and Ju¨rgen Ebel1 1

Botanisches Institut der Universita¨t, Mu¨nchen, Germany; 2Max-Planck-Institut fu¨r Biochemie, Martinsried, Germany; Institut fu¨r Systematische Botanik der Universita¨t, Mu¨nchen, Germany

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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 phenylpropanoids. Previous studies of 4CL appear to suggest that many isoenzymes are functionally equivalent in supplying substrates to various subsequent branches of phenylpropanoid 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 high similarity with 4CL3. The recombinant proteins expressed in Escherichia coli possessed highly divergent catalytic efficiency with various hydroxycinnamic acids. Remarkably, one isoenzyme (4CL1) was able to convert sinapate; thus the first cDNA encoding a 4CL that accepts highly substituted cinnamic acids is available for further studies on branches of

phenylpropanoid metabolism that probably lead to the precursors of lignin. Surprisingly, the activity levels of the four isoenzymes 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 the 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 soybean could be regulated at the level of catalytic specificity and the level of expression of the 4CL isoenzymes.

Phenylpropanoid compounds are major constituents of higher plants. They can serve as flower pigments, UV protectants, defence chemicals, signalling compounds, allelopathic agents, and as building units of the phenolic support polymer, lignin. Their synthesis is regulated both by developmental processes and by environmental cues and it proceeds via the general phenylpropanoid pathway and subsequent specialized branches of phenylpropanoid metabolism. Central to many of the biosynthetic pathways is the activation of differently substituted cinnamic acids to the corresponding CoA thioesters. This reaction 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) displayed different substrate affinities 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 ring-modifications on 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 of multiple 4CL in the former plants remains largely unknown. 4CL genes have been studied in a large variety of plants, where they comprise small gene families in most cases. In a number of plants, including parsley (Petroselinum crispum), loblolly pine (Pinus taeda), and potato (Solanum tuberosum), the genes encode identical or very similar proteins [7,15,16], whereas in other plants, such as tobacco (Nicotiana

Correspondence to J. Ebel, Botanisches Institut der Universita¨t Mu¨nchen, Menzinger Strasse 67, D-80638 Mu¨nchen, Germany. E-mail: [email protected] Abbreviation: 4CL, 4-Coumarate:coenzyme A ligase. Enzyme: 4-coumarate:CoA ligase (EC 6.2.1.12). Note: the nucleotide sequence data reported were deposited under GenBank accession nos AF279267 for Gm4CL1 cDNA, AF002259 for 4CL14 (Gm4CL2 cDNA), AF002258 for 4CL13 (Gm4CL3 genomic), and X69955 for 4CL16 (Gm4CL4 cDNA). (Received 23 October 2001, revised 28 December 2001, accepted 9 January 2002)

Keywords: 4-coumarate:CoA ligase; differential regulation; heterologous expression; plant defence; soybean (Glycine max L.).

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4-Coumarate:CoA ligase gene family from Glycine max (Eur. J. Biochem. 269) 1305

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 typical phenylpropanoids, or with pathways that are affected 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]. Conversely, aspen Pt4CL2 is thought to be involved in the biosynthesis of phenylpropanoids in lignin-deficient epidermal 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 precursor for syringyl units of angiosperm lignin. Nevertheless, recent findings indicate that the activated esters of sinapate, ferulate, and 5-hydroxyferulate are not likely to participate in monolignol biosynthesis [11,12,14]. However, 4CL isoforms from soybean, petunia, pea, and poplar have been found to convert sinapate 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 CoAactivation 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 point of phenylpropanoid metabolism in plants, the precise function of 4CL isoforms in providing CoA ester precursors for the synthesis of different classes of phenolic compounds with specialized functions remains, thus, largely controversial. In soybean, the production of phenylpropanoid compounds comprises one of the biochemical defence reactions that are activated upon challenge with the oomycete pathogen Phytophthora sojae or treatment with a b-glucan elicitor derived from the pathogen. The phenolic compounds that accumulate around infection sites or in elicitortreated cell cultures include pterocarpan phytoalexins [24], isoflavone conjugates [25,26], and wall-bound phenylpropanoid compounds [27]. Phytoalexin accumulation is preceded by the induced expression of many of the enzymes involved in the biosynthetic pathway, including 4CL [20]. Previous biochemical [1] and molecular studies [20] indicated that 4CL in soybean is encoded 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 of the encoded isozymes for differently substituted cinnamic acid substrates were found. Combined 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 conclusions that the 4CL isoenzymes in soybean serve different physiological functions. Phylogenetic comparison based on amino acid

sequences extends the recent classification [18] of 4CL isoforms within angiosperms.

EXPERIMENTAL PROCEDURES Plant material Soybean seeds (Glycine max L. cv. Harosoy 63) were 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 experiments, the taproots of 3-day-old seedlings were treated with a suspension of  104 zoospores in 200 lL sterile distilled water by dip inoculation; control seedlings 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 acid 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 to previously reported procedures [29]. Enzyme activity was determined spectrophotometrically according to the 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 substituted cinnamic acids at substrate concentrations of 500 lM 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 acid, ferulic acid, and 3,4-dimethoxycinnamic acid, and at 352 nm for sinapic acid [32]. Whereas 4-coumarate served as a substrate for all four isoenzymes, ferulate was a substrate for isoenzymes 1 and 2 under the conditions used, and 3,4-dimethoxycinnamate was converted exclusively by isoenzyme 1. By measuring relative V-values, the activity level of each 4CL isoenzyme could be estimated indirectly by using the above indicated 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 tested in a concentration range of 0.1–4 mM whereas 2.5–1000 lM 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 using the 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. Protein extracts were stored at )20 °C.

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1306 C. Lindermayr et al. (Eur. J. Biochem. 269) Table 1. Scheme for indirect calculations of 4CL isoenzyme activities.

Isoenzyme

Activity calculated Calculation procedure using for substrate various initial substrates

4CL1

Ferulate 4-Coumarate

4CL2

Ferulate

4CL3/4CL4

4-Coumarate 4-Coumarate

0.6 · 4CL1 activity for 3,4-dimethoxycinnamate 1.1 · 4CL1 activity for 3,4-dimethoxycinnamate Total activity for ferulate minus 4CL1 activity for ferulate 1.4 · 4CL2 activity for ferulate Total activity for 4-coumarate minus 4CL1 and 4CL2 activities, respectively, for 4-coumarate

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 bands 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 partial 4CL1 purification: buffer A, 0.2 M Tris/HCl pH 8.0, 14 mM 2-mercaptoethanol, 0.2 mM phenylmethanesulphonyl fluoride, 30% (v/v) glycerol; buffer B, 0.05 M Tris/HCl pH 7.9, 0.1 mM dithiothreitol, 0.2 mM phenylmethanesulphonyl fluoride, 30% (v/v) glycerol. All steps were carried out at 4 °C. Frozen soybean cells (300 g) were thawed and homogenized with 150 g quartz sand and 150 mL buffer A in a chilled mortar, stirred for 20 min with

0.1 g Dowex 1 · 2 (Serva; equilibrated with buffer A) per gram of cells, and centrifuged to remove Dowex 1 · 2 and cell debris. 4CL1 activity was precipitated from the supernatant with (NH4)2SO4 (38–72% saturation), dissolved in buffer B, and 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) which had been equilibrated 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 were 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 desalted by chromatography on Sephadex G-25M. Finally, 4CL1 was separated from 4CL2 by anion exchange chromatography on a Resource 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 peptide sequences: the N-terminal sequence consisted of APSPQEIIF, sequence S1 of (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 seedlings of G. max L. was isolated according to [37]. DNA was prepared according to [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 of pQE-31/Gm4CL3 were prepared as hybridization probes.

Table 2. Sequences of oligonucleotides. Restriction sites contained in the oligonucleotides are underlined. Designation

Sequence

4CL1-GSP1 4CL1-GSP2 4CL1-KpnI 4CL1-S1 4CL1-S2 4CL3-HindIII 4CL13–3¢KpnI 4CL13-EcoRI 4CL14-BamHI 4CL14-GSP1 4CL14-GSP2 4CL16-GSP1 4CL16-GSP2 4CL16-SphI Seq1

5¢-GTTGCGTAGGACGAGCAT-3¢ 5¢-CGGATGCCGATTTTGTGGAGG-3¢ 5¢-GCTGGTACCGCACCTTCTCCACAAG-3¢ 5¢-TCYGGRTCRTTNAGRTADCCTTTCAT-3¢ 5¢-TBACNCARTCNGCNTAYGTBGARAA-3¢ 5¢-GTTCTAAGCTTTTAAGGCGTCTGAGTGGC-3¢ 5¢-AGTTTCAGGGTCAACAACCCTG-3¢ 5¢-CTCGAATTCATGACAACGGTAGCTGCTTCTC-3¢ 5¢-CTCGGATCCATGGCTGATGATGGAAGCAG-3¢ 5¢-TCAGCGTCACCGTTATCCTC-3¢ 5¢-GTGAGAAATGGAGATGCTGC-3¢ 5¢-TGTTCCGGAGAGCCTCCTC-3¢ 5¢-CAACGGAAGCACGCATAGGAGCAC-3¢ 5¢-CACCGCATGCATAACTCTAGCTCCTTCTCTTG-3¢ 5¢-GTAAAACGACGGCCAGT-3¢

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4-Coumarate:CoA ligase gene family from Glycine max (Eur. J. Biochem. 269) 1307

Hybridization conditions were according to [39]. DNA fragments were labelled with [a-32P]dCTP (Amersham Pharmacia) using the random prime procedure (Primea-Gene, Promega). Membranes were washed at moderate stringency for 20 min twice in 1 · NaCl/Cit, 0.1% (w/v) SDS at 42 °C or at high stringency conditions for 30 min in 0.5 · NaCl/Cit, 0.1% (w/v) SDS at 65 °C, and autoradiographed between intensifying screens at )80 °C. When necessary, membranes were stripped by incubation in 0.1% SDS, 5 mM 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 that had been isolated previously [20]. Positive plaques were purified by four rounds of screening, and one 9.5-kb DNA clone (kEMBL/Gm4CL3) was isolated. Restriction maps of the genomic clone kEMBL/Gm4CL3 and subclones were constructed by single and multiple enzyme digestions of the clones ligated into pBluescriptIIKS and SK vectors [40].

Table 3. Summary of 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 Gm4CL1 cDNA with modified initiator codon (see pZL1/Gm4CL1) Partial Gm4CL2 cDNA [20]

pQE-30/Gm4CL1 pBluescriptKSII/ Gm4CL14 pBluescriptKSII/ Gm4CL2 pQE-30/Gm4CL2

pTZ19R/Gm4CL13 pTZ19R/Gm4CL3 pTrcHisB/Gm4CL3 pQE-31/Gm4CL3 pTZ19R/Gm4CL16 pTZ19R/Gm4CL4

cDNA synthesis and selection pQE-30/Gm4CL4

Fusion of partial Gm4CL2 cDNA and 5¢-end fragment recovered from 5¢-RACE Full-length Gm4CL2 cDNA; 5¢-noncoding nucleotides were eliminated by introducing a BamHI restriction site directly upstream of the initiator codon Partial Gm4CL3 cDNA [20] Fusion of partial Gm4CL3 cDNA and 5¢-end fragment amplified by PCR Full-length Gm4CL3 cDNA Full-length Gm4CL3 cDNA Partial Gm4CL4 cDNA [20] Fusion of partial Gm4CL4 cDNA and 5¢-end fragment recovered from 5¢-RACE; initiator codon was eliminated by introducing a SphI restriction site Full-length Gm4CL4 cDNA with modified initiator codon (see pTZ19R/Gm4CL4) Genomic clone of Gm4CL3 (9.5 kb)

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 temperature (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 isolated and was shown to be almost full length.

was performed with an annealing temperature of 55 °C for 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.

Completion of cDNA sequences

Construction of E. coli expression plasmids

5¢-RACE was used to complete the open reading frames of the partial cDNAs encoding Gm4CL1 (see above), Gm4CL2, and Gm4CL4 [20] (Table 3). Amplification was performed in the presence of the respective nested genespecific primers (Table 2) and the universal amplification primer UAP (Gibco/BRL) after reverse transcription of soybean cell culture RNA (1 lg each) using the gene-specific oligonucleotides Gm4CL1-GSP1, Gm4CL14-GSP1, and Gm4CL16-GSP1, respectively, and tailing with terminal transferase and dCTP according to the manufacturer’s instructions (Gibco/BRL). The following annealing conditions were used during PCR: increasing from 54.5 to 58 °C during 35 cycles with Gm4CL1-GSP2 + UAP, 55 °C for 30 cycles using Gm4CL14-GSP2 + UAP after four rounds of unidirectional amplification at 58 °C in the presence of solely the UAP oligonucleotide, and 60 °C with the primers Gm4CL16-GSP2 + UAP. The partial cDNA clone Gm4CL13, encoding the 4CL3 isozyme, was completed by RT/PCR using the genomic sequence for the generation of oligonucleotide primers (Gm4CL13–3¢KpnI and Gm4CL13-EcoRI, Table 2). PCR

For heterologous expression, either the pTrcHis (Invitrogen), or the pQE (Qiagen) vector series were used. A summary of recombinant plasmids is given in Table 3. The introduction of the Gm4CL1 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 using the 4CL1-KpnI oligonucleotide and the vector-binding oligonucleotide Seq1 as primers. The modified 5¢-RACE product was inserted into pZL1 containing the incomplete Gm4CL1 cDNA using KpnI and SalI. The complete open reading frame was transferred into pQE-30 using KpnI and HindIII. For the construction of pBluescriptKSII/Gm4CL2, the respective 5¢ fragment was released from the cloning vector by SalI-restriction and inserted into the single SalI site of the partial cDNA (pBluescriptKSII/Gm4CL14). Deletion of the 5¢ noncoding region was achieved by PCR using 4CL14BamHI and 4CL14-GSP1 as primers and pBluescriptKSII/ Gm4CL2 as template. The amplified product was digested with BamHI/SalI and cloned together with the SalI/

kEMBL/Gm4CL3

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1308 C. Lindermayr et al. (Eur. J. Biochem. 269)

HindIII-restricted insert of pBluescriptKSII/Gm4CL14 into pQE-30, previously cut with BamHI and HindIII. The resulting construct pQE-30/Gm4CL2 contained an upstream in-frame extension of 12 codons, including six histidine codons. The 4CL3 5¢ fragment was ligated into pTZ19R/ Gm4CL13 using KpnI resulting in the full-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 of 45 and 20 codons, respectively, including six histidine codons each. In spite of different N-terminal extensions, the two expressed 4CL3 proteins showed the same enzymatic characteristics. For completion of the Gm4CL4 cDNA, the initiator codon of the 5¢-RACE product was eliminated by PCR using 4CL16-SphI and 4CL16-GSP2 as primers. The modified 5¢-RACE fragment was inserted into pTZ19R/ Gm4CL16 using SphI and SalI resulting in pTZ19R/ Gm4CL4. For heterologous expression the complete open reading frame was amplified by PCR using 4CL16-SphI and 4CL3-HindIII as primers and pTZ19R/Gm4CL4 as template. The PCR product was inserted into pQE-30 using SphI and HindIII restriction sites. Mutagenized and PCR-amplified fragments inserted into the expression vectors were controlled by 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 25 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 A600  0.5 was reached, induced with 1.5 mM isopropyl-b-D-thiogalactopyranoside, and incubated for 4 h at 37 °C. After centrifugation, the bacterial cells were resuspended in an appropriate volume of buffer [50 mM Tris/HCl pH 8.0, 14 mM 2-mercaptoethanol, and 30% (v/v) glycerol] and 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 (NH4)2SO4 to 75% saturation. The precipitate was collected by centrifugation, dissolved in buffer (see above), and the protein fraction was passed through a Sephadex G-25M column. The recombinant proteins were purified by immobilized metal chelate 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 mM imidazole. Analysis of DNA and protein sequences Double-stranded DNA was sequenced 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 from 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 numbers 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. perenne 4CL3 (AF052223), Nicotiana 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 (AF239685), Solanum tuberosum 4CL1 (M62755), S. tuberosum 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 using 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. In 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 constant, 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, the corresponding nucleotide sequences were aligned and evaluated in the same manner (data not shown).

RESULTS The prime objective of the current investigation was to extend the molecular survey of 4CL isoenzymes from soybean by: (a) completing the existing cDNAs [20]; and (b) by isolating lacking members of the gene family. Moreover, we aimed to disclose details of the catalytic capacity of the isoforms as well as of the differential regulation of the isozymes in relation to specialized branches of phenylpropanoid 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

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4-Coumarate:CoA ligase gene family from Glycine max (Eur. J. Biochem. 269) 1309

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 and 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 4CL4, respectively, the 5¢-RACE was 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 share a high identity at the deduced amino acid level (94%), whereas in all other cases the identity between the deduced 4CL isoforms is much lower ( 60%). A phylogenetic reconstruction of the known plant 4CLs revealed earlier that two major 4CL classes have evolved within the angiosperms [18]. The addition of 4CL sequences deposited in the databases since the earlier report as well as of those presented here into the protein alignment and the subsequent calculation of the most parsimonious phylogenetic tree (Fig. 1) confirmed the previous observation of the evolution of two major 4CL groups. According to the earlier designation, soybean 4CL1 and 4CL2 are members of the class I cluster, whereas 4CL3 and 4CL4 belong to the more divergent class II cluster (Fig. 1, upper and lower branch of the phylogram, respectively). It was shown previously that 4CL genes can be regulated at the transcriptional level by both, infection of soybean seedlings with Phytophthora sojae zoospores and elicitation 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 comprising 700 bp of the Gm4CL16 cDNA (partial clone of Gm4CL4). The determination of the complete sequence revealed that the clone

represented the gene corresponding to the Gm4CL3 cDNA, which is 93% identical to Gm4CL4. The 4CL3 gene of soybean was characterized by six exons ranging in size 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] and in potato [7], the 4CL1 gene in rice [46], and the three 4CL genes in Arabidopsis [18]. It is interesting to note that, for the increased number of 4CL genes analysed so far, the number and the positions of the introns is increasingly variable. The number of introns in the coding region that are unrelated with regard to sequence 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 probes for 4CL1 and 4CL2, respectively, or with a probe which was not able to distinguish between the 4CL3 and 4CL4 genes, resulted in the detection 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 1 22 14

22

23

21

35

100 13

Nt4CL1 Nt4CL 100 7 Nt4CL2 16 2 61 Pc4CL1 57 1 23 13 Pc4CL2 100 83 31 Vp4CL 62 Le4CL1 58 Gm4CL2 26 62 70 31 Ri4CL1 63 74 Popt4CL1 26 Poph4CL1 26 42 Poph4CL2 98 39 Ri4CL2 39 At4CL1 60 46 100 At4CL2 97 Gm4CL1 25

99

56

60 30

19

11

4CL1

4CL2

4CL3

4CL1



4CL2



4CL3



Identity matrix 63 58 65 62 – 61 63 – –

Amino acid residues Molecular mass (kDa)

546 59.4

547 60.2

570 61.8

4CL4

Gm4CL4 Gm4CL3 48 19 53 Popt4CL2 57 57 32 Ri4CL3 78 85 Le4CL2 69 At4CL3 36 Lp4CL1 52 Os4CL2 42 Lp4CL2 60 Lp4CL3 Os4CL1 45

100

52

87 107

76 53

100

51

52 45 58

100 1

Isoenzyme

11

17

55

Table 4. Comparison of the soybean 4CL cDNAs and encoded isoenzymes. The identity matrix was calculated in pair-wise alignments using BioEdit [42] and is given in each case as percentage identity of the amino acid (upper line) and the nucleotide sequence (open reading frame only, lower line, italic).

St4CL1

100 1 St4CL2

100 94

100

2

Pt4CL1 Pt4CL2

50 changes

58 61 60 62 94 93 562 61.0

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. The protein alignment resulted in one most parsimoniuos tree with a minimal length of 2155 steps and a consistency index of 0.599 (RI, 0.625). 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.

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1310 C. Lindermayr et al. (Eur. J. Biochem. 269) Gm 4CL1 B

E

Gm 4CL2 H

B

E

1

Gm 4CL3/4 H

B

E

H

kb 10 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 the restriction endonucleases BamHI (B), EcoRI (E), and HindIII (H), separated in triplicate on a 0.6% agarose gel and transferred to a nylon membrane. The complete open reading frame of the Gm4CL1 cDNA, a HindIII-fragment of pQE-30/ Gm4CL2, and a BamHI/HindIII-fragment of pQE-31/Gm4CL3 were used as hybridization probes using high stringency conditions. Positions of DNA standards are given on the right.

Gm4CL1 and Gm4CL2 cDNAs. However, the single fragments detected for 4CL1 by BamHI or EcoRI restriction, respectively, and the double signal for 4CL2 after restriction with EcoRI, which is compatible 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 by a more complex genomic 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 to roots and hypocotyls, while 4CL1 and 4CL2 mRNA amounts were highest in hypocotyls and stems and also in young roots (Fig. 3). Only low levels of the latter two mRNAs were observed in 12-day-old roots. In shoot tips and leaves, no mRNA representing any of the four 4CL isoforms could be detected under the experimental conditions used. Differential regulation of 4CL transcript and enzyme activity levels Treatment of soybean cells with Phytophthora sojae crude elicitor resulted in differential changes of the activities of the

2

3

4

5

6

7

8

Gm 4CL1

1.8 kb

Gm 4CL2

1.8 kb

Gm 4CL3/4

1.9 kb

28S rRNA

2.3 kb

Fig. 3. Spatial expression pattern of the 4CL mRNAs in soybean seedlings. Total RNA (20 lg each), isolated from different plant tissues, was separated on 1.2% agarose gels, blotted onto nylon membranes, and hybridized with gene-specific 4CL probes. Blots were washed under high stringency conditions. Hybridization with a 28S rRNA probe demonstrated equal loading. RNA was isolated from 3- (1) and 12-day (2) -old roots, from hypocotyls (3), first (4) and second (5) internodium, from 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 the right.

4CL isoenzymes. At two growth stages of the cell suspension culture, representing cultures at 1 day after inoculation (stage I) and at the end of the linear growth phase (stage II), respectively [29], the 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 for stage I not shown). Conversely, the activity level of 4CL1 was strongly reduced 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 in soybean cell cultures was also assessed by 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 challenged with elicitor, differential changes in mRNA levels occurred that reflected those found for isoenzyme activity levels (Fig. 4A). An analysis similar to that shown for elicitor-treated cell cultures was performed with roots of 3-day-old soybean seedlings following infection with zoospores of Phytophthora sojae (Fig. 4C). Under the experimental conditions used, the levels of 4CL1 and 4CL2 mRNAs were low and remained unaffected. In contrast, 4CL3/4 mRNA levels were detectable in untreated tissues and increased strongly after infection (Fig. 4C). Functional analysis of recombinant 4CL To elucidate the biochemical functions of the soybean 4CL gene family members, the substrate specificity of the heterologously expressed 4CL isoenzymes was examined and compared with that of the previously isolated isoenzymes [1]. The four cDNAs were expressed in E. coli as

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4-Coumarate:CoA ligase gene family from Glycine max (Eur. J. Biochem. 269) 1311

Fig. 4. Elicitation of soybean cell cultures and seedlings. (A) Soybean cell cultures of growth stage II [29] were treated with Phytophthora 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 in crude protein extracts and discriminated by calculating the contribution of the isoenzymes to the overall 4CL activity by using isoenzyme-specific substrates. Data points shown are mean values of two independent experiments showing similar results. (B) Differential expression of individual members of the 4CL gene family was assayed in soybean cell cultures after elicitor treatment. Soybean cell cultures were treated as in (A) and harvested at the times indicated. Total RNA (20 lg each) from elicitor-treated (E) and untreated (C) cell cultures was separated on a 1.2% agarose gel and blotted onto a nylon membrane. The blot was hybridized 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 Phytophthora sojae was analysed by Northern blotting as described in (B). Roots of soybean (cv. Harosoy 63) seedlings were treated with Phytophthora sojae (race 1) zoospores by dip inoculation (E) and harvested at the times indicated. Control seedlings were placed in sterile water (C). Hybridization with soybean 28S rRNA was used to confirm equal loading. The sizes of the hybridizing RNA species are shown on the right.

inducible fusion proteins containing N-terminal His6-tags. After immobilized metal affinity chromatography, the four proteins revealed the expected relative molecular masses in SDS/polyacrylamide gels. Immunoblot analysis demonstrated that the recombinant proteins interacted with an antiserum raised against parsley 4CL [35], whereas no 4CL-like protein cross-reacted with the antiserum in bacterial extracts containing the empty expression vector (Fig. 5). The recombinant isoenzymes were tested for their relative abilities to use 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 ferulate but not sinapate and 3,4-dimethoxycinnamate. As summarized in Table 5, the Km and relative Vmax values for these two 4CL isoforms closely resembled those found previously for partially purified ligase 1 and 2 [1]. Similar experiments with recombinant 4CL3 and 4CL4 demonstrated that 4-coumarate and caffeate were most efficiently

Fig. 5. Immunoblot analysis of recombinant soybean 4CL. The four isoforms were expressed as His6-tagged fusion proteins and purified by immobilized metal chelate affinity chromatography. The purified proteins were separated by SDS/PAGE and transferred to nitrocellulosic filters. For immunodetection, antiserum raised against parsley 4CL [35] combined with goat antirabbit IgG conjugated to alkaline phosphatase was used. Purified protein extract from bacteria carrying the empty expression vector (pQE-30) served as a control. The relative molecular masses of protein standards are shown on the right.

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 Km values for the catalytic action of the four heterologously produced soybean 4CL isoforms for

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1312 C. Lindermayr et al. (Eur. J. Biochem. 269)

Table 5. Substrate specificity of recombinant soybean 4CL expressed in E. coli. The Km and Vmax values of recombinant 4CL1, 4CL2, 4CL3 and 4CL4 were determined using Lineweaver–Burk plots with at least five data points. Each acid was assayed at the long-wave absorbance maximum of its CoA ester [32] in the spectrophotometrical test. Relative Vmax values were obtained by setting Vmax 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

Km(lM)

Relative Vmax (% of coumarate)

Relative Vmax/Km (lM)1)

Gm4CL1

Cinnamate 4-Coumarate Caffeate Ferulate Sinapate 3,4-Dimethoxycinnamate Cinnamate 4-Coumarate Caffeate Ferulate Sinapate 3,4-Dimethoxycinnamate Cinnamate 4-Coumarate Caffeate Ferulate Sinapate 3,4-Dimethoxycinnamate Cinnamate 4-Coumarate Caffeate Ferulate Sinapate 3,4-Dimethoxycinnamate

4400 (1300) 22 (32) 33 (40) 8 (9) 11 (11) 83 (100) 1700 (4500) 42 (17) 13 (14) 140 (130) NC (NC) NC (NC) 1100 9 50 3100 NC NC 260 10 34 1300 NC NC

9 (3) 100 (100) 40 (56) 57 (56) 35 (46) 75 (89) 50 (23) 100 (100) 37 (87) 71 (96) – (–) – (–) 45 100 74 25 – – 20 100 50 30 – –

2.0 · 10)3 4.54 1.21 7.13 3.21 0.91 0.03 2.38 2.85 0.51 – – 0.04 11.12 1.48 8.1 · 10)3 – – 0.08 10.00 1.47 0.02 – –

Gm4CL2

Gm4CL3

Gm4CL4

4-coumarate and caffeate were similar to those reported for many purified plant 4CL, whereas differences of the substrate specificity (Vmax/Km) between members of the soybean 4CL protein family appeared to be pronounced and were not previously reported in several of the other plant 4CL analysed so far. 4CL1 accepted the broadest range of hydroxylated and O-methylated cinnamic acids (highest relative Vmax/Km value for ferulate followed by 4-coumarate and sinapate). 4CL2 used an intermediate range of substituted cinnamic acids, while 4CL3 and 4CL4 displayed the highest selectivity towards these acids. All ligases exhibited low affinity for cinnamate (Table 5). A major result 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 and unsubstituted cinnamic acids.

DISCUSSION The results of our studies demonstrate that 4CL in soybean is encoded by a small gene 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 four 4CL genes, supporting earlier notions on different physiological functions of members 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 amino acid sequences deduced from full-size Gm4CL2, Gm4CL3, and Gm4CL4 cDNAs confirmed the level of identity between the three soybean 4CL genes as determined earlier for the partial cDNAs [20]. The present work adds a further, and presumably last, member to the 4CL family in soybean. A positive detection in our earlier work [20] was not possible given that RNA samples from elicitor-treated cells were used. As demonstrated in this work, these RNA samples were an inadequate source for 4CL1 cDNA isolation due to the repression of 4CL1 mRNA expression in response to elicitor (Fig. 4A,B). A phylogenetic reconstruction (Fig. 1) illustrates the relationships of 4CL isoforms within the soybean gene family as well as within the angiosperms. As noted earlier, two classes of 4CL proteins have evolved [18]. Remarkably, 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 structures (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 isoforms which are activated strongly in elicited or infected tissues, whereas the Arabidopsis isoform 3 shows no regulation in response to pathogen challenge but to UV irradiation [18].

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4-Coumarate:CoA ligase gene family from Glycine max (Eur. J. Biochem. 269) 1313

Substrate specificity of all four recombinant soybean 4CL isoenzymes was analysed for a series of cinnamic acids 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 recombinant 4CL isoforms, Gm4CL1 and Gm4CL2, closely matched that of the known 4CL isoforms [1], whereas for Gm4CL3 and 4 there was no known counterpart. Although structural constraints in fusion proteins generated from cDNAs could affect substrate conversion, such an effect appears to be unlikely for the observed catalytic properties of recombinant 4CL3 or 4CL4. It appears more likely that 4CL3 and 4 had not been identified in earlier work because proteins had been extracted from unstressed tissues [1] or a differentiation between 4CL2 and 4CL3 or 4 in unfractionated protein extracts was not possible due to the lack of knowledge of their catalytic properties (Table 5) [20]. The analysis of the substrate specificity of the recombinant soybean 4CL expressed in E. coli (Table 5) revealed pronounced differences in the ability of the isoenzymes to utilize differently ring-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 precursor for the syringyl monolignol formation, for which a cDNA is available. Alternatively, woody angiosperms have been described to obviate the need for the activation of highly ring-substituted cinnamic acids as precursors for monolignol biosynthesis by using ring substitution-specific methylations acting on already activated metabolites [12]. Loss-of-function experiments in alfalfa (Medicago sativa) likewise indicated no necessity for CoAligase isozymes converting already highly substituted cinnamic 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 phenylpropanoids as presently thought [47]. The molecular characterization of 4CL isoforms expressing pronounced differences in the substrate specificity may facilitate studies on the active site of this class of enzymes to identify amino acids that are of functional importance. This may include amino acid motifs such as a putative AMPbinding domain [48], but also amino acids that are responsible for a broad or narrow specificity towards ringsubstituted cinnamates. The central position of 4CL in phenylpropanoid branch pathways therefore makes this enzyme a potentially valuable target for pathway or product engineering in higher plants. Attempts towards this goal have recently been reported for 4CL2 from Arabidopsis thaliana [49,50]. Another particularly striking observation is the differential expression of the four 4CL isoforms. Based on enzyme activity measurements, 4CL1 and 4CL2 are both expressed in the unstressed 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 transcript levels of Gm4CL3 and Gm4CL4. Even though some uncertainty remains about the

relative proportion of the expressed transcript levels corresponding 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 reduced by elicitor treatment, a behaviour which is reported only rarely for enzymes committed to the biosynthesis of plant protective compounds. A consequence of this differential elicitor responsiveness could be that the overall product profile of CoA esters of cinnamic acids in soybean cells is shifted after elicitation due to the large differences in substrate preference of the 4CL isoform 1 vs. 3 or 4. A similar consequence might apply 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 other enzymes of phenylpropanoid metabolism [51].

Fig. 6. Scheme illustrating the central position of hydroxycinnamate CoA esters and 4CL isoforms in the biosynthesis of various phenylpropanoid metabolites in soybean under different developmental and environmental conditions.

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 TATA box and other boxes (A, E, L, P, data not shown), which are conserved among several plant genes in phenylpropanoid metabolism (phenylalanine 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, including different branches of phenylpropanoid metabolism. Phenylpropanoid responses are temporally and spatially coordinated [26,28,55]. They lead to the massive deposition of cell wall phenolics, release of isoflavones from conjugates, and the production of the soybean phytoalexins, glyceollins. All together, these

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1314 C. Lindermayr et al. (Eur. J. Biochem. 269)

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 and distal cell responses of soybean to glucan 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 in the 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 substitutions at the level of conjugated intermediates, 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 cellular localization, and the dynamics of their synthesis. In any case, the coordinated regulation of 4CL3 or 4 with all other known enzymes of phytoalexin biosynthesis in soybean [24] indicate that at least these isoenzymes are involved in defence-related pathways, whereas 4CL1 and 2 may have different functions in phenylpropanoid metabolism of this plant.

8.

9.

10.

11.

12.

13.

14.

15.

16.

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. This work was supported by the Deutsche Forschungsgemeinschaft (grant Eb62/11-3), the Fonds der Chemischen Industrie, and by a fellowship of the state of Bavaria (to C. L.).

17.

18.

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