Plant Physiol. (1999) 121: 215-224

Tobacco O-Methyltransferases Involved in Phenylpropanoid Metabolism. The Different Caffeoyl-Coenzyme A/5-Hydroxyferuloyl-Coenzyme A 3/5-O-Methyltransferase and Caffeic Acid/5-Hydroxyferulic Acid 3/5-O-Methyltransferase Classes Have Distinct Substrate Specificities and Expression Patterns¹

Stéphane Maury, Pierrette Geoffroy, and Michel Legrand^*

Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique, Université Louis Pasteur, 12 rue du Général Zimmer, 67084 Strasbourg cedex, France

	ABSTRACT

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The biosynthesis of lignin monomers involves two methylation steps catalyzed by orthodiphenol-O-methyltransferases: caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferases (COMTs) and caffeoyl-coenzyme A (CoA)/5-hydroxyferuloyl-CoA 3/5-O-methyltransferases (CCoAOMTs). Two COMT classes (I and II) were already known to occur in tobacco (Nicotiana tabacum) and three distinct CCoAOMT classes have now been characterized. These three CCoAOMT classes displayed a maximum level of expression at different stages of stem development, in accordance with their involvement in the synthesis of lignin guaiacyl units. Expression profiles upon tobacco mosaic virus infection of tobacco leaves revealed a biphasic pattern of induction for COMT I, COMT II, and CCoAOMTs. The different isoforms were expressed in Escherichia coli and our results showed that CCoAOMTs and, more surprisingly, COMTs efficiently methylated hydroxycinnamoyl-CoA esters. COMT I was also active toward 5-hydroxyconiferyl alcohol, indicating that COMT I that catalyzes syringyl unit synthesis in planta may operate at the free acid, CoA ester, or alcohol levels. COMT II that is highly inducible by infection also accepted caffeoyl-CoA as a substrate, thus suggesting a role in ferulate derivative deposition in the walls of infected cells. Tobacco appears to possess an array of O-methyltransferase isoforms with variable efficiency toward the diverse plant o-diphenolic substrates.

	INTRODUCTION

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Lignin is a major cell wall polymer of vascular plants that provides mechanical strength and hydrophobicity to vascular vessels. It is a heteropolymer composed of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) lignin units derived from p-coumaryl, coniferyl, and sinapyl alcohols, respectively (Fig. 1). Lignin composition is known to change during plant development and under the influence of environmental factors (Boudet et al., 1995; Campbell and Sederoff, 1996; Baucher et al., 1998; Whetten et al., 1998). The phenylpropanoid pathway that provides the lignin-building units called monolignols is strongly activated upon infection by pathogens or treatment with elicitors (Legrand, 1983; Pakush and Matern, 1991; Pakush et al., 1991; Jaeck et al., 1992). In infected plants, the deposition of phenylpropanoid compounds participates in cell wall reinforcement that restricts pathogen invasion (Nicholson and Hammerschmitt, 1992; Kauss et al., 1993; Dixon and Paiva, 1995).

View larger version (32K): [in this window] [in a new window]   Figure 1. OMT-catalyzed reactions in the general phenylpropanoid pathway. Nomenclature of lignin units is indicated in parentheses. 1, Phe ammonia-lyase; 2, cinnamate 4-hydroxylase; 3, p-coumarate 3-hydroxylase; 4, ferulate 5-hydroxylase; 5, coumarate CoA ligase; 6, coumaroyl-CoA 3-hydroxylase; 7, cinnamoyl-CoA reductase; 8, cinnamyl alcohol dehydrogenase. Methyl groups incorporated by OMTs are surrounded by gray areas. COMT and/or CCoAOMT are indicated according to substrate specificity assayed in vitro; brackets indicate that their role in vivo is questionable (Atanassova et al., 1995). Ferulate 5-hydroxylase substrate specificity is unknown (Chapple et al., 1992; Meyer et al., 1998). In some species, coumarate CoA ligase has very little activity with sinapic acid as the substrate (Lee and Douglas, 1996; Allina et al., 1998).

Most of the enzymes involved in monolignol biosynthesis have been characterized (Fig. 1). In particular, several caffeic acid 3-O-methyltransferases (COMTs) from dicots have been shown to catalyze the methoxylation of caffeic and 5-hydroxyferulic acids in vitro and were believed to be involved in guaiacyl and syringyl unit synthesis in vivo. However, caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), an enzyme that converts caffeoyl-CoA to feruloyl-CoA, has been characterized from parsley cell suspensions treated with an elicitor preparation (Kühnl et al., 1989; Pakush et al., 1989; Schmitt et al., 1991). The enzyme was thought to be involved in plant defense reactions by synthesizing wall-bound forms of ferulic acid. More recently, CCoAOMT has been proposed as the key element of an alternative pathway for lignin biosynthesis in zinnia cells during tracheary element formation (Ye et al., 1994; Ye and Varner, 1995).

Lignin analysis of transgenic plants or mutants down-regulated for COMT expression disclosed a decreased S to G ratio and the appearance of a new unit, the 5-hydroxyguaiacyl unit (Fig. 1) (Lapierre et al., 1988; Atanassova et al., 1995; Van Doorsselaere et al., 1995). These data demonstrated that in vivo, COMT activity controls the second methylation step leading to the syringyl unit, but left open the question of the redundancy between COMTs and CCoAOMTs with respect to their function in the first methylation step leading to the guaiacyl unit. Two classes of COMTs have been isolated and cloned from tobacco mosaic virus (TMV)-infected tobacco (Nicotiana tabacum) leaves (Legrand et al., 1978; Jaeck et al., 1992; Pellegrini et al., 1993): class I COMTs are highly expressed in lignified tissues, whereas class II COMTs are barely expressed in healthy tissues but are strongly stimulated upon infection. Recently, four CCoAOMT cDNA clones were isolated from tobacco leaves, and sequence comparison has shown that they belong to the same class of CCoAOMT (Martz et al., 1998).

We describe the cloning of two other CCoAOMTs, which, in view of the phylogenic data, are likely to belong to distinct gene classes defined as class 2 and class 3. To clarify the functions of the numerous OMT isoforms occurring in tobacco, we have compared their expression profiles during stem development and TMV infection and their affinities for various o-diphenolic substrates. The gene expression patterns of class 2 and 3 CCoAOMTs were found to be clearly different from those of class 1 CCoAOMT genes at various stages of stem development and during the hypersensitive response of tobacco leaves to TMV. Using specific antibodies, two CCoAOMT proteins were revealed in variable amounts in different plant tissues. Seven CCoAOMTs belonging to the three classes, one COMT I, and one COMT II were expressed in Escherichia coli. A preference for CoA esters was found for all enzymes tested, and was particularly pronounced with CCoAOMT isoforms. COMT I was demonstrated to efficiently methylate 5-hydroxyferulate, 5-hydroxyferuloyl-CoA, and 5-hydroxyconiferyl alcohol in vitro. These data indicate that the synthesis of lignin syringyl units that is catalyzed by COMT I in vivo (Atanassova et al., 1995) may proceed through various metabolic routes (Fig. 1). COMT II was also demonstrated to be very active toward caffeoyl-CoA as a substrate, suggesting that COMT II, which is known to be highly inducible upon infection or elicitor treatment, might catalyze the synthesis of ferulic derivatives whose deposition within the cell wall builds up a mechanical barrier, thus restricting pathogens (Nicholson and Hammerschmitt, 1992; Matern et al., 1995).

	MATERIALS AND METHODS

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Plant Material and Treatments

Cloning of New Members of the Different Tobacco CCoAOMT Classes

Sequence Comparison

Heterologous Expression in E. coli Cells, Purification of Recombinant Proteins, and Production of Polyclonal Antibodies

The active purified CCoAOMT-1 (class 1) recombinant protein was used to raise polyclonal antibodies. About 50 to 100 µg of the purified protein was emulsified with 300 µL of either Freund's complete adjuvant for the first injection of antigen, or incomplete adjuvant for all the following boost injections, and was administrated in five intramuscular injections at 5-week intervals. Ten days after each boost immunization, serum was collected. After clot removal, the serum was clarified by centrifugation and stored in small aliquots at 20°C.

SDS-PAGE and Immunoblotting

RNA Analysis

Chemical Synthesis of Substrates, Assays of Enzyme Activities, and TLC Analyses

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	RESULTS

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Different CCoAOMT Isoforms Are Expressed in Tobacco

View larger version (10K): [in this window] [in a new window]   Figure 2. Phylogenic tree of CCoAOMT protein sequences of the tobacco cv Samsun NN. 1, 2, 2tr, 3, 4, 5, and 6 correspond to the different CCoAOMT proteins.

View larger version (19K): [in this window] [in a new window]   Figure 3. Characterization of recombinant and native isoforms of CCoAOMTs. CCoAOMT recombinant proteins from E. coli cells were purified using a glutathione-agarose affinity matrix. Plant extracts were prepared from healthy tissues or from TMV-infected leaves. Proteins were separated on a SDS-polyacrylamide gel, and, after blotting, CCoAOMT isoforms were detected with polyclonal antibodies raised against tobacco recombinant CCoAOMT-1 (class 1). Lane 1, Recombinant CCoAOMT of class 1; lane 2, recombinant CCoAOMT of class 2; lane 3, recombinant CCoAOMT of class 3; lanes 4 to 6, native isoforms extracted from vascular tissues of stem (lane 4), healthy leaves (lane 5), and 45-h TMV-infected leaves (lane 6). The molecular mass of proteins is shown on the right.

The apparent molecular mass of class 1 and 2 recombinant proteins was 27 kD, close to the value deduced from the nucleotide sequences. Surprisingly, class 3 recombinant protein migrated as a 32-kD species, whereas its calculated mass was not significantly different from that of the two other enzymes. Two main bands of about 27 and 32 kD were also detected in plant extracts, indicating the same kinds of differences between native and recombinant proteins. These data suggest that the 27-kD protein originates from class 1 and 2 CCoAOMT genes, and that class 3 genes encode the 32-kD protein. However, sequence analysis of proteins purified from plant extracts would be needed to confirm this assumption. It is noteworthy that two protein bands were also revealed in tobacco stems with antibodies prepared against zinnia recombinant CCoAOMT (Zhong et al., 1998).

The amounts of the two CCoAOMT isoforms varied in the different tobacco tissues analyzed (Fig. 3, lanes 4, 5, and 6), accumulating in vascular tissues (lane 4) but being only slightly detectable in healthy tobacco leaves (lane 5). Upon infection, the 27-kD species was strongly induced (lane 6). These data indicate that CCoAOMTs have differential patterns of expression in tobacco. An additional band of slightly smaller mass appeared in infected material but was not studied further.

OMT Expression during Stem Development

View larger version (33K): [in this window] [in a new window]   Figure 4. CCoAOMT and COMT I expression during stem development. Extracts were prepared from stem tissues harvested at different internodes numbered from the bottom to the top of the stem and were immunoblotted with antibodies recognizing CCoAOMTs and COMT I (A) or tested for enzymatic activities in the presence of caffeoyl-CoA or caffeic acid (B).

The expression profiles of the three CCoAOMT classes during stem development were studied by RT-PCR using primers specific for each type of sequences and shown to be strikingly different (Fig. 5). The expression level of class 2 (Fig. 5B) steadily increased from the bottom to the top of the plants, whereas class 1 (Fig. 5A) transcripts mostly accumulated in the bottom and middle parts of the stems. Class 3 (Fig. 5C) displayed an intermediate profile. These data indicate that each CCoAOMT class may be involved at specific stages of lignification. A similar observation has been made for two lignin-peroxidase genes in Populus kitakamiensis (Osakabe et al., 1995). These differential regulations may correlate with variations in lignin composition that are known to occur during development (Lewis and Yamamoto, 1990; Baucher et al., 1998).

View larger version (17K): [in this window] [in a new window]   Figure 5. Differential expression of the CCoAOMT classes during stem development. CCoAOMT expression was analyzed by reverse transcriptase-PCR using total RNA extracted from different stem internodes numbered from the bottom to the top.

OMT Expression in TMV-Infected Leaves

View larger version (29K): [in this window] [in a new window]   Figure 6. Kinetics of CCoAOMT and COMT amounts and enzymatic activities in TMV-infected tobacco leaves. Extracts were prepared from leaf tissue harvested at different times after infection (0, 16, 32, 48, 64, 72, and 96 h). A, Immunoblots with antibodies recognizing the different isoforms of CCoAOMTs and COMTs. B, Enzymatic activities were measured in the presence of caffeoyl-CoA or catechol.

The expression profiles of each CCoAOMT class are presented in Figure 7. Time-course studies of individual gene expression revealed a biphasic pattern of induction similar for the three classes but with a much higher amplitude in class 1 genes (Fig. 7A). These patterns are consistent with those observed at the protein and activity levels (Fig. 6) and suggest that CCoAOMT expression is controlled at the RNA level. These data contrast with those reported in alfalfa cell suspensions, where the accumulation of OMT transcripts after treatment by a fungal elicitor did not lead to increased extractable enzyme activities (Ni et al., 1996).

View larger version (19K): [in this window] [in a new window]   Figure 7. Differential expression of the CCoAOMT classes during the hypersensitive reaction of tobacco to TMV. CCoAOMT expression was analyzed by reverse transcriptase-PCR using total RNA extracted from TMV-infected leaves at various times after inoculation.

Comparison of Substrate Specificities of CCoAOMTs and COMTs

View larger version (31K): [in this window] [in a new window]   Figure 8. Substrate specificity of CCoAOMT and COMT recombinant proteins. The kinetic values of recombinant tobacco OMTs (seven CCoAOMTs and two COMTs) were determined by the Lineweaver-Burk method at a saturating concentration of S-adenosyl-L-Met, toward caffeic acid, 5-hydroxyferulic acid, the corresponding CoA esters, chlorogenic acid, 5-hydroxyconiferyl alcohol, protocatechuic aldehyde, and catechol. The calculated Vmax to Km ratios represent the mean value of four replicates. Vmax is expressed in nkat × 10 1 g1 of purified protein and Km in micromolar.

Among the seven CCoAOMTs tested, only CCoAOMT-3 (class 1) readily methylated free acids (Fig. 8, A and B). Although activity levels were low, TLC analysis confirmed the identity of the reaction products (data not shown). In all cases, CoA esters were better substrates of CCoAOMTs than the free forms. Caffeoyl-CoA was more efficiently methylated than 5-hydroxyferuloyl-CoA by all isoforms except CCoAOMT-2tr (class 1) and CCoAOMT-5 (class 2) (Fig. 8, C and D). CCoAOMT-6 (class 3) appeared to accept almost exclusively caffeoyl-CoA as a substrate. These data are in good agreement with the implication of the CCoAOMT in the synthesis of guaiacyl units in vivo (Fig. 1). The weak but significant activity of the 10-kD species encoded by CCoAOMT-2tr toward 5-hydroxyferuloyl-CoA, together with the occurrence of a 10-kD immunoreactive band in plant extracts (data not shown), supports the functionality of the CCoAOMT-2tr-encoded protein. With the exception of CCoAOMT-5 (class 2), which displayed some activity against protocatechuic aldehyde, CCoAOMTs do not accept chlorogenic acid or non-phenylpropanoid compounds as substrates (Fig. 8, E, G, and H).

With the exception of chlorogenic acid, COMT I was the sole enzyme that proved efficient against all substrates tested. In particular, this is the first report of the substrate specificity of a purified OMT toward 5-hydroxyconiferyl alcohol in vitro. Our results show that only class I COMT can efficiently methylate this substrate, as shown in Figure 8F. With all of the substrates tested, similar results were obtained with COMT I preparations purified from plant extracts (data not shown), suggesting that no posttranslational modifications are involved in substrate specificity. It is particularly striking that V_max/K_m values measured for COMT I were even higher with CoA esters than those calculated for free forms. It is also important to stress that the formation of feruloyl-CoA or sinapoyl-CoA after the incubation of COMTs in the presence of caffeoyl-CoA or 5-hydroxyferuloyl-CoA, respectively, was confirmed by TLC analysis (data not shown).

The preference for CoA esters versus free acid forms was even more pronounced for class II COMT, whose activity against free caffeic acid and 5-hydroxyferulic acid was undetectable under the conditions used (Fig. 8, A-D). These data confirm that catechol is the best substrate for COMT II, as was shown previously (Legrand et al., 1978). The activity of class II COMT against caffeoyl-CoA suggests that in infected leaves, where COMT II is strongly induced, this enzyme preferentially contributes to the synthesis of ferulic derivatives that are known to accumulate upon infection.

	DISCUSSION

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We have shown previously that tobacco possesses two classes of COMTs (class I and II) that have been purified and cloned (Hermann et al., 1987; Jaeck et al., 1992; Pellegrini et al., 1993). Substrate specificity studies of enzyme preparations purified from plant extracts (Legrand et al., 1978; Colendaveloo et al., 1981) and differential patterns of expression of the two COMT classes in tobacco tissues (Pellegrini et al., 1993; Jaeck et al., 1996) have suggested a specific role for class I enzyme in lignin biosynthesis and the involvement of class II COMT in the production of defense-related compounds. Lignin analysis of plants silenced in COMT expression (Atanassova et al., 1995) demonstrated that in planta COMT I is involved in the second methylating step leading to the syringyl unit, and that CCoAOMT activity is likely responsible for the synthesis of the lignin guaiacyl unit via the feruloyl-CoA ester (Fig. 1). Southern-blot experiments have indicated that several gene classes encode CCoAOMTs of tobacco, and four members of class 1 have been characterized (Martz et al., 1998).

In the present study, our major objective was to better define the role of the different tobacco OMTs. We show that tobacco possesses three CCoAOMT classes and have compared their expression patterns during development and defense responses. Furthermore, heterologous expression of seven distinct CCoAOMT cDNAs, together with cDNAs of COMTs I and II, enabled us to compare substrate specificities of the nine purified recombinant proteins. This led to the very surprising finding that caffeoyl-CoA and 5-hydroxyferuloyl-CoA are the best substrates for all enzymes, even for COMT I (recombinant and native enzymes), which is also very active against free acids (Fig. 8). COMT I activity against 5-hydroxyconiferyl alcohol (Fig. 8) was demonstrated for the first time and raises the possibility that the second methylation step of the pathway catalyzed by COMT I (Atanassova et al., 1995) can occur at the level of 5-hydroxyferulic acid, the corresponding CoA ester, or 5-hydroxyconiferyl alcohol (Fig. 1). The fact that in some species p-coumarate CoA ligases have been reported to have low activity toward sinapic acid (Lee and Douglas, 1996; Allina et al., 1998) argues in favor of the methylation at the level of the CoA ester or the alcohol.

Recent experiments using labeled precursors have demonstrated that coniferyl alcohol may be the precursor of lignin syringyl units in vivo (Matsui et al., 1994; Chen et al., 1999), in agreement with the efficient methylation of 5-hydroxy-coniferyl alcohol by COMT I (see Fig. 1). Recombinant class I COMTs from other plant species have been shown to accept both free acids and CoA esters, but free acids were preferentially accepted compared with cognate CoA esters (Meng and Campbell, 1996, 1998; Inoue et al., 1998). Recently, a COMT from a gymnosperm species, loblolly pine, was reported to share about 60% homology with COMTs of angiosperms and to accept hydroxycinnamoyl-CoA esters and free acids as substrates (Li et al., 1997). In fact, such multifunctionality appears to be a common feature of plant COMTs, with the notable exception of the zinnia enzyme, which did not methylate CoA esters (Ye and Varner, 1995).

A preference for CoA esters versus free acids was also observed for COMT II. Therefore, COMT II, which was known to accept a wide variety of phenolic substrates and was thought to participate in the synthesis of lignin-like compounds (Legrand et al., 1978), may also be involved in the increased deposition of hydroxymethoxycinnamate esters in the walls of plant cells responding to infection or elicitor treatment (Nicholson and Hammerschmitt, 1992; Kauss et al., 1993; Dixon and Paiva, 1995).

It has been reported that CCoAOMTs are encoded by one to two members in parsley (Grimmig and Matern, 1997), alfalfa (Inoue et al., 1998), aspen (Meng and Campbell, 1998), grapevine (Busam et al., 1997b), and five to 10 members in zinnia (Ye et al., 1994). Similar genomic complexity associated with distinct regulation of the different members of a gene family (Liang et al., 1989; Shufflebottom et al., 1993; Osakabe et al., 1995; Allina et al., 1998) and distinct substrate specificity of the different isoforms (Goffner et al., 1998; Hu et al., 1998) have also been described for different phenylpropanoid enzymes. These observations could explain, at least in part, the complex distribution of the phenylpropanoid compounds throughout the plant.

Our results suggest a role for each class of CCoAOMT genes at a specific stage(s) of lignification. The isolation of typical member(s) of each CCoAOMT class would be the first step toward the characterization of regulatory elements and transcriptional factors involved in spatiotemporal control of CCoAOMT gene expression. Recent studies have demonstrated that MYB-like factors control phenylpropanoid gene expression (Rushton and Somssich, 1998; Tamagnone et al., 1998) and are induced in TMV-infected tobacco leaves (Yang and Klessig, 1996). In situ hybridization experiments have demonstrated the accumulation of COMT I transcripts mainly in young xylem cells of tobacco stems and in the epidermis of TMV-infected leaves (Jaeck et al., 1996). In contrast, COMT II expression was undetectable in healthy tobacco (Pellegrini et al., 1993), but after infection by TMV, COMT II transcripts accumulated in all cell types of leaf tissues (L. Pellegrini, unpublished data) as observed for Phe ammonia-lyase transcripts (Pellegrini et al., 1994).

Infection of tobacco leaves by TMV induced COMT I, COMT II, and CCoAOMT expression at the protein and activity levels. Detailed kinetics studies uncovered two peaks of induction: an early one that was also detectable in wounded leaves, and a second one emerging at the time of necrotic lesion appearance. Moreover, the analysis of individual expression of CCoAOMT classes demonstrated that the three CCoAOMT classes were induced with similar kinetics in infected leaves, and that CCoAOMT transcripts of class 1 accumulated predominantly (Fig. 7A). Thus, the coordinated expression of CCoAOMTs and COMTs may provide the hypersensitively reacting plant cells with a variety of metabolites needed for wall reinforcement (Lagrimini, 1991; Campbell and Ellis, 1992a, 1992b; Nicholson and Hammerschmitt, 1992; Kauss et al., 1993). The fact that all tobacco OMTs accept at least one hydroxycinnamoyl-CoA ester as a substrate fosters the view that feruloyl-CoA and sinapoyl-CoA play a central role in the esterification process of cell wall polymers (Grisebach, 1981; Legrand, 1983; Schmitt et al., 1991; Ishii, 1997).

Recently, transgenic tobacco plants with reduced CCoAOMT expression have been shown to display a marked decrease in lignin content with no abnormal visible phenotype (Zhong et al., 1998). Our unpublished results (S. Maury, P. Geoffroy, and M. Legrand), however, demonstrate that CCoAOMT inhibition may affect plant growth and resistance to pathogens. Analysis of phenolic content of such plants should bring new insights into the functions of phenylpropanoids in plant physiology and stress response.

	FOOTNOTES

   Received March 19, 1999; accepted May 27, 1999.

	ACKNOWLEDGMENTS

We thank Dr. B. Fritig for helpful discussions and continuous interest. We are grateful to Prof. Kazuhiko Fukushima for providing samples of 5-hydroxyconiferyl alcohol -D-glucoside. We thank P. Keltz and R. Wagner for taking good care of tobacco plants, M. Meyer and B. Jessel for technical assistance in antibody production, and P. Hammann for DNA sequencing.

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