Immunolocalization of two ligninO-methyltransferases in stems of alfalfa (Medicago sativa L.)

Caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) catalyze parallel reactions that are believed to be involved in the biosynthesis of lignin monomers. Antisera specific for alfalfa (Medicago sativa L.) COMT or CCOMT were raised against the enzymes expressed inEscherichia coli, and were used for immunolocalization studies in lignifying alfalfa stem tissue. Both COMT and CCOMT were localized to xylem parenchyma cells, as assessed by light microscopy and immunocytochemistry. Electron microscopy revealed that both enzymes were located in the cytoplasm of xylem parenchyma cells, and to a lesser extent, in the cytoplasm of phloem cells. There was no significant difference in the localization pattern of COMT and CCOMT, suggesting that the two enzymes may be part of a metabolic grid leading to production of lignin monomers in lignifying tissue of mature alfalfa stem internodes.     

Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases

Lignin inhibits forage digestibility by ruminant animals, and lignin levels and the proportion of dimethylated syringyl (S) lignin monomers increase with progressive maturity in stems of forage crops. We generated transgenic alfalfa (Medicago sativa L.) with reduced lignin content and altered lignin composition. Down-regulation of caffeic acid 3-O-methyltransferase (COMT) reduces lignin content, accompanied by near total loss of S lignin, whereas down-regulation of caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) reduces lignin content without reduction in S lignin. These changes are not accompanied by altered ratios of cell wall polysaccharides. Analysis of rumen digestibility of alfalfa forage in fistulated steers revealed improved digestibility of forage from COMT down-regulated plants, but a greater improvement in digestibility following down-regulation of CCoAOMT. The results indicate that both lignin content and composition affect digestibility of alfalfa forage, and reveal a new strategy for forage quality improvement by genetic manipulation of CCoAOMT expression.     

Both caffeoyl Coenzyme A 3-<Emphasis Type="Italic">O</Emphasis>-methyltransferase 1 and caffeic acid <Emphasis Type="Italic">O</Emphasis>-methyltransferase 1 are involved in redundant functions for lignin,flavonoids and sinapoyl malate biosynthesis in Arabidopsis

Two methylation steps are necessary for the biosynthesis of monolignols, the lignin precursors. Caffeic acid O-methyltransferase (COMT) O-methylates at the C5 position of the phenolic ring. COMT is responsible for the biosynthesis of sinapyl alcohol, the precursor of syringyl lignin units. The O-methylation at the C3 position of the phenolic ring involves the Caffeoyl CoA 3-O-methyltransferase (CCoAOMT). The CCoAOMT 1 gene (At4g34050) is believed to encode the enzyme responsible for the first O-methylation in Arabidopsis thaliana. A CCoAOMT1 promoter-GUS fusion and immunolocalization experiments revealed that this gene is strongly and exclusively expressed in the vascular tissues of stems and roots. An Arabidopsis T-DNA null mutant named ccomt 1 was identified and characterised. The mutant stems are slightly smaller than wild-type stems in short-day growth conditions and has collapsed xylem elements. The lignin content of the stem is low and the S/G ratio is high mainly due to fewer G units. These results suggest that this O-methyltransferase is involved in G-unit biosynthesis but does not act alone to perform this step in monolignol biosynthesis. To determine which O-methyltransferase assists CCoAOMT 1, a comt 1 ccomt1 double mutant was generated and studied. The development of comt 1 ccomt1 is arrested at the plantlet stage in our growth conditions. Lignins of these plantlets are mainly composed of p-hydroxyphenyl units. Moreover, the double mutant does not synthesize sinapoyl malate, a soluble phenolic. These results suggest that CCoAOMT 1 and COMT 1 act together to methylate the C3 position of the phenolic ring of monolignols in Arabidopsis. In addition, they are both involved in the formation of sinapoyl malate and isorhamnetin.     

O-Methylation of benzaldehyde derivatives by "lignin specific" caffeic acid 3-O-methyltransferase

Although S-adenosyl-l-methionine (SAM) dependent caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) is one of the key enzymes in lignin biosynthesis, the present work demonstrates that alfalfa COMT methylates benzaldehyde derivatives more efficiently than lignin pathway intermediates. 3,4-Dihydroxy, 5-methoxybenzaldehyde and protocatechuic aldehyde were the best in vitro substrates for OMT activity in extracts from developing alfalfa stems, and these compounds were preferred over lignin pathway intermediates for 3-O-methylation by recombinant alfalfa COMT expressed in Escherichia coli. OMT activity with benzaldehydes was strongly reduced in extracts from stems of transgenic alfalfa down-regulated in COMT. However, although COMT down-regulation drastically affects lignin composition, it does not appear to significantly impact metabolism of benzaldehyde derivatives in alfalfa. Structurally designed site-directed mutants of COMT showed altered relative substrate preferences for lignin precursors and benzaldehyde derivatives. Taken together, these results indicate that COMT may have more than one role in phenylpropanoid metabolism (but probably not in alfalfa), and that engineered COMT enzymes could be useful for metabolic engineering of both lignin and benzaldehyde-derived flavors and fragrances.     

Structure and expression of the lignin O-methyltransferase gene from Zea mays L.

The isolation and characterization of cDNA and homologous genomic clones encoding the lignin O-methyltransferase (OMT) from maize is reported. The cDNA clone has been isolated by differential screening of maize root cDNA library. Southern analysis indicates that a single gene codes for this protein. The genomic sequence contains a single 916 bp intron. The deduced protein sequence from DNA shares significant homology with the recently reported lignin-bispecific caffeic acid/5-hydroxyferulic OMTs from alfalfa and aspen. It also shares homology with OMTs from bovine pineal glands and a purple non-sulfur photosynthetic bacterium. The mRNA of this gene is present at different levels in distinct organs of the plant with the highest accumulation detected in the elongation zone of roots. Bacterial extracts from clones containing the maize OMT cDNA show an activity in methylation of caffeic acid to ferulic acid comparable to that existing in the plant extracts. These results indicate that the described gene encodes the caffeic acid 3-O-methyltransferase (COMT) involved in the lignin biosynthesis of maize.     

Radiochemical high-performance liquid chromatographic assay for the determination of catechol O-methyltransferase activity towards various substrates

A new chromatographic catechol O-methyltransferase (COMT) assay based on S-adenosyl- -[methyl-¹⁴C]methionine and on-line radioactivity detection was developed. With minor modifications in the mobile phase composition the methylation velocities for 30 structurally diverse compounds including simple catechols, neurotransmitters, catecholestrogens and catecholic drugs could be measured using human and rat recombinant soluble COMT. The enzymes showed very similar substrate selectivities. The radiochemical method was validated using 3,4-dihydroxybenzoic acid as a model substrate and it was shown that accurate and reproducible methylation velocity values could be achieved for both of the catecholic hydroxyls. The method proved to be suited for determining the enzyme kinetic parameters and can probably be further used for gathering enzyme kinetic data on differentially substituted catechols in order to construct proper structure-activity relationships for COMT.     

Reduced lignin in transgenic plants containing a caffeic acidO-methyltransferase antisense gene

Lignin is a major structural polymer of secondarily thickended plant vascular tissue and fibres, imparting mechanical strength to stems and trunks and hydrophobicity to conducting vessels. Constitutive expression of a lucerne caffeic acid 3-O-methyltransferase antisense RNA in transgenic tobacco leads to a significant reduction in lignin content, particularly in the younger parts of the stems, without apparent alterations in lignin monomer composition. These observations open up the possibility of genetically manipulating plants with reduced lignin for improved processing and biomass digestibility.     

植物咖啡酸-O-甲基转移酶的研究进展

单木质素醇(H型、G型和S型)是构成植物木质素和木脂素的基本单元,其组成的不同直接决定木质素和木脂素的化学多样性和生物活性差异。咖啡酸-O-甲基转移酶(caffeic acid O-methyltransferase, COMT)可催化苯丙素类化合物羟基上氧原子的甲基化,在不同类型单木质素醇的构成中起决定作用,是木质素和木脂素生物合成途径的关键酶。2010年的相关综述主要对COMT的基因特征和在木质素生物合成中的调控作用作了介绍,文中聚焦了近十多年来COMT的最新研究进展,从基因特征、表达特征、结构特征和调控作用几个方面进行全面综述,并对COMT的研究和应用前景进行展望。     

Determination of differential activities of soluble and membrane-bound catechol-O-methyltransferase in tissues and erythrocytes

Catechol-O-methyltransferase (COMT) exists as two isoenzymes, a membrane-bound form (MB–COMT) and a soluble form (S–COMT), with different roles in the metabolism of catecholamines and other catechol compounds. This report documents an HPLC assay for separate estimation of S–COMT and MB–COMT activity and examines activities of the two isoezymes among different rat tissues and in human and rat erythrocytes. Activities of MB–COMT and S–COMT varied widely among tissues. There were higher activities of S–COMT than MB–COMT in all tissues except the adrenal medulla where MB–COMT was the predominant isoenzyme, consistent with the importance of this tissue and MB–COMT for the O-methylation of catecholamines. MB–COMT and S–COMT in rat and human erythrocytes showed divergent levels and patterns of activity. The assay represents a rapid and accurate method for quantifying MB–COMT and S–COMT in various tissues and examining the relative roles of COMT isoenzymes in the metabolism of catechol compounds in health and disease.     

Xylem parenchyma cells deliver the H<Subscript>2</Subscript>O<Subscript>2</Subscript> necessary for lignification in differentiating xylem vessels

Lignification in Zinnia elegans L. stems is characterized by a burst in the production of H₂O₂, the apparent fate of which is to be used by xylem peroxidases for the polymerization of p-hydroxycinnamyl alcohols into lignins. A search for the sites of H₂O₂ production in the differentiating xylem of Z. elegans stems by the simultaneous use of optical (bright field, polarized light and epi-polarization) and electron-microscope tools revealed that H₂O₂ is produced on the outer-face of the plasma membrane of both differentiating (living) thin-walled xylem cells and particular (non-lignifying) xylem parenchyma cells. From the production sites it diffuses to the differentiating (secondary cell wall-forming) and differentiated lignifying xylem vessels. H₂O₂ diffusion occurs mainly through the continuous cell wall space. Both the experimental data and the theoretical calculations suggest that H₂O₂ diffusion from the sites of production might not limit the rate of xylem cell wall lignification. It can be concluded that H₂O₂ is produced at the plasma membrane in differentiating (living) thin-walled xylem cells and xylem parenchyma cells associated to xylem vessels, and that it diffuses to adjacent secondary lignifying xylem vessels. The results strongly indicate that non-lignifying xylem parenchyma cells are the source of the H₂O₂ necessary for the polymerization of cinnamyl alcohols in the secondary cell wall of lignifying xylem vessels.     

Modification of lignin biosynthesis in transgenic Nicotiana through expression of an antisense O-methyltransferase gene from Populus

An aspen lignin-specific O-methyltransferase (bi-OMT; S-adenosyl-l-methionine: caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase, EC 2.1.1.68) antisense sequence in the form of a synthetic gene containing the cauliflower mosaic virus 35S gene sequences for enhancer elements, promoter and terminator was stably integrated into the tobacco genome and inherited in transgenic plants with a normal phenotype. Leaves and stems of the transgenes expressed the antisense RNA and the endogenous tobacco bi-OMT mRNA was suppressed in the stems. Bi-OMT activity of stems was decreased by an average of 29% in the four transgenic plants analyzed. Chemical analysis of woody tissue of stems for lignin building units indicated a reduced content of syringyl units in most of the transgenic plants, which corresponds well with the reduced activity of bi-OMT. Transgenic plants with a suppressed level of syringyl units and a level of guaiacyl units similar to control plants were presumed to have lignins of distinctly different structure than control plants. We concluded that regulation of the level of bi-OMT expression by an antisense mechanism could be a useful tool for genetically engineering plants with modified lignin without altering normal growth and development.Abbreviations OMT O-methyltransferase - bi-OMT bispecific O-methyltransferase - CAD cinnamyl alcohol dehydrogenase - Ptomt1 Populus tremuloides bi-OMT cDNA clone     

Targeted gene disruption of the avermectin B O-methyltransferase gene in Streptomyces avermitilis

The biological activity of avermectin B components is superior to that of avermectin A components, which are derived from avermectin B by avermectin B 5-O-methyltransferase. Gene disruption, targeting avermectin B 5-O-methyltransferase gene in Streptomyces avermitilis, was carried out to obtain a strain of avermectin B producer. Phenotype analysis of the mutant with the disrupted O-methyltransferase gene showed that only avermectin B components were produced with a significant increase in production     

Molecular characterization of a brown midrib3 deletion mutation in maize

The caffeic acid O-methyltransferase (COMT) gene plays an important role in the synthesis of lignin. We have used the polymerase chain reaction in conjuction with genomic analysis to characterize deletion mutations of this gene in maize. In addition, we have analyzed and compared regions of the COMT gene from three distinct heterotic groups. Both PCR and Southern analysis indicate that the active wild-type COMT gene can be polymorphic. We suggest that the intron domain of at least one heterotic inbred can contribute to the alteration of the wild-type gene. In addition, multiple deletion mutations have occurred at this locus. We have found a previously uncharacterized deletion mutation in which segments of both the intron and exon have been deleted and replaced by other sequences. Precise knowledge of its sequence has allowed us to develop an assay by which we can follow this mutation in a breeding program.     

Pharmacogenomics: Catechol O-Methyltransferase to Thiopurine S-Methyltransferase

1. Pharmacogenomics is the study of the role of inheritance in variation in the drug response phenotype—a phenotype that can vary from adverse drug reactions at one end of the spectrum to lack of therapeutic efficacy at the other.2. The thiopurine S-methyltransferase (TPMT) genetic polymorphism represents one of the best characterized and most clinically relevant examples of pharmacogenomics. This polymorphism has also served as a valuable “model system” for studies of the ways in which variation in DNA sequence might influence function.3. The discovery and characterization of the TPMT polymorphism grew directly out of pharmacogenomic studies of catechol O-methyltransferase (COMT), an enzyme discovered by Julius (Julie) Axelrod and his coworkers.4. This review will outline the process by which common, functionally significant genetic polymorphisms for both COMT and TPMT were discovered and will use these two methyltransferase enzymes to illustrate general principles of pharmacogenomic research—both basic mechanistic and clinical translational research—principles that have been applied to a series of genes encoding methyltransferase enzymes.     

Characterization of a caffeic acid 3-O-methyltransferase from wheat and its function in lignin biosynthesis

Caffeic acid 3-O-methyltransferase (COMT) catalyzes the multi-step methylation reactions of hydroxylated monomeric lignin precursors, and is believed to occupy a pivotal position in the lignin biosynthetic pathway. A cDNA (TaCM) was identified from wheat and it was found to be expressed constitutively in stem, leaf and root tissues. The deduced amino acid sequence of TaCM showed a high degree of identity with COMT from other plants, particularly in SAM binding motif and the residues responsible for catalytic and substrate specificity. The predicted TaCM three-dimensional structure is very similar with a COMT from alfalfa (MsCOMT), and TaCM protein had high immunoreactive activity with MsCOMT antibody. Kinetic analysis indicated that the recombinant TaCM protein exhibited the highest catalyzing efficiency towards caffeoyl aldehyde and 5-hydroxyconiferaldehyde as substrates, suggesting a pathway leads to S lignin via aldehyde precursors. Authority of TaCM encoding a COMT was confirmed by the expression of antisense TaCM gene in transgenic tobacco which specifically down-regulated the COMT enzyme activity. Lignin analysis showed that the reduction in COMT activity resulted in a marginal decrease in lignin content but sharp reduction in the syringl lignin. Furthermore, the TaCM protein exhibited a strong activity towards ester precursors including caffeoyl-CoA and 5-hydroxyferuloyl-CoA. Our results demonstrate that TaCM is a typical COMT involved in lignin biosynthesis. It also supports the notion, in agreement with a structural analysis, that COMT has a broad substrate preference.     

Gas chromatographic–mass spectrometric procedures for determination of the catechol-O-methyltransferase (COMT) activity and for detection of unstable catecholic metabolites in human and rat liver preparations after COMT catalyzed in statu nascendi derivatization using S-adenosylmethionine

A procedure is presented for determination of the catechol-O-methyltransferase (COMT) activity in liver cytosolic preparations using 3,4-dihydroxyphenethylamine as substrate and by quantifying the product 3-methoxy-4-hydroxyphenethylamine (3-MHP). For quantification of 3-MHP in liver cytosolic preparations a gas chromatographic–mass spectrometric procedure after liquid–liquid extraction and acetylation was established and validated. The intra- and inter-day accuracy and precision were better than 15% and 20%, respectively. Extraction efficiency and selectivity were also sufficient. For in statu nascendi derivatization of unstable catecholic metabolites in liver microsome preparations, cytosolic preparations with COMT activities of at least 1 nmol product/min/mg protein were used after addition of S-adenosylmethionine. Such catecholic metabolites, which are claimed to be responsible for toxic effects in vivo, e.g., neurotoxicity or carcinogenesis, must not be overlooked in in vitro metabolism studies. Using this trick, gas chromatography–mass spectrometry (GC–MS) was suitable for the determination of catecholic metabolites in human and rat liver preparations after the same sample preparation as for 3-MHP quantification. The applicability was exemplified for the antidepressant paroxetine.     

The seasonal activity and the effect of mechanical bending and wounding on the PtCOMT promoter in Betula pendula Roth

In this study, 900-bp (signed as p including nucleotides –1 to –886) and partly deleted (signed as dp including nucleotides –1 to –414) COMT (caffeate/5-hydroxyferulate O-methyltransferase) promoters from Populus tremuloides Michx. were fused to the GUS reporter gene, and the tissue-specific expression patterns of the promoters were determined in Betula pendula Roth along the growing season, and as a response to mechanical bending and wounding. The main activity of the PtCOMTp- and PtCOMTdp-promoters, determined by the histochemical GUS assay, was found in the developing xylem of stems during the 8th–13th week and in the developing xylem of roots in the 13th week of the growing season. The GUS expression patterns did not differ among the xylem cell types. The PtCOMT promoter-induced GUS expression observed in phloem fibres suggests a need for PtCOMT expression and thus syringyl (S) lignin synthesis in fibre lignification. However, the PtCOMTdp-promoter induced GUS expression in stem trichomes, which may contribute to the biosynthesis of phenylpropanoid pathway-derived compounds other than lignin. Finally, a strong GUS expression was induced by the PtCOMT promoters in response to mechanical stem bending but not to wounding. The lack of major differences between the PtCOMTp- and PtCOMTdp-promoters suggests that the deleted promoter sequence (including nucleotides −415 to −886) did not contain a significant regulatory element contributing to the GUS expression in young B. pendula trees.