Caffeoyl-coenzyme A (CoA) O-methyltransferase (CCoAOMT) has been proposed to be involved in an alternative methylation pathway of lignin biosynthesis. However, no direct evidence has been available to confirm that CCoAOMT is essential for lignin biosynthesis. To understand further the methylation steps in lignin biosynthesis, we used an antisense approach to alter O-methyltransferase (OMT) gene expression and investigated the consequences of this alteration. We generated transgenic tobacco plants with a substantial reduction in CCoAOMT as well as plants with a simultaneous reduction in both CCoAOMT and caffeic acid O-methyltransferase (CAOMT). Lignin analysis showed that the reduction in CCoAOMT alone resulted in a dramatic decrease in lignin content. The reduction in CCoAOMT also led to a dramatic alteration in lignin composition. Both guaiacyl lignin and syringyl lignin were reduced in the transgenic plants. However, guaiacyl lignin was preferentially reduced, which resulted in an increase in the S/G (syringl/guaiacyl) ratio. We have also analyzed lignin content and composition in transgenic plants having a simultaneous reduction in both CCoAOMT and CAOMT. The reduction in both OMTs resulted in a further decrease in total lignin content. This is in sharp contrast to the effect that resulted from the reduction in CAOMT alone, which only decreased the syringl lignin unit without a reduction in overall lignin content. These results unequivocally demonstrate that methylation reactions in lignin biosynthesis are catalyzed by both CCoAOMT and CAOMT.

Caffeoyl-coenzyme A (CoA) O-methyltransferase (CCoAOMT) has been proposed to be involved in an alternative methylation pathway of lignin biosynthesis. However, no direct evidence has been available to confirm that CCoAOMT is essential for lignin biosynthesis. To understand further the methylation steps in lignin biosynthesis, we used an antisense approach to alter O-methyltransferase (OMT) gene expression and investigated the consequences of this alteration. We generated transgenic tobacco plants with a substantial reduction in CCoAOMT as well as plants with a simultaneous reduction in both CCoAOMT and caffeic acid O-methyltransferase (CAOMT). Lignin analysis showed that the reduction in CCoAOMT alone resulted in a dramatic decrease in lignin content. The reduction in CCoAOMT also led to a dramatic alteration in lignin composition. Both guaiacyl lignin and syringyl lignin were reduced in the transgenic plants. However, guaiacyl lignin was preferentially reduced, which resulted in an increase in the S/G (syringl/guaiacyl) ratio. We have also analyzed lignin content and composition in transgenic plants having a simultaneous reduction in both CCoAOMT and CAOMT. The reduction in both OMTs resulted in a further decrease in total lignin content. This is in sharp contrast to the effect that resulted from the reduction in CAOMT alone, which only decreased the syringl lignin unit without a reduction in overall lignin content. These results unequivocally demonstrate that methylation reactions in lignin biosynthesis are catalyzed by both CCoAOMT and CAOMT.

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We report the purification of hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT) to apparent homogeneity in 12% yield from tobacco (Nicotiana tabacum L. cv. Xanthi) cell-suspension cultures elicited with a commercial preparation of pronase. The purification procedure employs only four chromatography steps and takes advantage of the fact that the transferase binds tightly both to phenyl-Sepharose and to hydroxyapatite. The native enzyme has a pI of 5.2 and consists of two identical or very similar subunits of approximately 24 kDa. The purified enzyme can synthesise a wide range of amides due to its relatively low specificity for cinnamoyl-CoA derivatives and hydroxyphenethylamines, but its best substrates are tyramine and feruloyl-CoA. THT follows Michaelis-Menten kinetics in the presence of low concentrations of feruloyl-CoA but negative cooperativity occurs when this concentration increases above 2.5 microM, resulting in a marked decrease of the affinity for tyramine. Large deviations from Michaelis-Menten kinetics are also observed when 3-methoxytyramine is used as acyl acceptor. The activity of tobacco THT is not affected by the addition of CaCl2 or MgCl2 but its maximal velocity is increased up to twofold by addition of ethanol to the assay mixture. It is inhibited in vitro by L-tyrosine benzyl ester, which binds reversibly to the tyramine-binding site. Experiments performed using L-tyrosine benzyl ester and caffeoyl-CoA as inhibitors confirm that feruloyl-CoA is the first substrate to add to the transferase in an ordered bi-bi mechanism. Part of the amino acid sequence of the transferase, elucidated by microsequencing of tryptic peptides, is also described.