Substrate preferences of O-methyltransferases in alfalfa suggest new pathways for 3-O-methylation of monolignols

Measurement of relative O-methyltransferase activities against all potentia l substrates in the monolignol pathway in developing alfalfa stem extracts revealed activities in the order: caffeoyl CoA > caffeoyl alcohol > 5-hydro xyferulic acid > caffeoyl aldehyde > 5-hydroxyconiferyl alcohol, 5-hydroxyf eruloyl CoA > 5-hydroxyconiferaldehyde > caffeic acid. Maxims for all activ ities occurred in the seventh internode. In stem extracts from transgenic a lfalfa with antisense downregulated caffeoyl CoA O-methyltransferase (CCoAO MT), activities with all substrates except for the two coenzyme A esters we re unaffected. In contrast, downregulation of caffeic acid O-methyltransfer ase (COMT) reduced activities against the non-esterifed substrates in the o rder: 5-hydroxyconiferyl alcohol > 5-hydroxyferulic acid and caffeoyl alcoh ol > caffeoyl aldehyde > caffeic acid > 5-hydroxyconiferaldehyde. Recombina nt COMT expressed in Escherichia coil exhibited the highest V-max/K-m value s with 5-hydroxyconiferaldehyde and caffeoyl aldehyde, and the lowest with caffeic acid. These results indicate that COMT is unlikely to methylate caf feic acid during lignin biosynthesis in vivo, and provide enzymatic evidenc e for an alternative pathway to monolignols involving methylation of caffeo yl aldehyde and/or caffeoyl alcohol by COMT. The concept of independent pat hways to guaiacyl and syringyl monolignols is discussed.