Secondary xylem-specific expression of caffeoyl-coenzyme A 3-O-methyltransferase plays an important role in the methylation pathway associated with lignin biosynthesis in loblolly pine

Two types of structurally distinct O-methyltransferases mediate the methyla tion of hydroxylated monomeric lignin precursors in angiosperms. Caffeate 3 -O-methyltransferase (COMT; EC 2.1.1.68) methylates the free acids and caff eoyl CoA 3-O-methyltransferase (CCoAOMT; EC 2.1.1.104) methylates coenzyme A esters. Recently, we reported a novel hydroxycinnamic acid/hydroxycinnamo yl CoA ester O-methyltransferase (AEOMT) from loblolly pine differentiating xylem that was capable of methylating both acid and ester precursors with similar efficiency. In order to determine the possible existence and role o f CCoAOMT in lignin biosynthesis in gymnosperms, a 1.3 kb CCoAOMT cDNA was isolated from loblolly pine that showed 79-82% amino acid sequence identity with many angiosperm CCoAOMTs. The recombinant CCoAOMT expressed in Escher ichia coli exhibited a significant methylating activity with hydroxycinnamo yl CoA esters whereas activity with hydroxycinnamic acids was insignificant . Moreover, 3.2 times higher catalytic efficiency for methylating caffeoyl CoA over 5-hydroxyferuloyl CoA was observed which could serve as a driving force towards synthesis of guaiacyl lignin. The secondary xylem-specific ex pression of CCoAOMT was demonstrated using RNA blot analysis, western blot analysis, and O-methyltransferase enzyme assays. In addition, Southern blot analysis indicated that CCoAOMT may exist as a single-copy gene in lobloll y pine genome. The transgenic tobacco plants carrying loblolly pine CCoAOMT promoter-GUS fusion localized the site of GUS activity at the secondary xy lem tissues. These data suggest that CCoAOMT, in addition to AEOMT, plays a n important role in the methylation pathway associated with lignin biosynth esis in loblolly pine.