5-Hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of monolignol biosynthesis in angiosperms

S-Adenosyl-L-methionine-dependent caffeate O-methyltransferase (COMT, EC 2. 1.1.6) has traditionally been thought to catalyze the methylation of caffea te and 5-hydroxyferulate for the biosynthesis of syringyl monolignol, a lig nin constituent of angiosperm wood that enables efficient lignin degradatio n for cellulose production. However, recent recognition that coniferyl alde hyde prevents 5-hydroxyferulate biosynthesis in lignifying tissue, and that the hydroxylated form of coniferyl aldehyde, 5-hydroxyconiferyl aldehyde, is an alternative COMT substrate, demands a re-evaluation of the role of CO MT during monolignol biosynthesis, Based on recombinant aspen (Populus trem uloides) COMT enzyme kinetics coupled with mass spectrometry analysis, this study establishes for the first time that COMT is in fact a 5-hydroxyconif eryl aldehyde O-methyltransferase (AldOMT), and that 5-hydroxyconiferyl ald ehyde is both the preferred AldOMT substrate and an inhibitor of caffeate a nd 5-hydroxyferulate methylation, as measured by K-m and K-i values. 5-Hydr oxyconiferyl aldehyde also inhibited the caffeate and 5-hydroxyferulate met hylation activities of xylem proteins from various angiosperm tree species, The evidence that syringyl monolignol biosynthesis is independent of caffe ate and 5-hydroxyferulate methylation supports our previous discovery that coniferyl aldehyde prevents ferulate B-hydroxylation and at the same time e nsures a coniferyl aldehyde 5-hydroxylase (CAld5H)-mediated biosynthesis of 5-hydroxyconiferyl aldehyde. Together, our results provide conclusive evid ence for the presence of a CAld5H/AldOMT-catalyzed coniferyl aldehyde 5-hyd roxylation/methylation pathway that directs syringyl monolignol biosynthesi s in angiosperms.