DEVELOPMENTAL EXPRESSION AND SUBSTRATE SPECIFICITIES OF ALFALFA CAFFEIC ACID 3-O-METHYLTRANSFERASE AND CAFFEOYL COENZYME-A 3-O-METHYLTRANSFERASE IN RELATION TO LIGNIFICATION

The biosynthesis of monolignols can potentially occur via two parallel pathways involving free acids or their coenzyme A (CoA) esters. Caffe ic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransf erase (CCOMT) catalyze functionally identical reactions in these two p athways, resulting in the formation of mono- or dimethoxylated lignin precursors. The activities of the two enzymes increase from the first to the sixth internode in stems of alfalfa (Medicago sativa L.), prece ding the deposition of lignin. Alfalfa CCOMT is highly similar at the amino acid sequence level to the CCOMT from parsley, although it conta ins a six-amino acid insertion near the N terminus. Transcripts encodi ng both COMT and CCOMT are primarily localized to vascular tissue in a lfalfa stems. Alfalfa CCOMT expressed in Escherichia coli catalyzes O- methylation of caffeoyl and 5-hydroxyferuloyl CoA, with preference for caffeoyl CoA. It has low activity against the free acids. COMT expres sed in E. coli is active against both caffeic and 5-hydroxyferulic aci ds, with preference for the fatter compound. Surprisingly, very little extractable O-methyltransferase activity versus 5-hydroxyferuloyl CoA is present in alfalfa stem internodes, in which relative O-methyltran sferase activity against 5-hydroxyferulic acid increases with increasi ng maturity, correlating with increased lignin methoxyl content.