Developmental expression and substrate specificities of alfalfa caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase in relation to lignification

摘要

The biosynthesis of monolignols can potentially occur via two parallel pathways involving free acids or their coenzyme A (CoA) esters. Caffeic acid 3-O-methyltransferase (COMT) [caffeate O-methyltransferase] and caffeoyl CoA 3-O-methyltransferase (CCOMT) catalyse functionally identical reactions in these two pathways, resulting in the formation of mono- or dimethoxylated lignin precursors. The activities of these two enzymes increased from the first to the sixth internode in stems of alfalfa (Medicago sativa), preceding the deposition of lignin. Alfalfa CCOMT was highly similar at the amino acid sequence level to the CCOMT from parsley, although it contained a six-amino acid insertion near the N terminus. Transcripts encoding both COMT and CCOMT were primarily localized to vascular tissue in alfalfa stems. Alfalfa CCOMT expressed in Escherichia coli catalysed O-methylation of caffeoyl and 5-hydroxyferuloyl CoA, with preference for caffeoyl CoA. It had low activity against the free acids. COMT expressed in E. coli was active against both caffeic and 5-hydroxyferulic acids, with preference for the latter compound. Surprisingly, very little extractable O-methyltransferase activity versus 5-hydroxyferuloyl CoA was present in alfalfa stem internodes,in which relative O-methyltransferase activity against 5-hydroxyferulic acid increased with increasing maturity, correlating with increased lignin methoxyl content.