Elicitor-Inducible Caffeoyl-Coenzyme A 3-O-Methyltransferase from Petroselinum crispum Cell Suspensions

Abstract

Parsley (Petroselinum crispum) cell cultures respond rapidly to treatment with fungal elicitor by the accumulation of coumarin phytoalexins in the culture fluid and by incorporation of ferulic esters into their cell walls. S-Adenosyl-l-methionine:trans-caffeoyl-CoA 3-O-methyltransferase activity, specifically involved in the formation of ferulic esters, is induced under these conditions. Such an inducible methyltransferase activity has been found in plant cells of various species. The methyltransferase was purified to homogeneity from parsley cells that had been treated for 12 hours with crude cell wall elicitor from Phytophthora megasperma f. sp. glycinea. It consists of two very similar or identical subunits of approximately 24 kilodaltons, which are N-terminally blocked. Attempts to generate antisera against the native enzyme in rabbit or mouse failed, but an antiserum, cross-reactive in enzyme-linked immunosorbent assays, was raised in mouse by intraperitoneal injection of the heat-denatured enzyme. Roughly 33% of the amino acid sequence was elucidated by microsequencing of tryptic peptides of the methyltransferase. The parsley enzyme may be related to adenine-specific methyltransferases known from bacterial sources. Antiserum generated in rabbit against a synthetic decapeptide, as inferred from one of the tryptic peptides and conjugated to ovalbumin, specifically cross-reacted with the methyltransferase protein in Western blots developed after SDS-polyacrylamide electrophoresis. This serum did not react, however, with native parsley methyltransferase.

Genome-Wide Characterization of the Lignification Toolbox in Arabidopsis

“…Pakusch et al, 1991;Chen et al, 2000); corresponding promoter elements were identified in CCoAOMT1, CCoAOMT2 and CCoAOMT3 (supplemental Table VIs). CCoAOMT3 has an extended N-terminal sequence, not shared by any of the other CCoAOMTs, predicted to be an ER-targeting peptide.…”

Modifications in Lignin and Accumulation of Phenolic Glucosides in Poplar Xylem upon Down-regulation of Caffeoyl-Coenzyme A O-Methyltransferase, an Enzyme Involved in Lignin Biosynthesis

“…The transgenic poplars were screened by protein gel blot analysis for a reduced or increased CCoAOMT protein level in the stem xylem. This analysis was chosen to screen the transgenic lines because bispecific COMT has been shown to Omethylate cinnamoyl-CoA esters too (12,32) and, consequently, to interfere with the classical CCoAOMT activity assay (33). The antibodies used were generated against a recombinant poplar CCoAOMT protein and reacted with the two poplar CCoAOMT isoforms (34).…”

Kinetic Characterization of Caffeoyl-Coenzyme A-Specific 3-O-Methyltransferase from Elicited Parsley Cell Suspensions

“…, and some homology to bacterial AMTs was noticed (15). This methyltransferase was shown to possess a remarkably narrow substrate specificity for caffeoyl-CoA, and the adenine moiety of CoA was assumed as a cause of homology with adenine-specific DNA-methyltransferases from bacterial sources, which had been proposed earlier to have evolved by gene duplication (7,12,15).…”

“…(14,15), and some homology to bacterial AMTs was noticed (15). This methyltransferase was shown to possess a remarkably narrow substrate specificity for caffeoyl-CoA, and the adenine moiety of CoA was assumed as a cause of homology with adenine-specific DNA-methyltransferases from bacterial sources, which had been proposed earlier to have evolved by gene duplication (7, 12,15).In the context of phenylpropanoid biosynthesis, in particular flavonoids and ferulic acid, various OMTs have been studied (6, 16). However, only few have been purified to homogeneity and there is little information on their kinetic mechanisms.…”

“…The small inhibitory constant determined in vitro for feruloyl-CoA suggests that, in vivo, the enzyme activity is also under tight control by the steady-state product concentration in addition to the rate of transcription that becomes affected upon elicitor challenge. (14,15), and some homology to bacterial AMTs was noticed (15). This methyltransferase was shown to possess a remarkably narrow substrate specificity for caffeoyl-CoA, and the adenine moiety of CoA was assumed as a cause of homology with adenine-specific DNA-methyltransferases from bacterial sources, which had been proposed earlier to have evolved by gene duplication (7, 12,15).…”