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

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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.