STRESS RESPONSES IN ALFALFA (MEDICAGO-SATIVA L) .9. ISOFLAVONE O-METHYLTRANSFERASE ACTIVITIES IN ELICITOR-TREATED CELL-SUSPENSION CULTURES OF MEDICAGO-SATIVA

Treatment of alfalfa cell suspension cultures with elicitor preparations from baker's yeast or from cell walls of Colletotrichum lindemuthianum resulted in a ca 200-fold induction of isoflavone O-methyltransferase (IOMT) activity. The elicited cultures contained O-methyltransferase activity against isoflavone, isoflavan and pterocarpan substrates. These activities could be separated into two distinct fractions by ion-exchange chromatography. The major IOMT activity (IOMT II) was purified to homogeneity by a combination of anion exchange chromatography, hydrophobic interaction chromatography and chromatofocussing. It is a monomeric enzyme of subunit M(r) 41 000 which could be photoaffinity labelled with tritiated SAM. IOMT II converted the isoflavone daidzein to its 7-O-methyl ether isoformononetin, with K(m) values of 20-mu-M for daidzein and 150-mu-M for SAM and a pH optimum of 8.5. Both IOMT II and the less abundant IOMT species (IOMT I) exhibited greatest activity with 6,7,4'-trihydroxyisoflavone as methyl acceptor. IOMT I, but not IOMT II, also catalysed the A-ring methylation of the pterocarpan phytoalexin medicarpin. Isoflavone 4'-OMT activity, which is believed necessary for the formation of the B-ring methoxy substituent of medicarpin, was present at very low activity in extracts from the cultures and was only weakly induced by elicitor.