Medicarpin, the major phytoalexin in alfalfa, is synthesized via the 
isoflavonoid branch of phenylpropanoid metabolism. The methyl group at the 9 
position of medicarpin is generally accepted to arise via the methylation of the 
4' position (B-ring) of daidzein. Surprisingly, the 
isoflavone-O-methyltransferase (IOMT), which is induced along with other enzymes 
involved in medicarpin biosynthesis, methylates the A-ring 7-hydroxyl group of 
daidzein in vitro, a reaction that probably does not occur in vivo. Utilizing 
internal amino acid sequence information from purified alfalfa IOMT, we have 
isolated three full-length IOMT cDNA clones. A search of the protein databases 
revealed sequence similarities to O-methyltransferases from various sources. The 
highest match (50.5% identity) was found between IOMT8 and 6a-hydroxymaackiain 
3-O-methyltransferase from Pisum sativum. The molecular weight of alfalfa IOMT 
expressed in Escherichia coli was similar to that of purified IOMT from alfalfa 
cell cultures (41 kDa by SDS-PAGE). The recombinant enzyme catalyzed the 
O-methylation of A-ring hydroxyl group(s) of isoflavones, and could also 
methylate the pterocarpan (+) 6a-hydroxymaackiain. Alfalfa contains multiple 
IOMT genes, and closely related sequences are present in the genomes of chickpea 
and cowpea, species that also produce B-ring methylated isoflavonoids in vivo. 
Northern blot analysis indicated that IOMT transcripts are rapidly induced 
following elicitation, prior to the increase in IOMT activity and medicarpin 
accumulation. The possible role of the isoflavone 7-OMT in the synthesis of 
formononetin in vivo is discussed.