The major phytoalexin in alfalfa is the isoflavonoid (-)-medicarpin (or 6aR, 
11aR)-medicarpin. Isoflavone reductase (IFR), the penultimate enzyme in 
medicarpin biosynthesis, is responsible for introducing one of two chiral 
centers in (-)-medicarpin. We have isolated a 1.18 kb alfalfa cDNA (pIFRalf1) 
which, when expressed in Escherichia coli, converts 2'-hydroxyformononetin 
stereospecifically to (3R)-vestitone, as would be predicted for IFR from 
alfalfa. The calculated molecular weight of the polypeptide (35,400) derived 
from the 954 bp open reading frame compares favorably to estimated Mrs 
determined for IFR proteins purified from other legumes. The transcript (1.4 kb) 
is highly induced in elicited alfalfa cell cultures. The kinetics of induction 
are consistent with the appearance of IFR activity, the accumulation of 
medicarpin, and the observed induction of other enzymes in the pathway. Low 
levels of IFR transcripts were found in healthy plant parts (roots and nodules) 
which accumulate low levels of a medicarpin glucoside. IFR appears to be encoded 
by a single gene in alfalfa. The cloning of IFR opens up the possibility of 
genetic manipulation of phytoalexin biosynthesis in alfalfa by altering 
isoflavonoid stereochemistry.