STRESS RESPONSES IN ALFALFA (MEDICAGO-SATIVA L) .11. MOLECULAR-CLONING AND EXPRESSION OF ALFALFA ISOFLAVONE REDUCTASE, A KEY ENZYME OF ISOFLAVONOID PHYTOALEXIN BIOSYNTHESIS

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 (pIFRalfl) 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 estimate M(r)s 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.