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 (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.
Paiva
N L
NL
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73402.
Edwards
R
R
Sun
Y J
YJ
Hrazdina
G
G
Dixon
R A
RA
D000595:Amino Acid Sequence; D001483:Base Sequence; D001578:Benzopyrans / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression / Q000502:physiology; D007529:Isoflavones / Q000378:metabolism; D007700:Kinetics; D000455:Medicago sativa / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010088:Oxidoreductases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010936:Plant Extracts / Q000096:biosynthesis*; D010940:Plant Proteins / Q000235:genetics*; D036343:Pterocarpans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012717:Sesquiterpenes; D013729:Terpenes; D001616:beta-Galactosidase / Q000235:genetics; D000094162:Phytoalexins
D001578:Benzopyrans; D007529:Isoflavones; D010936:Plant Extracts; D010940:Plant Proteins; D036343:Pterocarpans; D011993:Recombinant Fusion Proteins; D012717:Sesquiterpenes; D013729:Terpenes; C047353:medicarpin; D010088:Oxidoreductases; C071072:vestitone reductase; D044925:Oxidoreductases Acting on CH-CH Group Donors; D001616:beta-Galactosidase; D000094162:Phytoalexins