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Stress responses in alfalfa (Medicago sativa L.) 11. Molecular cloning and expression of alfalfa isoflavone reductase, a key enzyme of isoflavonoid phytoalexin biosynthesis

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Abstract

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 (35400) derived from the 954 bp open reading frame compares favorably to estimated M rs 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.

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Author notes

  1. Robert Edwards

    Present address: Department of Biological Sciences, University of Durham, DH1 3HP, Durham, UK

Authors and Affiliations

  1. Plant Biology Division, The Samuel Roberts Noble Foundation, P.O. Box 2180, 73402, Ardmore, OK, USA

    Nancy L. Paiva, Robert Edwards & Richard A. Dixon

  2. Institute of Food Science, Cornell University, 14456, Geneva, NY, USA

    Yuejin Sun & Geza Hrazdina

Authors
  1. Nancy L. Paiva
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  2. Robert Edwards
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  3. Yuejin Sun
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  4. Geza Hrazdina
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  5. Richard A. Dixon
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Paiva, N.L., Edwards, R., Sun, Y. et al. Stress responses in alfalfa (Medicago sativa L.) 11. Molecular cloning and expression of alfalfa isoflavone reductase, a key enzyme of isoflavonoid phytoalexin biosynthesis. Plant Mol Biol 17, 653–667 (1991). https://doi.org/10.1007/BF00037051

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  • DOI: https://doi.org/10.1007/BF00037051

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