Many plant species, in response to stresses, accumulate low molecular weight secondary metabolites called phytoalexins. Pea (Pisum sativum ) makes a pterocarpanoid phytoalexin called pisatin which is relatively unique among pterocarpans because its stereochemical configuration is different at two adjacent carbons from the corresponding carbons in pterocarpan phytoalexins synthesized by alfalfa, soybean, clover and other legumes. Previous research demonstrated that an (-) isoflavanone-synthesizing isoflavone reductase (EFR) is induced during (+) pisatin biosynthesis and the final step in the biosynthesis is the methylation of (+) cis-6a-hydroxymaackiain (HMK) by 6a-hydroxymaackiainmethyltransferase (HMM). And, contrary to a predominant model of (+) pisatin biosynthesis, the 6a-OH of pisatin was shown to involve oxygen from H₂O rather than O₂. This work describes the role of (-) isoflavanone (sophorol) in (+) pisatin biosynthesis. Radioactive tracer techniques were used both in vivo and in vitro to analyze metabolism of (-) sophorol and related isoflavonoids. I have found that, in vivo, the incorporation of (-) sophorol into (+) pisatin is more efficient than the incorporation of (+) sophorol and (+) maackiain, suggesting that the normal biosynthetic route to (+) pisatin utilizes (-) and not (+) sophorol and does not use maackiain. (+) Sophorol is not metabolized in vitro by pea protein extracts, although isoflavene, 7,2 '-Dihydroxy-4',5'-methylenedioxyisoflavanol (DMDI) and a novel diastereomer of HMK, trans-HMK, accumulate when (-) sophorol is used as substrate. A cDNA from pea, which encodes sophorol reductase (SOR), was cloned by homology to an alfalfa cDNA coding for isoflavanone reductase. The SOR cDNA was found to be transcribed in response to CuCl₂ treatment of pea seedlings, as was previously found for cDNAs of IFR and HMM, which are involved in pisatin biosynthesis. The SOR cDNA gene product specifically reduces (-) and not (+) sophorol in vitro. DMDI, the product formed by the activity from the recombinant protein, is incorporated in vivo into (+) pisatin. My current model of (+) HMK synthesis proposes that (-) sophorol and (3R) DMDI are normal in vivo pathway intermediates. However, trans-HMK is likely an artifact as it is a poor pisatin intermediate in vivo and is also a poor substrate in vitro for HMM.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/282830 |
| Date | January 1998 |
| Creators | DiCenzo, Gregory Lawrence |
| Contributors | VanEtten, Hans |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Dissertation-Reproduction (electronic) |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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