Persistent Link:
http://hdl.handle.net/10150/282830
Title:
Elucidation of late steps in pisatin biosynthesis
Author:
DiCenzo, Gregory Lawrence
Issue Date:
1998
Publisher:
The University of Arizona.
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.
Abstract:
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.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Agriculture, Plant Pathology.; Chemistry, Biochemistry.; Biology, Plant Physiology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Plant Diseases
Degree Grantor:
University of Arizona
Advisor:
VanEtten, Hans

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleElucidation of late steps in pisatin biosynthesisen_US
dc.creatorDiCenzo, Gregory Lawrenceen_US
dc.contributor.authorDiCenzo, Gregory Lawrenceen_US
dc.date.issued1998en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © 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.en_US
dc.description.abstractMany 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.en_US
dc.description.noteDigitization note: p. 75 missing from paper original; appears to be pagination error rather than missing content.-
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectAgriculture, Plant Pathology.en_US
dc.subjectChemistry, Biochemistry.en_US
dc.subjectBiology, Plant Physiology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplinePlant Diseasesen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorVanEtten, Hansen_US
dc.identifier.proquest9912137en_US
dc.identifier.bibrecord.b39124411en_US
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