Study of the (+)-Pisatin Biosynthetic Pathway by RNAi and Development of a Novel Method to Elicit the Production of Plant Secondary Metabolites

Persistent Link:
http://hdl.handle.net/10150/193608
Title:
Study of the (+)-Pisatin Biosynthetic Pathway by RNAi and Development of a Novel Method to Elicit the Production of Plant Secondary Metabolites
Author:
Kaimoyo, Evans
Issue Date:
2005
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:
(+)-Pisatin, ([+]-[6aR,11aR]-6a-hydroxy-3-methoxy-8,9-methylenedioxypterocarpan) is the major phytoalexin of the garden pea (Pisum sativum L.). Despite being the first phytoalexin to be chemically characterized, its biosynthesis remains to be fully elucidated. RNA-mediated genetic interference (RNAi) was used to gain further insights into the (+)-pisatin biosynthetic pathway. The expression of three genes, isoflavone reductase (IFR) catalyzing the reduction of 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone to (-)-sophorol, sophorol reductase (SOR) involved in reducing (-)-sophorol to (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanol and hydroxymaackiain-3-O methyltransferase (HMM) involved in methylation of (+)-6a-hydroxymaackiain to (+)-pisatin was silenced. The genes are transcriptionally co-regulated during (+)-pisatin biosynthesis, with the IFR and SOR proposed to function upstream of the HMM gene.Hairy roots expressing the HMM RNAi construct, deficient in (+)-pisatin biosynthesis were identified. However, these did not accumulate (+)-6a-hydroxymaackiain, precursor to (+)-pisatin. Instead they accumulated 2,7,4'-trihydroxyisoflavanone, daidzein, liquiritigenin and isoformononetin. The amino acid sequence of HMM is very similar to that of another methyltransferase, hydroxyisoflavanone-4'-O-methyltransferase (HI4MOT), found in most legumes. HI4?MOT catalyzes the methylation of 2,7,4'-trihydroxyisoflavanone (THI) to 2,7-dihydroxy-4'-methoxyisoflavanone, one of the earliest enzymatic steps in isoflavonoid biosynthesis. In pea, HI4OMT may be the same enzyme as "HMM" catalyzing the methylation of both THI and (+)-6a-hydroxymaackiain. Preventing the methylation of THI could divert pea intermediates to the production of daidzein and isoformononetin instead of (+)-pisatin.None of the transgenic hairy roots expressing the IFR RNAi construct were totally deficient in (+)-pisatin biosynthesis. However, all produced reduced amounts of (+)-pisatin, with one culture accumulating 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone, the substrate for IFR. Hairy roots expressing the SOR RNAi construct deficient in (+)-pisatin biosynthesis were identified. These accumulated (-)-sophorol, the substrate for SOR. These data provide evidence for the involvement of these genes and the intermediates with (-)-optical activity in (+)-pisatin biosynthesis.The elicitation of the biosynthesis of secondary metabolites in plant cell and tissue cultures by electric current was explored. Electric current was demonstrated to elicit the biosynthesis of secondary metabolites in pea hairy and intact roots, seedling, root and cell suspension cultures of various plant species. Electric current has potential for use as an elicitor of secondary metabolites in basic and commercial research ventures.
Type:
text; Electronic Dissertation
Keywords:
Plant Pathology
Degree Name:
PhD
Degree Level:
doctoral
Degree Program:
Plant Pathology; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
VanEtten, Hans D.

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleStudy of the (+)-Pisatin Biosynthetic Pathway by RNAi and Development of a Novel Method to Elicit the Production of Plant Secondary Metabolitesen_US
dc.creatorKaimoyo, Evansen_US
dc.contributor.authorKaimoyo, Evansen_US
dc.date.issued2005en_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.abstract(+)-Pisatin, ([+]-[6aR,11aR]-6a-hydroxy-3-methoxy-8,9-methylenedioxypterocarpan) is the major phytoalexin of the garden pea (Pisum sativum L.). Despite being the first phytoalexin to be chemically characterized, its biosynthesis remains to be fully elucidated. RNA-mediated genetic interference (RNAi) was used to gain further insights into the (+)-pisatin biosynthetic pathway. The expression of three genes, isoflavone reductase (IFR) catalyzing the reduction of 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone to (-)-sophorol, sophorol reductase (SOR) involved in reducing (-)-sophorol to (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanol and hydroxymaackiain-3-O methyltransferase (HMM) involved in methylation of (+)-6a-hydroxymaackiain to (+)-pisatin was silenced. The genes are transcriptionally co-regulated during (+)-pisatin biosynthesis, with the IFR and SOR proposed to function upstream of the HMM gene.Hairy roots expressing the HMM RNAi construct, deficient in (+)-pisatin biosynthesis were identified. However, these did not accumulate (+)-6a-hydroxymaackiain, precursor to (+)-pisatin. Instead they accumulated 2,7,4'-trihydroxyisoflavanone, daidzein, liquiritigenin and isoformononetin. The amino acid sequence of HMM is very similar to that of another methyltransferase, hydroxyisoflavanone-4'-O-methyltransferase (HI4MOT), found in most legumes. HI4?MOT catalyzes the methylation of 2,7,4'-trihydroxyisoflavanone (THI) to 2,7-dihydroxy-4'-methoxyisoflavanone, one of the earliest enzymatic steps in isoflavonoid biosynthesis. In pea, HI4OMT may be the same enzyme as "HMM" catalyzing the methylation of both THI and (+)-6a-hydroxymaackiain. Preventing the methylation of THI could divert pea intermediates to the production of daidzein and isoformononetin instead of (+)-pisatin.None of the transgenic hairy roots expressing the IFR RNAi construct were totally deficient in (+)-pisatin biosynthesis. However, all produced reduced amounts of (+)-pisatin, with one culture accumulating 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone, the substrate for IFR. Hairy roots expressing the SOR RNAi construct deficient in (+)-pisatin biosynthesis were identified. These accumulated (-)-sophorol, the substrate for SOR. These data provide evidence for the involvement of these genes and the intermediates with (-)-optical activity in (+)-pisatin biosynthesis.The elicitation of the biosynthesis of secondary metabolites in plant cell and tissue cultures by electric current was explored. Electric current was demonstrated to elicit the biosynthesis of secondary metabolites in pea hairy and intact roots, seedling, root and cell suspension cultures of various plant species. Electric current has potential for use as an elicitor of secondary metabolites in basic and commercial research ventures.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectPlant Pathologyen_US
thesis.degree.namePhDen_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplinePlant Pathologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairVanEtten, Hans D.en_US
dc.contributor.committeememberGang, David R.en_US
dc.contributor.committeememberPierson, Leland S.en_US
dc.contributor.committeememberHawes, Martha C.en_US
dc.contributor.committeememberJorgensen, Richard A.en_US
dc.identifier.proquest1434en_US
dc.identifier.oclc137355635en_US
All Items in UA Campus Repository are protected by copyright, with all rights reserved, unless otherwise indicated.