Identification and Characterization of Genes Involved in Regulation of Ascorbate Metabolic Pathway(s) in Arabidopsis thaliana

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dc.contributor.authorZhang, Wenyanen
dc.contributor.committeechairChevone, Boris I.en
dc.contributor.committeecochairNessler, Craig L.en
dc.contributor.committeememberGrabau, Elizabeth A.en
dc.contributor.committeememberHess, John L.en
dc.contributor.committeememberBeers, Eric P.en
dc.contributor.departmentPlant Pathology, Physiology, and Weed Scienceen
dc.date.accessioned2014-03-14T20:07:32Zen
dc.date.adate2007-03-27en
dc.date.available2014-03-14T20:07:32Zen
dc.date.issued2007-02-01en
dc.date.rdate2008-03-27en
dc.date.sdate2007-02-15en
dc.description.abstractVitamin C (ascorbic acid, AsA), an important primary metabolite of plants, functions as an antioxidant, an enzyme cofactor, and a cell-signaling modulator in a wide array of crucial physiological processes including biosynthesis of the cell wall, secondary metabolites and phytohormones, stress resistance, photoprotection, cell division, senescence, and growth. To identify genes that may regulate vitamin C levels in plants, about 3000 activation-tagged Arabidopsis lines were treated with ozone, which is a power oxidizing agent. Two mutants were selected for identification of potential genes involved in the regulation of vitamin C synthesis. A putative F-box gene, VCF1, and a purple acid phosphatase, AtPAP15, were identified for further characterization. Two homozygous SALK T-DNA knockouts in the open reading frame (ORF) of VCF1 exhibited high tolerance to ozone when treated with 450 ppb for 3 hours and the AsA levels of these mutants were 2 to 3 fold higher than wild-type (wt) plants. Developmental studies, using RT-PCR, indicated that foliar expression of the VCF1 gene increased with plant age from 1 to 5 weeks, whereas AsA decreased during this same period. The expression of VCF1 was higher under a low-light condition in which AsA was reduced considerably. The AsA levels in two VCF1 overexpressing lines were only 50 to 70% of wt plants. These results suggested that the putative F-box gene functions as a negative regulator of leaf ascorbate content. Overexpression of AtPAP15 with the CaMV 35S promoter resulted in up to 3-fold higher AsA levels than wt plants, where two independent SALK T-DNA insertion mutants in AtPAP15 had 50% less AsA than wt plants. Enzyme activity of bacterially expressed GST:AtPAP15 was greatest with phytate as a substrate indicating that AtPAP15 is a phytase. Phytase catalyzes hydrolysis of phytate (myo-inositol hexakisphosphate) to yield myo-inositol and free phosphate. Thus, AtPAP15 may regulate AsA levels by controlling the input of myo-inositol into this branch of AsA biosynthesis in Arabidopsis. AtPAP15 was expressed in all tested organs in wt plants and suggests that the enzyme may have functions other than phytate degradation during seed germination.en
dc.description.degreePh. D.en
dc.identifier.otheretd-02152007-110933en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-02152007-110933/en
dc.identifier.urihttp://hdl.handle.net/10919/26211en
dc.publisherVirginia Techen
dc.relation.haspartThesis_Zhang.pdfen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectRT-PCRen
dc.subjectGUSen
dc.subjectTAIL-PCRen
dc.subjectactivation taggingen
dc.subjectAtPAP15en
dc.subjectVCF1en
dc.subjectascorbateen
dc.subjectArabidopsisen
dc.titleIdentification and Characterization of Genes Involved in Regulation of Ascorbate Metabolic Pathway(s) in Arabidopsis thalianaen
dc.typeDissertationen
thesis.degree.disciplinePlant Pathology, Physiology, and Weed Scienceen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

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