Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1997 Use of pyrimidine pathway to produce mutant plants that fail to respond to wound induction Lan Zhou Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biochemistry Commons, Molecular Biology Commons, and the Plant Sciences Commons Recommended Citation Zhou, Lan, "Use of pyrimidine pathway to produce mutant plants that fail to respond to wound induction " (1997). Retrospective Theses and Dissertations. 11583. https://lib.dr.iastate.edu/rtd/11583 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. 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This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 II ! Graduate College Iowa State University This is to certify that the doctoral dissertation of Lan Zhou has met the dissertation requirements of Iowa State University Signature was redacted for privacy. Major Professor Signature was redacted for privacy. Iflg'M^or Pre Signature was redacted for privacy. For th^^ra^ate College iii i DEDICATION To my parents, !l{enjie Zfiou and. Shufang ZRang, for their (ove and understanding. To my mentor, T)r. ^Bert W. ThomBurg, for his guidance and encouragement. IV T.ABLE OF CONTENTS LIST OF NOMENCLATURE vi ABSTRACT ix CHAPTER L GENERAL INTRODUCTION I Introduction 1 Dissertation Organization 5 References 5 CHAPTER 2. A STRATEGY FOR SELECTING MUTANTS DEFECTIVE IN WOUND INDUCTION IN ARABIDOPSIS THALIANA 12 Abstract 12 Introduction 12 Experimental Procedures 17 Results 23 Discussion 26 Acknowledgments 28 References 28 CHAPTER 3. ALTERATION OF NUCLEOTIDE POOLS IN TRANSGENIC PLANTS BY EXPRESSION OF DICTYOSTEUUM DISCOIDEUM UMP SYNTHASE IN PLANTS 45 Abstract 45 Introduction 46 Experimental Procedures 47 Results 52 Discussion 56 References 57 CHAPTER 4. CLONING, EXPRESSION IN £. CO LI AND CHARACTERIZATION OF ARABIDOPSIS THALIANA UMP/CMP KINASE 66 Abstract 66 Introduction 67 Experimental Procedures 70 Results 74 Discussion 83 Acknowledgments 87 References 87 CHAPTER 5. EFFECT OF MUTATIONS OF CONSERVED RESIDLIES IN THE PHOSPHATE-BINDING SEQUENCE IN ARABIDOPSIS THALIANA 108 Abstract 108 Introduction 109 Experimental Procedures 110 Results 112 Discussion 114 Acknowledgments 117 References 117 V CHAPTER 6. GENERAL CONCLUSIONS 125 APPENDIX A. VECTORS USED IN TfflS WORK 128 APPENDIX B. OLIGONUCLEOTIDES USED IN TfflS WORK 136 ACKNOWLEDGMENTS 137 I VI LIST OF NOMENCLATUEIE ABA: abscisicacid APjA: P', P'-di(adenosine-5')-pentaphosphate CAT assay: chloramphenicol acetyl transferase assay CD: circular dichroism CMP: cytosine monophosphate codA\ gene encoding cytosine deaminase EMS: ethylmethane sulfonate 5FC: 5-fluorocytosine 5F0A: 5-fluoroorotic acid 5FU: 5-fluorouracil GST: glutathione-S-transferase HPLC: high performance liquid chromatography IPTG: isopropyl-P-D-thiogalactoside JA: jasmonic acid k^^.: catalytic constant kDa: kilodalton Km: Michaelis constant MI plants: plants germinated from Ml seeds Ml seeds: seeds after EMS treatment M2 plants: plants germinated from M2 seeds M2 seeds: seeds produced by M1 plants M3 plants: plants germinated from M3 seeds M3 seeds: seeds produced by M2 plants MALDI: matrix-assisted laser desorption ionization MS medium: Murashige and Skoog medium VII NDP: nucleoside diphosphate NMP: nucleoside monophosphate NTP: nucleoside triphosphate OMP: orotidine monophosphate Q: 5' untranslated tobacco mosaic virus leader sequence PEI: polyethyleneimine pGA482: a plant transformation binary vector pinl: gene encoding proteinase inhibitor II pNE3: a construct containing cauliflower mosaic virus 35S promoter, TMV Q. sequence and a bacterial cociA gene PE^P: phosphoribosyl pyrophosphate pRT291: a construct containing pinl promoter, a Dictyosteliiim discoideum LIMP synthase gene and pinl terminator, expresses UMP synthase in a wound-inducible manner in transgenic plants (Tr291) pRT292: a construct containing cauliflower mosaic virus 35S promoter, a Dictyosteliiim discoideum UMP synthase gene and pinl terminator, expresses UMP synthase constitutively in transgenic plants (Tr292) pRT349; a construct containing pinl promoter, a bacterial codA gene and pinl terminator. expresses codA in a wound-inducible manner in transgenic plants (Tr349) pRT354: a construct containing Arabidopsis thaliana nitrilase 1 promoter, a bacterial codA gene and pinl terminator, expresses codA constitutively in transgenic plants (Tr354) pRT380: a bacterial expression vector pGEX-4T-3 containing Arabidopsis thaliana UMP/CMP kinase cDNA PVDF: polyvinylidene difluoride PYR5-6: gene encoding UMP synthase RPA: RNase protection assay VIII SDS PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis T5 plants: the fifth generation of transgenic plants made by self-pollination TLC: thin layer chromatography TMV: tobacco mosaic virus Tr25: N. tabacum transformed by the construct pRT45 containing pinl promoter, a chloramphenicol acetyl transferase gene and pinl terminator, expresses CAT in a wound-inducible manner UMP: uridine monophosphate UMP'^: Dicryosteliurn discoideum gene encoding UMP synthase UMPS: UMP synthase UP5A: P'-(5'-adenosyl) P'-(5'-uridyl)-pentaphosphate ura3 mutant: a Saccharomyces cerevisiae cell without OMP decarboxylase activity ura5 mutant: a Saccharomyces cerevisiae cell without orotate phosphoribosyltransferase activity ura6 mutant: a Saccharomyces cerevisiae cell without UMP kinase activity ix ABSTRACT To understand how proteinase inhibitor genes, as a part of plant defense system, are activated following wounding, we have developed a molecular genetic approach to directly select for mutants blocked in wound response in Arcibidopsis thaliana. The wound-inducible promoter from the potato Proteinase Inhibitor II gene {pinl) was linked to a bacterial negative marker gene codA encoding for cytosine deaminase and transformed into Arcibidopsis thaliana. The cytosine deaminase activity was detected in the leaves of the transgenic plants (Tr349) induced by sucrose, ABA, methyl jasmonate and mechanical wounding. Methyl jasmonate was the best for the pinl induction in the seedlings among all the wound signal chemicals tested. Wild type seedlings had a dose-dependent sensitivity to 5FU. The seedlings were completely killed at a level of 100 |ig/mL and 130 |ig/mL 5FU for Arcibidopsis and N. tabacLim, respectively. 5FC at a concentration of 1 mg/mL had no effect on the seedlings of wild type of Arabidopsis or N. tabacum, or on Arabidopsis transgenic Tr349 seedlings without pinl induction. However, 5FC had a dose-dependent effect on the seedlings of transgenic Tr349 with the pinl induction by methyl jasmonate. No seedlings could survive above the concentration of 500 |J.g/mL 5FC. Homozygous Tr349 seeds were mutagenized by EMS. M2 seeds were collected and plated out on selective media containing 50 |J.g/mL kanamycin + 25 |jM methyl jasmonate + 500 |ig/mL 5FC. The 110 seedlings from M2 seeds survived on selective media. M3 seeds were collected and rescreened on selective media to eliminate false positives. Four M2 mutant lines that lost cytosine deaminase activity in M2 and M3 plants have been isolated. Because of the long timeframes involved in the work with transgenic plants several smaller projects were also conducted. Due