Expression of Genes Encoding Acetyl-Coa Carboxylase, Biotin Synthase, and Acetyl-Coa Generating Enzymes in Arabidopsis Thaliana Jinshan Ke Iowa State University

Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1997 Expression of genes encoding acetyl-CoA carboxylase, biotin synthase, and acetyl-CoA generating enzymes in Arabidopsis thaliana Jinshan Ke Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Botany Commons, and the Molecular Biology Commons Recommended Citation Ke, Jinshan, "Expression of genes encoding acetyl-CoA carboxylase, biotin synthase, and acetyl-CoA generating enzymes in Arabidopsis thaliana " (1997). Retrospective Theses and Dissertations. 11996. https://lib.dr.iastate.edu/rtd/11996 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. 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UMI A Bell & Howell Infonnation Company 300 North Zeeb Road, Ann Aibor MI 48106-1346 USA 313/761-4700 800/521-0600 I Expression of genes encoding acetyl-CoA carboxylase, biotin synthase, and acetyl-CoA generating enzymes in Arabidopsis thaliana by Jinshan Ke A dissertation submitted to the graduate faculty in partial fulfillment of the requirement for the degree of DOCTOR OF PHILOSOPHY Major; Botany (Physiology and Molecular Biology) Major Professor: Eve Syrkin Wurtele Iowa State University Ames, low^a 1997 Copyright © Jinshan Ke. 1997. All rights reserved DMI Nxunber: 9814656 Copyright 1997 by Ke, Jinshan All rights reserved. UMI Microform 9814656 Copyright 1998, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 Graduate College Iowa State University This is to certify that the Doctoral dissertation of Jinshan Ke has met the dissertation requirements of Iowa State University Signature was redacted for privacy. Committee Member Signature was redacted for privacy. Committee Member Signature was redacted for privacy. Committee Member Signature was redacted for privacy. Committee Member Signature was redacted for privacy. Maj^r Professor Signature was redacted for privacy. the Major Program Signature was redacted for privacy. uate College Ill TABLE OF CONTENTS GENERAL INTRODUCTION I Acetyl-CoA Carboxylase I Heteromeric Acetyl-CoA Carboxylase 2 Homomeric Acetyl-CoA Carboxylase 3 Regulation of Acetyl-CoA Carboxylase 4 Biotin and Biotin Synthase 5 Dissertation Organization 7 References 7 CHAPTER 1: COORDINATE EXPRESSION OF GENES ENCODING HETEROMERIC ACCASE IN ARABIDOPSIS 13 ABSTRACT 13 INTRODUCTION 13 MATERIALS AND METHODS 15 Materials 15 In Situ Hybridization 15 RNA Blot Analysis 16 RESULTS 17 DISCUSSION 18 REFERENCES 20 CHAPTER 2: STRUCTURE OF THE CACI GENE AND IN SITU CHARACTERIZATION OF ITS EXPRESSION: THE ARABIDOPSIS THALIANA GENE CODING FOR THE BIOTIN-CONTAINING SUBUNIT OF THE PLASTIDIC ACETYL-CoA CARBOXYLASE 33 ABSTRACT 33 INTRODUCTION 34 MATERIALS AND METHODS 35 Materials 35 Isolation and Characterization of Macromolecules 36 iv In Situ Techniques 36 RESULTS 38 Isolation and Characterization of the Arabidopsis CACl Gene 38 BCC Subunit is Located in the Stroma of Plastids 40 Cellular Distribution of the BCC Subunit and CACl mRNA in Siliques and Leaves of Arabidopsis 41 DISCUSSION 42 ACKNOWLEDGEMENTS 45 REFERENCES 45 CHAPTER 3: EXPRESSION OF THE GENES ENCODING HOMOMERIC ACETYL-CoA CARBOXYLASE IN ARABIDOPSIS THALIANA 61 ABSTRACT 61 INTRODUCTION 62 MATERIALS AND METHODS 64 Materials 64 In Situ Techniques 64 RNA Blot Analysis 64 RESULTS 65 DISCUSSION 66 REFERENCES 69 CHAPTER 4: EXPRESSION OF THE GENES ENCODING BIOTIN SYNTHASE IN ARABIDOPSIS THALIANA 85 ABSTRACT 85 INTRODUCTION 85 MATERIALS AND METHODS 87 Materials 87 In Situ hybridization 87 RNA Blot Analysis 87 V RESULTS 88 Distribution of Biotin Synthase mRNA in Developing Flowers and Siliques of Arabidopsis 88 Distribution of Biotin Synthase mRNA in Developing Leaves and Shoot Meristems 89 DISCUSSION 89 ACKNOWLEDGEMENTS 92 REFERENCES 92 GENERAL CONCLUSIONS 105 APPENDIX A: EXPRESSION OF THE GENES ENCODING ATP CITRATE LYASE IN ARABIDOPSIS 108 APPENDIX B: EXPRESSION OF A GENE ENCODING THE Ei b SUBUNIT OF PYRUVATE DEHYDROGENASE IN ARABIDOPSIS 122 APPENDIX C: EXPRESSION OF THE GENE ENCODING ACETYL-CoA SYNTHETASE IN ARABIDOPSIS 125 APPENDIX D: EXPRESSION OF THE EMB3 GENE IN CARROT {DAUCUS CAROTA) 130 APPENDIX E: BIOCHEMICAL AND MOLECULAR BIOLOGICAL CHARACTERIZATION OF CAC2: ARABIDOPSIS THALIANA GENE CODING FOR THE BIOTIN CARBOXYLASE SUBUNIT OF THE PLASTIDIC ACETYL-CoA CARBOXYLASE 134 ACKNOWLEDGMENTS 177 1 GENERAL INTRODUCTION Acetyl-CoA Carboxylase (ACCase) Acetyl-CoA carboxylase (ACCase), a biotin containing enzyme, catalyzes the formation of malonyl-CoA from acetyl-CoA and bicarbonate (Harwood. 1988). The carboxyiation reaction actually proceeds in two steps as follows; (BCCP) Biotin Carboxylase (EC) Enzyme-biotin + HCO3 + ATP >-Enzyme-biotin-C02 +ADP +Pi Carboxyltransferase (CT) Enzyme-biotin-C02 + Acetyl-CoA > Enzyme-biotin +• Malonyl-CoA There are structurally distinct forms of ACCase in living cells. In Escherichia coli. ACCase contains three separable protein components, a 5l-kD biotin carboxylase (BC) encoded by the accC gene, a 17-kD biotin carboxyl carrier protein (BCCP) encoded by the accB gene, and a 130-kD carboxyltransferase (CT) composed of a (accA ) and (3 (accD) subunits (Li and Cronan. 1992a, 1992b). This type of ACCase is referred to as the "prokaryotic" or "heteromeric form". In contrast, in animal, ftmgi and yeast cells, ACCase is composed of a single polypeptide of 200- to 280-kD. containing domains of BC. BCCP and CT (Harwood. 1996). This type of ACCase is called the "eukaryotic" or "homomeric" form. Prior to 1993. it was confusing as to which type of ACCase existed in plant cells. It was thought by many that only a eukaryotic form of ACCase existed in plant cells (Harwood. 1988). The different molecular weights of protein bands on SDS/PAGE yielded by the purified ACCase from plants were unexplainable (Stumpf, 1980). The results were interpreted by many as the severe degradation of ACCase by endogenous proteinases during isolation (Harwood, 1988); however, others disagreed (Nikolau et al.. 1984; Wurtele and Nikolau. 1990). In spinach chloroplasts. three to four different subunits of ACCase. ranging from 47-kD to 120-kD. were isolated (Kannagara and Stumpf. 1972: Mohan and Kekwick. -> 1980). These are roughly comparable to the sizes of the four bacterial ACCase subunits. ACCases from 220- to 240-ldD were also isolated from plants (Harwood, 1988). These results, together with the requirement for malonyl-CoA in both the plastids and the cytosol. and the existence of multiple biotin containing polypeptides in plants (Nikolau et al., 1985), led to the hypothesis that two structurally distinct ACCases are present in plants; one in plastids and the other in the cytosol (Pollard and Stumpf, 1980; Nikolau et al., 1984). The plastidic ACCase is responsible for synthesis of fatty acids and oils; the cytosolic ACCase is used for making secondary products such as flavonoids and phytoalexins (Nikolau et al., 1984; Wurtele and Nikolau, 1990). Heteromeric Acetyl-CoA Carboxylase Heteromeric ACCase is a multiple protein complex, consisting of dissociable subunits of biotin carboxylases, biotin carboxyl carrier protein and a and P subimits of carboxyltransferase. This type of ACCase is in plastids of plant cells and generates malonyl- CoA for de novo fatty acid biosynthesis. Direct evidence for a heteromeric ACCase in plants emerged from findings on pea; the pea plastid genome was found to contain a gene similar in sequence to accD of E. coli. (Li and Cronan, 1992a, 1992b; Sasaki et al.. 1993). A DNA fragment of this putative CTp gene encoded in the pea chloroplast genome was expressed in E. coli, and antibodies were raised to the resultant protein. The antibodies were found to inhibit the activity of ACCase purified from chloroplasts. The antibodies also precipitated a protein complex containing

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