The Central Role of Acetyl-Coa in Plant Metabolism, As Examined Through Studies of ATP Citrate Lyase and the Bio1 Mutant of Arabidopsis Elizabeth K

Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2004 The central role of acetyl-CoA in plant metabolism, as examined through studies of ATP citrate lyase and the bio1 mutant of Arabidopsis Elizabeth K. Winters Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Genetics Commons, Molecular Biology Commons, and the Plant Sciences Commons Recommended Citation Winters, Elizabeth K., "The ec ntral role of acetyl-CoA in plant metabolism, as examined through studies of ATP citrate lyase and the bio1 mutant of Arabidopsis " (2004). Retrospective Theses and Dissertations. 1203. https://lib.dr.iastate.edu/rtd/1203 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]. The central role of acetyl-CoA in plant metabolism, as examined through studies of ATP citrate lyase and the biol mutant of Arabidopsis by Elizabeth K. Winters A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Plant Physiology Program of Study Committee: Eve Syrkin Wurtele, Co-major Professor Basil J. Nikolau, Co-major Professor Thomas Baum James T. Colbert David J. Oliver Mark Westgate Iowa State University Ames, Iowa 2004 UMI Number: 3158378 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. UMI UMI Microform 3158378 Copyright 2005 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Graduate College Iowa State University This is to certify that the doctoral dissertation of Elizabeth K. Winters has met the dissertation requirements of Iowa State University Signature was redacted for privacy. Co-major Professor Signature was redacted for privacy. Co-major Professor Signature was redacted for privacy. For the Major Program Ill TABLE OF CONTENTS ABSTRACT v CHAPTER 1. GENERAL INTRODUCTION 1 DISSERTATION ORGANIZATION 1 THE GVG PROMOTER SYSTEM 2 ATP CITRATE LYASE 3 BIOTIN 9 LITERATURE CITED 13 FIGURES 24 CHAPTER 2. STEROL-INDUCIBLE EXPRESSION DRIVEN BY THE GVG PROMOTER SHOWS A UNIQUE REPLICABLE PATTERN IN TRANSGENIC GL YCINE MAX 26 ABSTRACT 26 INTRODUCTION 27 MATERIALS AND METHODS 29 RESULTS 33 DISCUSSION 35 ACKNOWLEDGEMENTS 39 LITERATURE CITED 39 FIGURES 44 CHAPTER 3. OVEREXPRESSION OF ATP CITRATE LYASE IN ARABIDOPSIS REVEALS MULTIPLE LEVELS OF REGULATION 50 ABSTRACT 50 INTRODUCTION 51 MATERIALS AND METHODS 53 RESULTS 58 DISCUSSION 61 ACKNOWLEDGEMENTS 63 iv LITERATURE CITED 63 FIGURES AND TABLE 69 CHAPTER 4. CHANGES IN BIOTIN STATUS LEAD TO CHANGES IN GLOBAL GENE EXPRESSION 75 ABSTRACT 75 INTRODUCTION 76 RESULTS AND DISCUSSION 80 MATERIALS AND METHODS 90 ACKNOWLEDGMENTS 91 LITERATURE CITED 91 FIGURES AND TABLES 97 CHAPTER 5. GENERAL CONCLUSIONS 118 THE GVG PROMOTER IN SOYBEAN AND ARABIDOPSIS 118 REGULATION OF ACL 119 TRANSCRIPT PROFILING OF THE biol MUTANT 120 CURRENT CHALLENGES TO ANALYZING MICRO ARRAY DATA 122 APPENDIX. BIOTIN GENECHIP DATA 123 ACKNOWLEDGEMENTS 180 V ABSTRACT The acetyl-CoA/biotin network encompasses genes involved in acetyl-CoA generation and utilization and biotin synthesis, recovery, and transport, as well as biotin- containing enzymes involved in myriad metabolic processes. To expand our understanding of this network and address its control, plants with altered levels of ACL (ATP citrate lyase) and BIOl (encoding adenosylmethionine-8-amino-7-oxononanoate synthase), have been examined. An inducible promoter provides a powerful method to test the effects of alterations in expression of specific genes. The synthetic GVG promoter, reported to be sterol-inducible in Arahidopsis thaliana, was inducible in Glycine max (soybean) when used to control the GUS reporter gene, or theACLA or ACLB genes. It showed the same unique replicable pattern of expression in soybean and Arahidopsis, directing high expression primarily to the vasculature of the leaf and root. ACL is a heteromeric enzyme, responsible for production of cytosolic acetyl-CoA. The ACLA-1 and ACLB-2 genes have been over-expressed individually in Arahidopsis using the GVG promoter. Over-expression of ACL genes led to significantly increased ACLA and ACLB RNA accumulation. Over-expression of one subunit of ACL had no effect on mRNA level of the other subunit or on ACL enzyme activity. Transgenic lines over-expressing high levels of ACLA and ACLB RNA were crossed to yield plants containing both transgenes. ACLA and ACLB RNA levels were greatly increased in ACLA+ACLB plants. For the same plants, there was little or no increase in ACLA or ACLB protein from the wild-type level. ACL enzyme activity and phenotype remained unchanged. In contrast, ACL activity was slightly higher (approximately 1.5-fold increase) in cold-treated ACLA+ACLB plants than in cold-treated wild-type plants. These data suggest that ACL is regulated at the translational and/or post-translational levels, and that this regulation can be modulated environmentally. Global transcript profiling experiments utilizing the Arahidopsis biol mutant deficient in biotin synthesis reveal decreased accumulation ofRNAs encoding processes vi directly related to biotin synthesis and utilization in this mutant. Specifically, subunits of the biotin-containing enzymes acetyl-CoA carboxylase and methylcrotonyl-CoA carboxylase, and L-allo-threonine aldolase/lyase, the catalyst for an early step in biotin synthesis, have decreased transcript accumulation in the biol mutant when compared to wild-type. A number of mRNAs involved in defense responses, stress responses, and production of secondary metabolites show increased accumulation in the mutant plants. At the same time, there are decreases in particular mRNAs for the light reactions of photosynthesis and the TCA cycle. The presence of exogenous biotin caused changes in mRNA profiles in both the biol mutant and wild-type plants. 1 CHAPTER 1. GENERAL INTRODUCTION DISSERTATION ORGANIZATION The alternative paper format has been selected for this dissertation, which consists of five chapters. Where appropriate, formatting guidelines for the target journals have been incorporated into this document. Chapter 1 is a general introduction and literature review introducing the plant acetyl- CoA/biotin network and the GVG promoter system. Chapter 2, "Sterol-inducible expression driven by the GVG promoter shows a unique replicable pattern in transgenic Glycine max is a manuscript to be submitted to Plant Molecular Biology. Transformation constructs were produced and Arahidopsis were transformed by Hongjian Liang, a research associate in the laboratory of Dr. Eve S. Wurtele. Soybean transformation was performed by the staff of the Iowa State University Plant Transformation Facility, under the direction of Dr. Kan Wang. Both Hongjian Liang and Lankun Wu, a graduate student of Dr. Wurtele, screened a number of GVG-GUS Arahidopsis plants for GUS expression. Additional screening of GVG-GUS Arahidopsis was done, and the rest of the data presented were collected and analyzed by myself. Chapter 3, "Overexpression of ATP citrate lyase in Arahidopsis reveals multiple levels of regulation," is a manuscript to be submitted to Plant Molecular Biology. Transformation constructs were produced and Arahidopsis were transformed by Hongjian Liang. The rest of the data presented were collected and analyzed by myself. Chapter 4, "Changes in biotin status lead to changes in global gene expression," is part of a manuscript to be submitted to The Plant Cell. Plant material was grown and harvested by Dr. Ping Che and Dr. Cunxi Wang, two postdoctoral associates of Dr. Basil J. Nikolau. Dr. Cunxi Wang also extracted RNA. Normalizations of the Afiymetrix data were done by Dr. Carol Foster (postdoctoral associate of Dr. Wurtele), Dr. Heike Hofmann (Assistant Professor of Statistics), and Dr. Dianne Cook (Associate Professor of Statistics). I analyzed microarray data using GeneSpring, FC Modeler, and MapMan, developed hypotheses and proposed 2 conclusions, wrote the 'Results and Discussion' section, and extensively edited the introduction. Chapter 5 is a general conclusion summarizing and discussing the results from Chapters 2 through 4, and proposing future directions for this research. THE GVG PROMOTER SYSTEM Why an inducible promoter? In any plant species, use of an inducible promoter allows the study of a certain gene in a plant without the inevitable other compensatory effects masking the true impact of the transgene on metabolism. In addition, an inducible promoter can be used to study gene action in a directed manner, allowing targeting and modification of specific processes

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