Functional Characterization of Mitochondrial and Cytosolic Aldehyde Dehydrogenases in Maize (Zea Mays L) Feng Liu Iowa State University
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Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2002 Functional characterization of mitochondrial and cytosolic aldehyde dehydrogenases in maize (Zea mays L) Feng Liu 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 Liu, Feng, "Functional characterization of mitochondrial and cytosolic aldehyde dehydrogenases in maize (Zea mays L) " (2002). Retrospective Theses and Dissertations. 531. https://lib.dr.iastate.edu/rtd/531 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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ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 Functional characterization of mitochondrial and cytosolic aldehyde dehydrogenases in maize (,Zea mays L.) by Feng Liu A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Genetics Program of Study Committee: Patrick S. Schnable, Major Professor Herbert J. Fromm Alan Myers Basil Nikolau Roger P. Wise Iowa State University Ames, Iowa 2002 UMI Number: 3073466 UMT UMI Microform 3073466 Copyright 2003 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 iî Graduate College Iowa State University This is to certify that the doctoral dissertation of Feng Liu 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. Committeettee memberl Signature was redacted for privacy. Commit mbb Signature was redacted for privacy. Signature was redacted for privacy. r tlj Major Pr am iii TABLE OF CONTENTS LIST OF TABLES v LIST OF FIGURES vi CHAPTER 1. GENERAL INTRODUCTION I Significance and Rationale 1 Dissertation Organization 1 Introduction 2 References 7 CHAPTER 2. CHARACTERIZATION OF THE ALDEHYDE DEHYDROGENASE GENE FAMILIES OF ZEA MA YS AND ARABIDOPSIS 17 Abstract 17 Abbreviations 18 Introduction 19 Methods 21 Results 27 Discussion 34 Acknowledgments 38 References 39 CHAPTER 3. MITOCHONDRIAL ALDEHYDE DEHYDROGENASE ACTIVITY IS REQUIRED FOR MALE FERTILITY IN MAIZE 54 Abstract 54 Introduction 55 Results 58 Discussion 69 Methods 75 Acknowledgments 85 References 86 CHAPTER 4. FUNCTIONAL SPECIALIZATION OF MAIZE (ZEA MAYS L.) MITOCHONDRIAL ALDEHYDE DEHYDROGENASES 105 Abstract 105 Introduction 106 Results 109 Discussion 122 Materials and Methods 131 Acknowledgements 141 Literature Cited 141 iv CHAPTERS. MAIZE CYTOSOLIC ALDEHYDE DEHYDROGENASES EXHIBIT SPECIFICITY TO ALDEHYDES PRODUCED VIA LIPID PEROXIDATION 167 Abstract 167 Introduction 168 Results 174 Discussion 185 Methods 191 Acknowledgments 200 References 200 CHAPTER 6. GENERAL CONCLUSIONS 226 General Conclusions 226 References 232 ACKNOWLEDGEMENTS 236 V LIST OF TABLES TABLE Page 2.1 Comparison of Amino Acid Identity and Similarity for the Maize and Arabidopsis ALDHs 46 3.1 Markers for Cell Fractionation 96 3.2 Maize mtALDH Assay 97 3.3 Correlation between Anther Arrest and rJ2 Genotype in Progeny from the Test Cross: (N) rfl-m/rjl-m x rf2-m/Rf2 97 4.1 Purification of recombinant RF2A and RF2B 152 4.2 Kinetic analyses of RF2A and RF2B 153 4.3 Lipid peroxidation assay of maize spikelets 156 4.4 ALDH assays of maize mitochondrial preparations 156 5.1 Markers for cell fractionation 213 5.2 Purification of recombinant RF2C and RF2D 214 5.3 Kinetic analyses of RF2C and RF2D 215 vi LIST OF FIGURES Figure Page 1.1 Acetaldehyde generated via ethanolic fermentation pathway 12 1.2 Generation of aldehyde products via ROS-mediated lipid peroxidation 13 1.3 IAA biosynthesis in plants via L-tryptophan 14 1.4 Toxicity of aldehydes 15 1.5 Aldehyde detoxification reactions 16 2.1 Phylogenetic tree of selected ALDHs 47 2.2 The rf2a, rj2c, and AtALDHla genes can complement an E. coli ALDH mutant 48 2.3 RF2B, RF2C, RF2D, AtALDH2a and AtALDH 1 a all exhibit ALDH activity 49 2.4 Gene structures of the ALDH genes of maize and Arabidopsis 50 2.5 Genetic mapping of maize ALDH genes 51 2.6 ALDH gene family protein alignment 52 2.7 Residues predicted to be responsible for functional differences between mtALDHs and cALDHs 53 3.1 Immunoblot analysis of RF2 protein accumulation 98 3.2 RF2 Protein accumulates in mitochondria 99 3.3 Immunolocalization of the RF2 protein 100 3.4 ALDH assay of E. coli-expressed recombinant RF2 100 3.5 The rf2 gene can complement an £. coli o/</-deficient mutant 101 vii 3.6 Mitochondrial ALDH assay 101 3.7 Structure of the RF2 protein 102 3.8 Anther arrest in rf2 plants carrying N or T cytoplasm. 103 3.9 Microscopic observation of anther arrest 104 4.1 In vitro import of RF2B precursor protein into mitochondria 157 4.2 Accumulation of RF2A and RF2B in mitochondria 158 4.3 RNA gel-blot analyses of rfla and rflb transcripts 159 4.4 Immunolocalization of R.F2A and R.F2B proteins 160 4.5 Purification of recombinant RF2A and RF2B from E. coli 161 4.6 Biochemical characterizations of RF2A and RF2B 162 4.7 Determination of ROS levels in maize anthers 163 4.8 Predicted 3-D structures of RF2A and RF2B 164 4.9 Amino acid alignment of grass mtALDHs 165 4.10 Phylogentic tree of plant and mammalian Family 1 and Family 2 ALDHs 166 5.1 Phylogenetic tree of plant and mammalian family 1, Family 2 and Family 3 ALDHs 217 5.2 Subcellular localization of RF2C and RF2D 218 5.3 Purification of recombinant RF2C and RF2D 219 5.4 Biochemical characterization of RF2C and RF2D 220 5.5 Predicted 3-D structures of RF2C and RF2D 221 5.6 ROS levels in response to exogenous ABA or wounding 222 5.7 Accumulation of rj2c and rfld transcripts and protein in response viii to application of ABA and wounding 223 5.8 Accumulation of rf2c and rf2d transcripts and protein in response to hypoxia and re-aeration 224 5.9 Role of Family I cALDHs in the plant defense system 225 I CHAPTER 1 GENERAL INTRODUCTION Significance and Rationale Aldehyde dehydrogenases (ALDHs, EC 1.2.1.3) exist in many, if not all, taxa, and play diverse roles in cellular function. ALDHs oxidize aldehydes to their corresponding carboxylic acid upon reducing NAD+ to NADH. Aldehydes are generally considered to be cytotoxic because their carbonyl groups can interact with many molecules including proteins and DNA, and alter functions of these molecules. A number of ALDHs have been well studied in animal systems and fungi, yet there has been little research on plant ALDHs. Although animals and plants share many metabolic pathways, considering the unique aspects of plant physiology and cellular function, it is necessary to study plant ALDHs to define their unique physiological functions in these organisms. Recently it has been shown that one of the maize mitochondrial ALDH is involved in pollen development, which is of great importance for both basic plant science research and agricultural application, because male sterile maize lines can facilitate the production of hybrid com. Dissertation organization This dissertation is divided into six chapters. Chapter 1 includes a general introduction to ALDHs and aldehydes generated by various biochemical pathways. This chapter also includes an introduction to maize cytoplasmic male sterility and the role a mitochondrial ALDH plays in fertility restoration. Chapter 2 is a paper published in Plant Molecular Biology, in which Feng Liu is a co- first author with David Skibbe and Tsui-Jun Wen. This paper described characterization of four maize ALDH genes and three Arabidopsis ALDH genes. David Skibbe characterized the maize rflc and rfld genes, which encode two cytosolic ALDHs; Tsui-Jun Wen 2 characterized three Arabidopsis ALDH genes. Feng Liu studied the maize rf2a and rf2b genes, which encode two mitochondrial ALDHs. Feng Liu initiated cDNA cloning of rJ2c, rf2d and three Arabidopsis ALDH genes, performed ALDH assay for all seven genes and established complementation conditions in E. coli. Although David Skibbe was the primary writer of the manuscript, Feng Liu also participated in its preparation. Chapter 3 is a paper published in the Plant Cell, which establishes that maize mitochondrial ALDH activity is required for normal pollen development. Xiangqin Cui collected the data involving anther arrest and interpreted the results, and prepared the predicted 3-D structure. Feng Liu collected all of the remaining data and was the primary writer of the manuscript.