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Molecular and Genetic Characterization of Rf2, A Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2001 Molecular and genetic characterization of rf2, a mitochondrial aldehyde dehydrogenase gene required for male fertility in maize (Zea mays L) Xiangqin Cui 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 Cui, Xiangqin, "Molecular and genetic characterization of rf2, a mitochondrial aldehyde dehydrogenase gene required for male fertility in maize (Zea mays L) " (2001). Retrospective Theses and Dissertations. 1037. https://lib.dr.iastate.edu/rtd/1037 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]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. 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 bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI 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. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 Molecular and genetic characterization of rf2, a mitochondrial aldehyde dehydrogenase gene required for male fertility in maize (Zea mays L.) by Xiangqin Cui A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Genetics Major Professor: Patrick S. Schnable Iowa State University Ames, Iowa 2001 UMI Number: 3016701 ® UMI UMI Microform 3016701 Copyright 2001 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ii Graduate College Iowa State University This is to certify that the Doctoral dissertation of Xiangqin Cui has met the dissertation requirements of Iowa State University Signature was redacted for privacy. Major professor Signature was redacted for privacy. For the Major Program Signature was redacted for privacy. Fopme Graduate College iii TABLE OF CONTENTS CHAPTER 1. GENERAL INTRODUCTION 1 General Introduction 1 Dissertation Organization 2 Literature Review 3 Literature Cited 13 CHAPTER 2. THE rj2 NUCLEAR RESTORER GENE OF MALE-STERILE, T-CYTOPLASM MIAZE 23 Abstract 23 Results and Discussion 24 References and Notes 28 CHAPTER 3. MITOCHONDRIAL ALDEHYDE DEHYDROGENASE ACTIVITY IS REQUIRED FOR MALE FERTILITY IN MAIZE 38 Abstract 38 Introduction 39 Results 42 Discussion 54 Methods 60 Acknowledgments 71 Literature Cited 72 CHAPTER 4. MUTATIONS OF THE MAIZE pdc3 GENE REDUCE TOLERANCE TO ANAEROBIC STRESSES BUT DO NOT AFFECT MALE FERTILITY 94 Abstract 94 iv Introduction 95 Results 98 Discussion 107 Materials and Methods 110 Acknowledgments 117 Literature Cited 118 CHAPTER 5. ALTERNATIVE TRANSCRIPTION INITIATION SITES AND POLYADENYLATION SITES ARE RECRUITED DURING MU SUPPRESSION AT THE rf2 LOCUS OF MAIZE 135 Abstract 135 Introduction 136 Materials and Methods 141 Results 150 Discussion 160 Acknowledgements 167 Literature Cited 167 CHAPTER 6. GENERAL CONCLUSIONS 188 General Conclusions 188 Future Research 190 Literature Cited 193 ACKNOWLEDGEMENTS 194 1 CHAPTER 1. GENERAL INTRODUCTION General Introduction Maize, commonly known as corn, is a major agricultural crop (Dowswell et al., 1996; Salvador, 1997). Hybrid seeds have played an important role in increasing corn yields during the 20th century (Bauman et al., 1986). The production of hybrid seed often involves the hand emasculation of female parents, which is a labor-intensive process. In the late 1960's, this process was largely (85%) replaced by using the T-cytoplasm male sterility (cms-T) system to induce male sterility in the female parents of commercial hybrids (Ullstrup, 1972). However, plants that carry T cytoplasm are highly susceptible to two fungal strains, Mycosphaerella zeae-maydis and race T of Cochiliobolus heterostrophus. The latter was directly responsible for the loss of 710 million bushels of corn in the United States during the southern corn leaf blight epidemic in 1970 (Tatum, 1971; Hooker, 1972; Ullstrup, 1972; Wise et al., 1999a). Since the epidemic, extensive research has been conducted on the cms-T system to address the underlying mechanism of male sterility and fungus susceptibility. It has been demonstrated that both male sterility and fungal susceptibility are due to a T-cytoplasm- specific protein, URF13. URF13 is encoded by the T-cytoplasm mitochondrial genome and accumulates in the mitochondria inner membrane, where it increases membrane permeability (reviewed by Pring and Lonsdale, 1989; Levings, 1990,1993; Williams and Levings, 1992; Dewey and Korth, 1994; Wise et al., 1999a). 2 The cms-T system has two unlinked nuclear restorer genes, rfl and rfl. They work in concert to revert the phenotype of T-cytoplasm-induced male sterility; however, they do not diminish the susceptibility of T-cytoplasm maize to the disease-causing fungi (Duvick, 1965; Ullstrup, 1972). The functional Rfl allele is able to alter the accumulation of T-urfl3 transcripts and reduce the accumulation of URF13 by 80% (Forde and Leaver, 1980; Dewey et al., 1986; Dewey et al., 1987; Kennell et al., 1987; Wise et al., 1996). In contrast, the Rfl gene does not affect the accumulation of URF13, although it is necessary for the restoration of T-cytoplasm male sterility (Forde and Leaver, 1980; Dewey et al., 1987). Unlike rfl, most maize inbred lines have a functional Rfl allele (Duvick, 1965; Wise et al., 1999a). Due to its unique behavior, research on the rf2 gene will not only improve the understanding of cms-T related male sterility and its restoration, but also contribute to the knowledge of nuclear and mitochondrial genome interactions as well as anther development in general. Dissertation Organization This dissertation is organized into six chapters. Chapter 1 is a general introduction to the current body of knowledge on the cms-T system and its restorers. Chapters 2,3,4 and 5 are papers that have been published or submitted to scientific journals as a result of the dissertation research. Chapter 2 describes the cloning and sequence analysis of the rfl gene. Hypotheses on the mechanism of rfl function were formed based on the high degree of sequence similarity between RF2 and mammalian mitochondria aldehyde dehydrogenases. This paper is the 3 result of contributions from multiple authors. My contributions include the isolation and sequence analysis of the rJ2 cDNA and the mRNA accumulation survey of the mutant alleles. Chapter 3 describes the genetic and biochemical characterizations of rf2 and its encoded protein, RF2. This study demonstrates that RF2 is indeed a mitochondrial aldehyde dehydrogenase and that its activity is involved in anther development, even in plants that carry normal cytoplasm. The expression and biochemical analyses of RF2 were conducted by Feng Liu in the Schnable laboratory. The genetic characterization of rfl and the analyses of the rf2-R213 allele arose from my dissertation research. Chapter 4 describes experiments testing whether the RF2 protein acts in the fermentation pathway. Three genes coding for the rate-limiting enzyme of this pathway, PDC, were knocked out. Mutants from pdc3 were extensively characterized for their effects on anaerobic tolerance and male fertility. Assistance for sequencing the pdc genes and genetic crossing was received from other authors. Chapter 5 describes the characterization of the Mu suppression, which is associated with some of the rf2 mutant alleles when the Mu activity is low in the genome. All data in this paper except the sequencing of the rf2 genomic clones were directly derived from my dissertation research. Literature Review Maize anther development and male sterility Maize is a monoecious plant. Its male flowers develop in the tassel on the apex of the plant (Kiesselbach, 1999). Each tassel branch has hundreds of spikelets and each spikelet 4 consists of an upper and lower floret. Three anthers develop in each floret, while the pistil is degenerated in the early developmental stage. Although the upper and lower
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