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Genetic and Molecular Characterization of Maize Starch Debranching Enzymes Jason Robert Dinges Iowa State University

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Genetic and Molecular Characterization of Maize Starch Debranching Enzymes Jason Robert Dinges Iowa State University Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2003 Genetic and molecular characterization of maize starch debranching enzymes Jason Robert Dinges Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biochemistry Commons, Genetics Commons, and the Plant Sciences Commons Recommended Citation Dinges, Jason Robert, "Genetic and molecular characterization of maize starch debranching enzymes " (2003). Retrospective Theses and Dissertations. 576. https://lib.dr.iastate.edu/rtd/576 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]. Genetic and molecular characterization of maize starch debranching enzymes by Jason Robert Dinges 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: Alan M. Myers (Co-Major Professor) Martha G. James (Co-Major Professor) Philip W. Becraft M. Paul Scott Patrick S. Schnable Iowa State University Ames, Iowa 2003 UMI Number: 3085901 UMI UMI Microform 3085901 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 ii Graduate College Iowa State University This is to certify that the doctoral dissertation of Jason Robert Dinges has met the dissertation requirements of Iowa State University Signature was redacted for privacy. Co-maJor Professor Signature was redacted for privacy. Co-mar Profe Signature was redacted for privacy. F the Major Program iii TABLE OF CONTENTS LIST OF FIGURES v LIST OF TABLES vii ABBREVIATIONS viii ABSTRACT x CHAPTER 1. GENERAL INTRODUCTION 1 Literature Review 2 Experimental Approach 50 Dissertation Organization 51 References 52 CHAPTER 2. MOLECULAR STRUCTURE OF THREE MUTATIONS AT THE MAIZE sul LOCUS AND THEIR ALLELE-SPECIFIC PHENOTYPIC EFFECTS 66 Abstract 66 Introduction 67 Results 71 Discussion 86 Materials and Methods 92 Acknowledgements 97 References 98 CHAPTER 3. MUTATIONAL ANALYSIS OF THE PULLULANASE-TYPE STARCH DEBRANCHING ENZYME OF MAIZE INDICATES MULTIPLE FUNCTIONS IN STARCH METABOLISM 102 Abstract 102 Introduction 103 Results 106 Discussion 128 Materials and Methods 135 Acknowledgements 142 References 142 CHAPTER 4. GENETIC ANALYSIS INDICATES MAIZE PULLULANASE- AND ISOAMYLASE-TYPE STARCH DEBRANCHING ENZYMES HAVE PARTIALLY OVERLAPPING FUNCTIONS IN STARCH METABOLISM 146 Abstract 146 Introduction 147 Results and Discussion 150 Materials and Methods 159 iv References 160 CHAPTER 5. GENE EXPRESSION ANALYSIS OF MAIZE STARCH 163 DEBRANCHING ENZYMES BY REAL-TIME QUANTITATIVE PCR Abstract 163 Introduction 164 Results and Discussion 167 Conclusion 177 Materials and Methods 179 Acknowledgements 183 References 183 CHAPTER 6. SUMMARY AND GENERAL CONCLUSIONS 186 General Discussion 186 Recommendations for Future Research 191 References 193 APPENDIX. REVERSE GENETICS OF ZmIso2 and ZmIso3 194 ACKNOWLEDGEMENTS 197 V LIST OF FIGURES Figure 1.1 Hierarchical levels of starch structure 7 Figure 1.2 Sucrose metabolism in endosperm tissue 11 Figure 1.3 Reactions catalyzed by starch metabolizing enzymes 17 Figure 1.4 Pathway of starch degradation 29 Figure 1.5 Models of DBE function in starch synthesis 47 Figure 2.1 Kernel phenotypes of sul- mutants 72 Figure 2.2 RNA gel blot analysis of sul- alleles 73 Figure 2.3 PCR and RT-PCR analyses of sul-st 76 Figure 2.4 Nucleotide sequence analysis of the sul-st allele 79 Figure 2.5 HPAEC-PAD analysis of chain length distributions 83 Figure 2.6 Native PAGE/activity gels of starch modifying activities 85 Figure 3.1 Molecular structure of zpul- mutant alleles 107 Figure 3.2 zpul-204 is a null allele 109 Figure 3.3 Leaf starch content during the diurnal cycle 111 Figure 3.4 Sepharose CL2B separation of leaf starch 114 Figure 3.5 Chain length distributions of amylopectin 116 Figure 3.6 Analysis of WSP from zpul-204 endosperm 117 Figure 3.7 Germination analysis of zpul-204 kernels 119 Figure 3.8 Activity gel analysis of starch modifying enzymes 122 Figure 3.9 Synthetic phenotypes of DBE double mutants 124 Figure 4.1 Crossing scheme used to generate sul' zpul' double mutants 151 Figure 4.2 Kernel phenotypes of sul' zpul' double mutants 152 Figure 5.1 Alignment of maize isoamylase-type DBEs 169 Figure 5.2 Phylogenetic relationships of plant and bacterial DBEs 171 Figure 5.3 Relative quantitation of DBE gene expression 174 Figure A.1 Molecular structure of ZmIso2 and ZmIso3 mutant alleles 196 vi LIST OF TABLES Table 2.1 Carbohydrate analysis of W64A and sul- mutant kernels 82 Table 3.1 Carbohydrate content of destarched leaves 113 Table 3.2 Endosperm carbohydrate content 118 Table 3.3 Endosperm carbohydrate content in DBE double mutants 126 Table 3.4 Starch granule characteristics of DBE mutants 127 Table 4.1 Carbohydrate content of individual DBE mutant kernels 154 Figure 5.1 Primer sequences for real-time PCR 181 vii ABBREVIATIONS ADP Adenosine 5'-diphosphate ATP Adenosine 5'-triphosphate ADP-Glc Adenosine diphosphoglucose AGPase ADP-glucose pyrophosphorylase bp Base pair C Carbon DAP Days after pollination DBE Debranching enzyme DNA Deoxyribonucleic acid DTT Dithiothreitol DP Degree of polymerization DW Dry weight EDTA Ethylenediaminetetraacetic acid FACE Fluorophore associated capillary electrophoresis Fru-6-P Fructose-6-phosphate FBPase Fructose-1,6-bisphosphatase Fru Fructose FW Fresh weight Glc-l-P Glucose-1-phosphate Glc-6-P Glucose-6-phosphate GBSS Granule bound starch synthase Glc Glucose h Hours HPAEC High performance anion exchange chromatography kD Kilodalton min Minutes MOPS 3 -(N-morpholino)propanesulphonic acid viii MOS Maltooligosaccharides ran Nanometer nt Nucleotide PCR Polymerase chain reaction 3-PGA 3-phosophoglycerate PG Phytoglycogen Pi Inorganic phosphate PPi Inorganic pyrophosphate Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase RNA Ribonucleic acid SBE Starch branching enzyme ± SE ± Standard error of the mean SEM Scanning electron microscopy SDS Sodium dodecyl sulphate SS Soluble starch synthase Sue Sucrose Susy Sucrose synthase TEM Transmission electron microscopy TIR Terminal inverted repeat TPT Triose phosphate transporter Tris 3-N-tris(hyrdroxymethyl)aminomethane TUSC Trait utility system for corn U Units of enzyme activity UDP-Glc Uridine diphosphoglucose UGPase UDP-glucose pyrophosphorylase UTR Untranslated region v/v Volume by volume w/v Weight by volume w/w Weight by weight Starch is the primary carbohydrate reserve of plants, a significant source of food in the human diet, and an important industrial raw material. The arrangement of linear chains and branch linkages within amylopectin is responsible for the highly conserved architecture of the starch granule. This structure allows for efficient packaging of glucose and results in the unique functional properties of starch. It is known that starch synthases and branching enzymes catalyze a(l—»4) chain elongation and introduction of a(l—>6) branch linkages, respectively, but studies in several species suggests that starch debranching enzymes (DBE) are also involved in the process of starch synthesis. The overall goal of this research is to determine the role played in maize starch metabolism by each individual DBE. DBEs are broadly classified as either isoamylase- or pullulanase-type based upon their substrate specificity and sequence similarity to defined bacterial proteins. To determine the function(s) of the isoamylase-type DBE, an allelic series of mutations in the gene Sul was characterized. The molecular defects of the sul- alleles were associated with accumulation of phytoglycogen at the expense of granular starch. The phenotypic severity was also correlated to an alteration in the fine structure of the residual amylopectin. Thus, the SU1 isoamylase-type DBE functions directly in starch synthesis by removing branches from an amylopectin precursor. cDNAs coding for two other isoamylase-type DBE genes, ZmIso2 and ZmIso3, were cloned and characterized with respect to mRNA transcript accumulation. This analysis indicated metabolic specialization for some DBE isoforms either during starch catabolism or anabolism. To investigate the function(s) of the pullulanase-type DBE, reverse genetics was used to identify a transposable element insertion in the maize Zpul gene. X Characterization of this mutation demonstrated that pullulanase-type DBE is important for degradation of both leaf and endosperm starch. ZPUl also acts during starch synthesis significantly reducing phytoglycogen accumulation in some genetic backgrounds. This suggests that the biosynthetic function of ZPUl partially overlaps with the biosynthetic function of SU1. Together, these data indicate that isoamylase- and pullulanase-type DBEs are each involved in aspects of both starch synthesis and degradation. 1 CHAPTER 1. GENERAL INTRODUCTION Starch is the major carbohydrate storage form in higher plants. It serves a central role in providing energy in the absence of photosynthesis. Starch metabolism in maize, Arabidopsis, and other plants involves
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