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Chapter 1: Introduction MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Kerry A. Lucas Candidate for the Degree: Doctor of Philosophy __________________________________ Director (Dr. John W. Hawes) _________________________________ Reader (Dr. Mike W. Crowder) _________________________________ Reader (Dr. Gary A. Lorigan) _________________________________ Reader (Dr. Chris A. Makaroff) _________________________________ Graduate School Representative (Dr. Alfredo J. Huerta) ABSTRACT VALINE METABOLISM IN ARABIDOPSIS By Kerry A. Lucas Branched-chain amino acids (BCAAs) are essential amino acids, meaning mammals have the inability to synthesize them de novo and must therefore obtain them through dietary intake. They have been well studied in mammalian systems due to their key role in several metabolic and signaling pathways. Plants, on the other hand, have the ability to synthesize and degrade BCAAs, although the metabolism and importance of these amino acids is less well understood. It is known that mutations in the biosynthetic pathway results in herbicide resistant plants, whereas mutations in the degradation pathway result in an array of responses, from resistance to hormones, to embryo lethality. The largest questions concerning BCAA metabolism in plants involve understanding valine and propionyl-CoA metabolic pathways. It is believed they share a common pathway; however, the localization and actual enzymatic reactions remain controversial. In an effort to piece together this pathway, several techniques were employed. NMR spectroscopic results showed the peroxisomal metabolism of exogenous propionate through a pathway similar to valine degradation in the mitochondria. Reverse genetics using several knockout mutants in the valine degradation pathway, as well as measuring mRNA levels under several stress conditions, helped to resolve much of the ambiguity regarding this pathway. Based on the data presented in this dissertation, we are the first to provide evidence for the metabolism of propionyl-CoA by a modified β-oxidation pathway that utilizes both the mitochondria and the peroxisomes to produce acetyl-CoA. In addition, preliminary data are presented regarding the maintenance of BCAA homeostasis and evidence for the stimulation of protein synthesis. VALINE METABOLISM IN ARABIDOPSIS A DISSERTATION Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry and Biochemistry by Kerry A. Lucas Miami University Oxford, OH 2008 Dissertation Director: Dr. John W. Hawes Table of Contents Chapter 1: Introduction 1 1.1 Branched-chain Amino Acids 1 1.2 BCAA Metabolism in Mammalian Systems 1 1.2.2 BCAAs and Neurotransmitters 3 1.2.3 BCAAs and the Immune System 4 1.2.4 Metabolic Diseases associated with BCAAs 4 1.2.5 Reactive Intermediates 5 1.2.6 Propionyl-CoA Metabolism 5 1.3 BCAA Metabolism in Higher Plants 7 1.3.1 BCAA Synthesis 7 1.3.2 BCAA Degradation 11 1.3.3 Propionyl-CoA Metabolism in Plants 14 1.4 Sections of the Dissertation 16 1.5 References 17 Chapter 2: Peroxisomal Metabolism of Propionic Acid and Isobutyric Acid in A. thaliana 27 2.1 Summary 28 2.2 Introduction 29 2.3 Experimental Procedures 31 2.3.1 Materials 31 2.3.2 Cell Culture and Seedling Preparation and NMR Spectroscopy Procedure 31 2.3.3 End Point Assay for Quantitation of β-Hydroxyisobutyrate 32 2.3.4 Valine, Isobutyrate, and Propionate Growth Response 33 2.4 Results 33 2.4.1 Genes for a Modified Propionate β-Oxidation Pathway in Plants 33 2.4.2 Metabolism of 2-13C-Propionate in A. thaliana Seedlings and Suspension Cell Cultures 37 2.4.3 Effect of chy1 Mutation on Metabolism of Propionate and Isobutyrate 40 2.4.4 Metabolism of 2-13C-Propionate in Different Plants 43 ii 2.4.5 Metabolism of U-13C-Valine in A. thaliana Seedlings 43 2.4.6 Effects of Exogenous Isobutyrate, Propionate, and Valine on Seedling Growth of Wild-type and chy1-3 A. thaliana Seedlings 45 2.5 Discussion 49 2.6 Acknowledgements 52 2.7 References 53 Chapter 3: Functional Diversity of Mitochondrial β-Hydroxyacyl-CoA Hydrolases in A. thaliana 59 3.1 Summary 60 3.2 Introduction 61 3.3 Experimental Procedures 63 3.3.1 Materials 63 3.3.2 Plant Materials and Growth Conditions 63 3.3.3 T-DNA Insertion Mutations 64 3.3.4 Bioinformatic Analysis 64 3.3.5 Recombinant Hydrolases 64 3.3.6 Enzyme Activity 65 3.3.7 Meausring mRNA Levels 65 3.3.8 Complementation 66 3.3.9 Microscopy 66 3.4 Results and Discussion 67 3.4.1 Sequence Identity of Putative Mitochondrial Hydrolases 67 3.4.2 Activity of Recombinant Hydrolases 71 3.4.3 Mitochondrial CHY mRNA Levels of Seedling Growth and the Effects of Sucrose 72 3.4.4 Effects of Metabolites and Oxidative Stress on Mitochondrial CHY mRNA Levels 77 3.4.5 Effects of T-DNA Insertion Mutations 79 3.5 Conclusion 85 3.6 Acknowledgements 86 3.7 References 87 Chapter 4: Evidence for Propionate and Isobutyrate Metabolism involving Mitochondrial Methylmalonate Semialdehyde Dehydrogenase in A. thaliana Mitochondria 92 iii 4.1 Summary 93 4.2 Introduction 94 4.3 Experimental Procedures 97 4.3.1 Plant Materials and Growth Conditions 97 4.3.2 Measuring mRNA Levels 98 4.4 Results 98 4.4.1 AtMMSDH mRNA levels during germination 98 4.4.2 AtMMSDH Knockout Mutant Affects Seed Viability 100 4.4.3 Knockout Mutants Show Sensitivity to Peroxisomal Metabolites 104 4.5 Discussion 107 4.6 Acknowledgements 111 4.7 References 112 Chapter 5: The Biosynthesis of Leucine and Its Effects on Protein Synthesis in A. thaliana 118 5.1 Summary 119 5.2 Introduction 120 5.3.1 Plant Materials and Growth Conditions 124 5.3.2 NMR Sample Preparation and Procedure 124 5.3.3 Measuring mRNA Levels 125 5.4 Results and Discussion 125 5.4.1 The Effects of Sucrose on Leucine Biosynthesis 125 5.4.2 Stimulation of Protein Synthesis by Leucine 126 5.5 Conclusion 129 5.6 Acknowledgements 131 5.7 References 132 Chapter 6: Conclusion 137 6.1 Understanding Branched-Chain Amino Acids 137 6.2 BCAA Metabolism 137 6.3 Valine Degradation 138 6.4 Valine Metabolism in the Chloroplast 140 6.5 Leucine and Protein Synthesis 142 6.6 Future Directions 142 iv 6.6.1 Model for Valine and Propionyl-CoA Metabolism in Plants 142 6.6.2 Stimulation of Protein Synthesis 143 6.7 References 145 v List of Tables Table 1.1 Genes coding for enzymes of valine degradation. 12 Table 2.1 Genes coding for putative enzymes for catabolism of valine and propionyl-CoA in A. thaliana. 35 Table 3.1 Specific Activity of recombinant hydrolases. 73 Table 4.1 Mitochondrial localization prediction scores and potential cleavage sites for AtMMSDH. 109 vi List of Figures Figure 1.1 Branched-chain amino acid degradation pathways. 2 Figure 1.2 Structures of reactive intermediates in valine catabolism. 6 Figure 1.3 Propionyl-CoA metabolic pathways for mammals, bacteria, and plants. 8 Figure 1.4 BCAA biosynthetic pathway. 10 Figure 1.5 Possible routes for propionyl-CoA metabolism in plants. 15 Figure 2.1 Proposed pathways for metabolism of valine and propionyl-CoA in mitochondria and peroxisomes in plants. 34 Figure 2.2 13C-NMR analysis of metabolites produced from 2-13C-propionate in A. thaliana. 38 Figure 2.3 Time course of β-hydroxypropionate production. 39 Figure 2.4 HMQC analysis of standard β-hydroxypropionate and A. thaliana seedlings with 2- 13C-propionate. 41 Figure 2.5 β-Hydroxyisobutyrate accumulation in wild-type and chy1-3 A. thaliana seedlings. 42 Figure 2.6 Production of β-hydroxypropionate in various plants. 44 Figure 2.7 Pathway for the metabolism of valine to leucine. 46 Figure 2.8 13C NMR analysis of A. thaliana seedlings with U-13C-valine. 47 Figure 2.9 Effect of exogenous valine, isobutyrate and propionate on seedling growth. 48 Figure 3.1 Valine catabolic pathway. 62 Figure 3.2 Sequence comparison of A. thaliana CHY proteins with human HIBYL-CoA hydrolase. 68 Figure 3.3 Mitochondrial CHY mRNA levels during seedling growth. 74 Figure 3.4 Effects of sucrose on mitochondrial CHY mRNA levels. 76 Figure 3.5 Effect of metabolites and oxidative stress on mitochondrial CHY mRNA levels. 78 Figure 3.6 Location of T-DNA insertion sites for each mitochondrial CHY gene. 80 Figure 3.7 Light microscopy of A. thaliana siliques. 81 Figure 3.8 Confocal microscopy of chy4 seeds from a single heterozygous silique. 82 Figure 3.9 Valine resistance of chy5 mutant seedlings. 83 Figure 4.1 Valine degradation pathway in mitochondria and peroxisomes. 96 Figure 4.2 mRNA levels of MMSDH during germination and seedling establishment. 99 Figure 4.3 MMSDH mRNA levels in the presence and absence of sucrose. 101 vii Figure 4.4 MMSDH mRNA levels with GA and 2,4-D. 102 Figure 4.5 Location of T-DNA insertion and confirmation of homozygous knockout. 103 Figure 4.6 Growth development of mmsdh knockout seedlings. 105 Figure 4.7 mmsdh Knockout seedlings grown with valine, isobutyrate, and propionate. 106 Figure 4.8 Sequence alignment of MMSDH from A. thaliana, rice, human, and rat. 108 Figure 5.1 BCAA biosynthesis pathway. 121 Figure 5.2 TOR signaling pathway. 123 Figure 5.3 13C-NMR studies. 127 Figure 5.4 mRNA levels during leucine biosynthesis in response to sucrose. 128 Figure 5.5 GFP response to leucine. 130 Figure 6.1 Final model for valine and propionyl-CoA metabolism in plants. 141 viii Acknowledgements The success of this dissertation would not have been possible without the love and support of several people throughout the last five years.
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