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The Role of 3-Deoxy-D-Arabino-Heptulosonate 7- Phosphate Synthase 1 in Arabidopsis Thaliana Metabolism

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The Role of 3-Deoxy-D-Arabino-Heptulosonate 7- Phosphate Synthase 1 in Arabidopsis Thaliana Metabolism THE ROLE OF 3-DEOXY-D-ARABINO-HEPTULOSONATE 7- PHOSPHATE SYNTHASE 1 IN ARABIDOPSIS THALIANA METABOLISM by Jimmy Poulin A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Cell and System Biology University of Toronto © Copyright by Jimmy Poulin, 2011 The role of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase 1 in Arabidopsis thaliana metabolism Jimmy Poulin Master of Science Cell and System Biology University of Toronto 2011 Abstract The enzyme 3-deoxy-D-arabino-heptulusonate 7-phosphate synthase (DHS) catalyzes the first step of the shikimate pathway. In bacteria, the regulation of the pathway is mediated by allosteric inhibition of DHS by the aromatic amino acids tyrosine, phenylalanine and tryptophan. The regulation of the pathway in plants remains elusive but the aromatic amino acids are involved as suggested by the hypersensitivity of dhs1 knockout mutant to tyrosine. In this study the effects of the dhs1 mutation on endogenous levels of aromatic amino acids and of downstream metabolites are explored. HPLC analysis is used to measure levels of tyrosine and phenylalanine and 5-methyltryptophan sensitivity is used to probe levels of tryptophan. Additionally, the auxin content of whole seedlings was quantified by LC/MS and its local levels at the root apex are visualized with the DR5::GUS reporter system. ii Acknowledgements I could not have completed my master’s degree without the help of many resourceful individuals. First and foremost I would like to thank Dr. Dinesh Christendat for his supervision and guidance. I am also grateful for guidance received from Dr. Darrell Desveaux, Dr. Nicholas Provart, Dr. Daphne Goring and Dr. Geoff Fucile. I also received valuable advice and support from other members of the Christendat lab including James Peek, Dr. Christel Garcia and Kate Penney. iii Table of Contents Abstract......................................................................................................................................................ii Acknowledgements................................................................................................................................iii Table of Contents ...................................................................................................................................iv List of Tables ...........................................................................................................................................vi List of Figures..........................................................................................................................................vi List of Abbreviations ...........................................................................................................................viii Introduction.............................................................................................................................................. 1 Shikimate Pathway ............................................................................................................................... 1 DAHP Synthase .................................................................................................................................... 6 Regulation of AroAI DHS ..................................................................................................................... 8 Regulation of AroAII DHS.................................................................................................................... 9 Regulation of Arabidopsis thaliana DHS ......................................................................................... 10 Aromatic Amino Acid Biosynthesis................................................................................................... 13 Auxin Biosynthesis.............................................................................................................................. 16 Thesis Objective.................................................................................................................................. 18 Materials and Methods ........................................................................................................................ 19 Materials............................................................................................................................................... 19 Phylogenetic Analysis ........................................................................................................................ 19 Plant Growth Conditions and Root Length Assays........................................................................ 20 Genomic DNA Extraction................................................................................................................... 20 PCR Genotyping of T-DNA Lines ..................................................................................................... 21 Aromatic Amino Acid Treatment....................................................................................................... 22 Metabolite Extraction and Derivatization ......................................................................................... 23 HPLC Analysis..................................................................................................................................... 23 Auxin Quantification by LC-MS......................................................................................................... 24 GUS Staining....................................................................................................................................... 25 Microarray Data Analysis................................................................................................................... 26 Results..................................................................................................................................................... 28 AroAII-type DHS Gene Duplicate Retention in Higher Plants ...................................................... 28 iv Conservation of 3D Structure in MtDHS and AtDHS..................................................................... 31 Verification of Col-0 and dhs1 Genotypes....................................................................................... 33 Changes in Intracellular Levels of Aromatic Amino Acids in Atdhs1........................................... 34 Sensitivity of dhs1 to 5-Methyltryptophan Supplementation......................................................... 39 Auxin Levels in Col-0 and dhs1 Whole Seedlings ......................................................................... 45 Auxin Levels in Col-0 and dhs1 Seedling Roots ............................................................................ 46 Tyrosine Treatment of dhs1 Elicit Stress Responses.................................................................... 49 Discussion.............................................................................................................................................. 54 Diversification in the Regulatory Mechanisms of Bacterial and Plant AroAII Enzymes............ 55 Conservation of Structural Domains between Arabidopsis and M.tuberculosis DHS Enzymes ............................................................................................................................................................... 56 Levels of Aromatic Amino Acids are Disrupted in dhs1 Seedlings.............................................. 57 Changes in Auxin................................................................................................................................ 61 Tyrosine treatment of dhs1 knockout turns on Arabidopsis stress response and causes significant transcriptional changes in tryptophan and auxin biosynthetic genes ....................... 64 Proposed Model and Future Directions ........................................................................................... 65 Conclusion ............................................................................................................................................. 70 References.............................................................................................................................................. 71 v List of Tables Table 1 – Primers used for genotyping ........................................................................................22 Table 2 – PCR program used for genotyping...............................................................................22 Table 3 – Elution profile for HPLC separation of amino acid dansyl derivatives .......................24 Table 4 – Number of differentially expressed genes in dhs1 following treatment with tyrosine (Y), phenylalanine (F) or tryptophan (W) and between dhs1 and its non-transgenic sibling (NTS) ......................................................................................................................................................50 List of Figures Figure 1 – The 7 enzymatic steps of the shikimate pathway .........................................................4 Figure 2 – Shikimate pathway-derived secondary metabolites......................................................5 Figure 3 – Condensation reaction catalyzed by DHS ....................................................................7 Figure 4 – Organ specific map of DHS expression in Arabidopsis thaliana...............................12 Figure 5 – Hypersensitivity of Arabidopsis dhs1
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