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APPLICATIONS of RADIOTRACER in PLANT BIOLOGY a Dissertation Presented to the Faculty Of APPLICATIONS OF RADIOTRACER IN PLANT BIOLOGY _______________________________________ A Dissertation Presented to The Faculty of the Graduate School University of Missouri-Columbia _______________________________________________________ In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy _____________________________________________________ by LIHUI SONG Dr. Silvia Jurisson and Dr. Gary Stacey, Dissertation Supervisors December 2013 The undersigned, appointed by the dean of the Graduate School, have examined the dissertation entitled APPLICATIONS OF RADIOTRACER IN PLANT BIOLOGY presented by Lihui Song, a candidate for the degree of [doctor of philosophy of Chemistry], and hereby certify that, in their opinion, it is worthy of acceptance. Professor Silvia Jurisson Professor Gary Stacey Professor David Robertson Professor Timothy Glass Professor Richard Ferreri To My loves, I would like to dedicate this work to my dear family who have been loving me and supporting me unconditionally, even though I have not spent any New Year eves with them during the last five years. I also would like to dedicate this work to my loved fiancé, Noah Marchal, for his understanding, support, patience and help with revision during the dissertation writing process. ACKNOWLEDGEMENTS There are so many people who helped me create new neuron connections in my brain throughout my graduate career and all of them deserve more than just the ink on one page of my dissertation. First, I would like to thank my advisor, Dr. Silvia Jurisson, and my co-advisors, Dr. Gary Stacey and Dr. Richard Ferreri, for their guidance, patience, encouragement and revisions on my dissertation. Gary led me to the door of this research opportunity, Silvia welcomed me in, and Rich taught me hand by hand how to conduct radiotracer studies on plants. Without them, I would not have gained the knowledge and experience I had today and figured out what I love to do for my future career. I also would like to thank my dissertation committee members, Dr. Timothy Glass and Dr. David Robertson for their continuous support and their help with the revision of my dissertation. Another person I would like to thank here is Minviluz (Bing) G. Stacey. Although she is not in my dissertation committee, she taught me how to conduct plant biological studies and other important molecular biology techniques, especially helped me with the study mentioned in Chapter 2. In addition, I would like to thank Dr. Jurisson’s Group, Dr. Stacey’s Group and Dr. Ferreri’s Group. Some of them helped me a lot with experimental design and actual experiments, and some of them are my spiritual support. I have enjoyed every one of their companies during my graduate career. There are also facilities I would like to acknowledge for conducting analyses for my work. They are MU Research Reactor (David J. Robertson, James Guthrie and Gang Li) and Environmental Molecular Sciences Laboratory/ Pacific Northwest National ii Laboratory (EMSL/PNNL; Lizabeth Alexander) for the ICP-MS analyses; MU Molecular Cytology Core (Yan Liang) for providing the microscopic imaging services. At last, I would like to thank MU Chemistry Department and Department of Energy Biological and Environmental Research (DOE-BER) program for the financial supports. iii Table of Contents Acknowledgments ...................................................................................................................... ii List of Tables ........................................................................................................................... viii List of Figures ............................................................................................................................. x List of Schemes ........................................................................................................................ xiii List of Equations ..................................................................................................................... xiv Abstract ...................................................................................................................................... xv Chapter 1: Introduction 1.1 General Concepts of Radiotracer ...................................................................................... 1 1.2 Techniques used for Radiation Detection ....................................................................... 3 1.3 Advantages and Disadvantages of Radiotracer Studies ................................................ 6 1.4 Underlying Assumptions in Design of Radiotracer Experiments ............................... 6 1.4.1 Basic Assumptions ...................................................................................................... 6 1.4.2 Evaluation of the Feasibility of Radiotracer Experiments ..................................... 8 1.5 History of Plant Biological Studies using Radioactive Isotopes ................................. 9 1.6 Radiotracer Studies in This Dissertation ...................................................................... 10 References ............................................................................................................................... 12 Chapter 2: Accumulation of Heavy Metals by the Arabidopsis opt3-2 Mutant 2.1 Isotope Summary ............................................................................................................. 14 2.2 Background ....................................................................................................................... 14 2.3 Materials and Methods .................................................................................................... 16 2.3.1 Materials .................................................................................................................... 16 2.3.2 Plant Growth Conditions ......................................................................................... 16 iv 2.3.3 Determination of Chlorophyll Content .................................................................. 18 2.3.4 ICP-MS Analysis ...................................................................................................... 18 2.3.5 Radioactive 203Pb2+ Uptake Assay and Imaging .................................................. 19 2.3.6 Yeast Transformation and Growth Assays .......................................................... 19 2.3.7 Statistic Analysis ...................................................................................................... 20 2.4 Results ............................................................................................................................... 20 2.4.1 Toxic Effects of Heavy Metals on Plant Growth ................................................. 20 2.4.2 Heavy Metal Quantification in Plant Tissues by ICP-MS .................................. 21 2.4.3 Radioactive 203Pb2+ Uptake Assay ......................................................................... 22 2.4.4 IRT1 does not Mediate Pb or Hg Uptake .............................................................. 22 2.5 Discussion ......................................................................................................................... 23 References ............................................................................................................................... 33 Chapter 3: Dynamics of Sugar Metabolism and Re-location in Arabidopsis Starch 11 Mutants Using CO2 3.1 Isotope Summary ............................................................................................................. 36 3.2 Background ....................................................................................................................... 36 3.3 Materials and Methods .................................................................................................... 41 3.3.1 Materials .................................................................................................................... 41 3.3.2 Plant Growth Conditions ......................................................................................... 41 3.3.3 Starch Staining Assay .............................................................................................. 42 11 3.3.4 CO2 Production, Pulsing and Incubation Apparati ........................................... 42 3.3.5 Tissue Extraction and Soluble Sugar Analysis ..................................................... 43 3.3.6 Carbon Allocation .................................................................................................... 45 3.3.7 Phosphor Imaging .................................................................................................... 46 3.3.8 Root Morphology ..................................................................................................... 46 v 3.3.9 Root Elongation and Gravitropic Response .......................................................... 46 3.3.10 Statistical Analysis ................................................................................................. 46 3.4 Results ............................................................................................................................... 47 3.4.1 Plant Growth ............................................................................................................. 47 3.4.2 Plant Physiology ....................................................................................................... 47 3.4.3 Carbon Metabolism .................................................................................................
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