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Transport in Plants and in Root Nodules

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Transport in Plants and in Root Nodules DEVELOPMENT OF SPECIALIZED TRANSPORT PATHWAYS IN PISUM SATIVUM ROOT NODULES by Cherish A. Warner A dissertation submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biological Sciences Fall 2018 © 2018 Cherish A. Warner All Rights Reserved DEVELOPMENT OF SPECIALIZED TRANSPORT PATHWAYS IN PISUM SATIVUM ROOT NODULES by Cherish A. Warner Approved: __________________________________________________________ E. Fidelma Boyd, Ph.D. Chair of the Department of Biological Sciences Approved: __________________________________________________________ John Pelesko, Ph.D. Interim Dean of the College of Arts and Sciences Approved: __________________________________________________________ Douglas J. Doren, Ph.D. Interim Vice Provost for Graduate and Professional Education I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ D. Janine Sherrier, Ph.D. Professor in charge of dissertation I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Jeffrey L. Caplan, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Diane Herson, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Jia Song, Ph.D. Member of dissertation committee ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Janine Sherrier for her advising, encouragement and invaluable contribution on my thesis, my projects, and my research in both general and specific terms. I thank all the members of Delaware Biotechnology Institute for creating such an inclusive and wonderful supportive community. I thank the people of the Bioimaging Center of Delaware Biotechnology Institute, especially Dr. Jeffrey Caplan and Shannon Modla for providing outstanding training and assistance on the imaging instruments and techniques. I thank all my friends and family for their unwavering support. In others eyes I was the least likely to come this far but the most stubborn of us to do it. I want to dedicate this piece of writing to Ta and Gran (Norman and Loretta Gilbreath) whose work ethic and life story was inspirational in times of hardship. To you Pete, my best friend, I thank you for everything and love you with all my heart. I thank my Dad Doug, for always listening. I would like to thank my Mom Kathy, who raised three wonderful girls, myself and my sisters, Daisha and January. A special thanks to my second Dad, Steve Belcher who cared for me like I was his own. I love you all and couldn’t have done this without you. To my nephews Luis and Scott, and nieces August, Vivian and Lillian, I hope your dreams come true and I believe in you. I thank all my current and previous labmates for giving me friendship and support, both professionally and personally. I want to thank all those not named above. Your support and kindness made this possible. I’m tremendously grateful for your guidance and counsel during the course of my graduate studies. iv TABLE OF CONTENTS LIST OF TABLES ......................................................................................................... ix LIST OF FIGURES ........................................................................................................ x ABSTRACT ................................................................................................................. xii Chapter 1 INTRODUCTION .............................................................................................. 1 1.1 Statement of Research Question ................................................................ 1 1.2 The Importance of Legumes ...................................................................... 3 1.2.1 Feeding Mankind; Chemical versus biological nitrogen fixation .. 4 1.3 An overview of the L-R Symbiosis and Organ Formation ........................ 9 1.3.1 In the Beginning: Evolution of L-R Symbiosis ............................. 9 1.3.2 “Sweet talking”; Initiation of Symbiosis ..................................... 10 1.3.3 Organogenesis .............................................................................. 11 1.3.4 Maintenance of a Functional Root Nodule .................................. 13 1.4 Transport Pathways in Plants ................................................................... 14 1.4.1 Transport and Structure of Vascular System ............................... 14 1.4.2 Two types of ‘cell to cell’ transport in Plants and in Root Nodules ........................................................................................ 16 1.4.3 The Plant Wall Barrier ................................................................. 19 1.4.4 Formation and Structure of PD .................................................... 19 1.4.5 Mechanisms of Function .............................................................. 20 1.5 Transport and Metabolism To and From Infected Cells .......................... 22 1.6 Supporting Literature and Research Approach ........................................ 23 2 MATERIALS AND METHODS ...................................................................... 26 2.1 Materials .................................................................................................. 26 2.1.1 Bacterial Strains ........................................................................... 26 v 2.1.2 Plant Species ................................................................................ 26 2.1.3 Reagents and Enzymes ................................................................ 26 2.1.4 Microscopy Stains and Dyes ........................................................ 27 2.1.5 Standard Solutions ....................................................................... 28 2.2 Methods.................................................................................................... 29 2.2.1 Bacterial Culture .......................................................................... 29 2.2.2 Pisum sativum Plant Growth ........................................................ 30 2.2.2.1 Seed Germination.......................................................... 30 2.2.2.2 Pisum sativum Plant Growth ......................................... 30 2.2.3 Medicago truncatula Plant Growth ............................................. 32 2.2.3.1 Seed Germination.......................................................... 32 2.2.3.2 Plant Growth ................................................................. 33 2.2.3.3 Rhizobial Inoculation of Caisson .................................. 33 2.2.4 Harvest of Root Nodules .............................................................. 34 2.2.5 Optical Clearing of Nodule Tissue .............................................. 34 2.2.5.1 Sample Preparation and Infiltration with Clearing Reagent ......................................................................... 34 2.2.5.2 Data Acquisition for Confocal Microscopy .................. 35 2.2.5.3 Data Processing for Three-Dimensional Reconstruction and Segmentation................................. 36 2.2.6 Transmission Electron Microscopy- Plasmodesmata Quantification .............................................................................. 36 2.2.6.1 Sample Preparation ....................................................... 36 2.2.6.2 Data Acquisition ........................................................... 37 2.2.6.3 Plasmodesmata Scoring for Quantification .................. 38 2.2.7 Transmission Electron Microscopy- Plasmodesmata Tomography ................................................................................. 38 2.2.7.1 Sample Preparation ....................................................... 39 2.2.7.2 Data Acquisition ........................................................... 40 2.2.7.3 Tomogram Reconstruction and 3D Modeling .............. 40 2.2.8 Mapping of Uninfected Cell Arrays and Vasculature Tissue ...... 41 2.2.9 Dye Loading Technique ............................................................... 41 vi 3 SYMPLASMIC FLOW IN CENTRAL NODULE TISSUE............................ 43 3.1 Background .............................................................................................. 43 3.1.1 Nitrogen transport in determinate and indeterminate nodules ..... 46 3.1.2 Sucrose transport and tissue organization in determinate and indeterminate nodules .................................................................. 47 3.1.3 Other aspects of transport involved in nodulation ....................... 47 3.1.4 The role of PD in symplasmic transport ...................................... 48 3.1.5 Transport and Tissue Organization in the Indeterminate Nodule 50 3.2 Results .....................................................................................................
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