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Microtubule Function in Meristematic Cells and Chloroplast Avoidance Movement in Arabidopsis Thaliana

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Microtubule Function in Meristematic Cells and Chloroplast Avoidance Movement in Arabidopsis Thaliana MICROTUBULE FUNCTION IN MERISTEMATIC CELLS AND CHLOROPLAST AVOIDANCE MOVEMENT IN ARABIDOPSIS THALIANA A Thesis Submitted to the College of Graduate and Postdoctoral Studies In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In the Department of Biology University of Saskatchewan Saskatoon By YEN PHI LE Copyright Yen Phi Le, April 2019. All rights reserved. PERMISSION TO USE In presenting this thesis/dissertation in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis/dissertation in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis/dissertation work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis/dissertation or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis/dissertation. DISCLAIMER Reference in this thesis/dissertation to any specific commercial products, process, or service by trade name, trademark, manufacturer, or otherwise, does not constitute or imply its endorsement, recommendation, or favoring by the University of Saskatchewan. The views and opinions of the author expressed herein do not state or reflect those of the University of Saskatchewan, and shall not be used for advertising or product endorsement purposes. Requests for permission to copy or to make other uses of materials in this thesis/dissertation in whole or part should be addressed to: Head of the Department of Biology University of Saskatchewan 112 Science Place Saskatoon, Saskatchewan, S7N 5E2 Canada OR Dean College of Graduate and Postdoctoral Studies University of Saskatchewan 116 Thorvaldson Building, 110 Science Place Saskatoon, Saskatchewan, S7N 5C9 Canada i ABTRACT The microtubule (MT) cytoskeleton plays multiple functions in plant morphogenesis and development including cell division, expansion, hormonal signaling, and response to biotic and abiotic stress. During these diverse processes, the primary role of MTs is to modulate cell wall structure and to direct intracellular movement of organelles that mainly depend on MT organization. In meristematic cells, MT organization system is continuing by the MT organization at cell cortex called cortical MT (CMT) array and in cytoplasm called endoplasmic MT (EMT). The mechanism underlying microtubule organization in plants has been primarily studied using the CMTs array, which lines the plasma membrane and configures the cell wall properties that define plant cell shape. Much less is known about EMTs, which radiate from the nuclear envelope and attach end-on to the plasma membrane, and are abundant in meristematic cells. By studying two mutants having meristematic defects in root tip clasp-1 and knt1-2, I found that EMTs appeared to maintain cells in a meristematic state by providing a structural scaffold that stabilized the cytoplasm to counteract actomyosin-based cytoplasmic streaming forces, thereby preventing premature establishment of a central vacuole and rapid cell elongation. Besides actin filaments, the function of MT orientation in intracellular movement of organelles is necessary for many cellular functions. In plants which generate their own food supply and energy from light, water and carbon dioxide by photosynthesis, the photorelocation movement of chloroplast (CP) to optimize the photosynthesis efficiency is essential. CP movement is believed to be driven mainly by the actin cytoskeleton, but the participation of MTs is currently under debate. By using high-resolution live-cell imaging of MTs during the CP avoidance response, I found evidence that the CMT orientation is important for the CP movement. CP light avoidance movement was significantly affected by MT inhibitors, and changed corresponding to different CMT orientations. The movement of CPs was guided by minus-ends of treadmilling CMTs to travel from the periclinal wall to the anticlinal wall in long distance. It suggests that CMTs might function directly as tracks for stable and long-distance movement of CPs or indirectly by interacting with actin in CP movement. ii ACKNOWLEDGMENT The first person I would like to thank is my supervisor, Dr. Chris Ambrose. He taught me how to be independent in thinking and experimental designing as well as successfully turned me into a microscope nerd like him. My committee members, Dr. Carlos Carvalho, Dr. Hong Wang, Dr. Yangdou Wei, and Dr. Ken Wilson are so awesome. Their kindness makes my meetings with them always delightful and exciting. Their opinions and questions contributed a variety of ideas to my research. I would like to thank microscopy technicians, Dr. Guosheng Liu and Dr. Eiko Kawamura. Their knowledge of microscopes and their patience assisting me has helped me achieve my microscopy skills. Finally, yet importantly, I would like to thank my lab mates, Zhihai Chi, Liyong Chang, and Devon Ireland who always let me play music in the lab and never complained about that when I needed short breaks. In addition, I would like to thank the staff and faculty in the Department of Biology who have always been there to support me when I needed help. An NSERC Discovery Grant and the University of Saskatchewan provided funding for this project. iii TABLE OF CONTENTS PERMISSION TO USE.............................................................................................................. i ABTRACT................................................................................................................................. ii ACKNOWLEDGMENT ..........................................................................................................iii TABLE OF CONTENTS ......................................................................................................... iv LIST OF TABLES..................................................................................................................viii LIST OF FIGURES .................................................................................................................. ix LIST OF ABBREVIATIONS .................................................................................................. xi CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW .............................................. 1 1.0. Microtubule structure and dynamics .................................................................................. 1 1.0.1. Microtubule structure .................................................................................................. 1 1.0.2. MT dynamic instability ............................................................................................... 3 1.1. Regulators of MT dynamics and array orientation ............................................................. 6 1.2.1. MAPs ........................................................................................................................... 6 1.2.2. MAPs controlling MT nucleation and severing .......................................................... 9 1.2. MT organization and function in plant cells ..................................................................... 11 1.2.1. Cortical microtubule behaviors in plant cells ............................................................ 12 1.2.2. Mechanisms of CMT reorientation in response to external cues .............................. 14 1.2.3. CMT and EMT in root tips ........................................................................................ 15 CHAPTER 2. MATERIALS AND METHODS ......................................................................... 18 2.0. Plant materials and growth conditions.............................................................................. 18 2.1. Molecular biology and construct design ........................................................................... 18 2.2. Light treatment ................................................................................................................. 19 2.3. Drug treatment and staining ............................................................................................. 20 iv 2.4. Immunofluorescence ........................................................................................................ 20 2.5. Tissue Preparation and Microscopy ................................................................................. 21 2.6. Image Analysis ................................................................................................................. 21 CHAPTER 3. CLASP PROMOTES STABLE TETHERING OF ENDOPLASMIC MICROTUBULES TO THE CELL CORTEX TO MAINTAIN CYTOPLASMIC STABILITY AND PREVENT VACUOLATION IN ARABIDOPSIS MERISTEMATIC CELLS (Published in June 2018, partially modified herein). ..................................................................................... 23 3.0. Introduction and Goals ..................................................................................................... 23 3.1. Results .............................................................................................................................. 25 3.1.1. CLASP localizes
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