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Functional Analysis of the TETRASPANIN Gene Family in Plant Development and Growth

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Functional Analysis of the TETRASPANIN Gene Family in Plant Development and Growth Ghent University Faculty of Sciences Department of Plant Biotechnology and Bioinformatics Functional Analysis of the TETRASPANIN Gene Family in Plant Development and Growth This thesis is submitted as partial fulfillment of the requirements for the degree of Doctor of Philosophy (Ph.D.) in Sciences: Biochemistry and Biotechnology Feng WANG Promotor: Prof. Dr. Mieke Van Lijsebettens Co-promoter: Prof. Dr. Sofie Goormachtig VIB / Plant Systems Biology Technologiepark 927, B-9000 Gent, Belgium This work was conducted in the Department of Plant Systems Biology (PSB) of the Flanders Institute for Biotechnology (VIB). Feng WANG was supported by a CSC PhD fellowship grant of China. The author and promoter give the authorization to consult and copy parts of this work for personal use only. Every other use is subject to the copyright laws. Permission to reproduce any material contained in this work should be obtained from the author. Examination Committee Chair Prof. Dr. Geert De Jaeger Ghent University - Department of Plant Biotechnology and Bioinformatics VIB - Department of Plant Systems Biology Secretary Prof. Dr. Mieke Van Lijsebettens (Promoter) Ghent University - Department of Plant Biotechnology and Bioinformatics VIB - Department of Plant Systems Biology Members Prof. Sofie Goormachtig (Co-promoter) Ghent University - Department of Plant Biotechnology and Bioinformatics VIB - Department of Plant Systems Biology Prof. Kristiina Himanen (Reading Committee) University of Helsinki - Department of Agricultural Sciences Prof. Gerda Cnops Institute for Agricultural and Fisheries Research, 9000 Gent, Belgium. Prof. Filip Vandenbussche (Reading Committee) Ghent University - Department of Physiology Prof. Ive De Smet (Reading Committee) Ghent University - Department of Plant Biotechnology and Bioinformatics VIB - Department of Plant Systems Biology Prof. Klaas Vandepoele Ghent University - Department of Plant Biotechnology and Bioinformatics VIB - Department of Plant Systems Biology Prof. Tom Beeckman (Reading Committee) Ghent University - Department of Plant Biotechnology and Bioinformatics VIB - Department of Plant Systems Biology. CONTENTS SCOPE AND AIMS .............................................................................................................. 1 LIST OF ABBREVIATIONS .................................................................................................... 2 CHAPTER 1 TETRASPANIN GENES IN PLANTS ....................................................................................... 5 ABSTRACT ................................................................................................................................ 6 Membrane Proteins .................................................................................................................. 8 Evolutionary and Phylogenetic Studies of Tetraspanins ............................................................ 10 From Structure to Function ..................................................................................................... 17 A. Transmembrane Domains ....................................................................................................................... 18 B. Extracellular Loops................................................................................................................................... 18 C. Cytoplasmic Domains .............................................................................................................................. 19 Tetraspanin-enriched Microdomains ....................................................................................... 20 A. Cell Adhesion and Membrane Fusion ...................................................................................................... 21 B. Cell-to-cell/cell-to-environment Communication and Infections ............................................................ 22 ACKNOWLEDGEMENTS ........................................................................................................... 24 CHAPTER 2 EMBRYONIC AND VEGETATIVE TETRASPANIN GENE EXPRESSION PATTERNS IDENTIFY FUNCTIONS IN SPECIFIC TISSUES, DOMAINS AND CELL TYPES ............................................25 ABSTRACT .............................................................................................................................. 26 2.1 INTRODUCTION ................................................................................................................ 27 2.2 RESULTS ............................................................................................................................ 29 2.2.1 Generation of Promoter TET-reporter Gene Lines and Analysis in Embryo, Root, Leaf and Flower .. 29 2.2.2 Most Duplicated TET Genes Have Divergent Expression Patterns in Embryonic and Vegetative Development ............................................................................................................................................... 36 2.2.3 TET5 and TET6 Redundant Genes Have a Function in Growth Control .............................................. 39 2.3.4 TET2 in Stomatal Development and Function .................................................................................... 41 2.3.5 TET13 Has a Function in Primary and Lateral Root Development ...................................................... 45 2.3.6 Fluorescence Activated Cell Sorting of Synchronized tet13-1 Lateral Root Founder Cells for RNA-Seq ..................................................................................................................................................................... 50 2.3 DISCUSSION ...................................................................................................................... 52 2.4 MATERIALS AND METHODS ............................................................................................... 56 SUPPLEMENTAL DATA ............................................................................................................. 60 ACKNOWLEDGEMENTS ........................................................................................................... 67 CHAPTER 3 TETRASPANINS SUBCELLULAR LOCALIZATION ....................................................................69 ABSTRACT .............................................................................................................................. 70 3.1 INTRODUCTION ................................................................................................................ 71 3.2 RESULTS ............................................................................................................................ 72 3.2.1 Signal Peptide and Signal Anchor at the N-terminus of Tetraspanins................................................. 72 3.2.2 Tetraspanins Subcellular Localization Prediction ................................................................................ 72 3.2.3 Generation of TETRASPANIN Fluorescence Tag Fusion Transgenic Lines and Subcellular Localization of TETRASPANINS ......................................................................................................................................... 74 3.3 DISCUSSION ...................................................................................................................... 79 3.4 MATERIALS AND METHODS ............................................................................................... 81 SUPPLEMENTAL DATA ............................................................................................................. 83 CHAPTER 4 INFERRING TETRASPANINS FUNCTIONS BY LINKING EXPERIMENTAL DATA WITH BIOINFORMATIC DATA ......................................................................................................85 ABSTRACT .............................................................................................................................. 86 4.1 INTRODUCTION ................................................................................................................ 87 4.2 RESULTS ............................................................................................................................ 88 4.2.1 Meta-Analysis of TET Response to a Variety of Perturbations ............................................................ 88 4.2.2 Regulatory Element Analysis in TETs Noncoding Regions ................................................................... 91 A. The Regulatory Elements in TET1 Promoter and Intron Are Related to Light Regulation ................... 96 B. The Regulatory Elements in TET2 Promoter and Intron Are Related to Stress Response ................... 96 C. The Regulatory Elements in TET3 Promoter Are Related to ABA and Cold Response ........................ 96 D. The Regulatory Elements in TET4 Promoter Are Related to ABA Response and Tissue-Specific ........ 97 E. The Regulatory Elements in TET5 and TET6 Promoter Regions Are Related to Sugar Response ........ 97 F. TET8 and TET9 Have the Most Regulatory Elements and are Most Diverse ........................................ 98 4.2.3 Inference of TETs Functions by Transcription Factors-TETs Regulatory Network Analysis .................. 99 A. Generating a Gene Regulatory Network of Upstream Regulators of TETs .......................................... 99 B. The Regulatory Network Provides Insights into TETs Functions ......................................................... 99 C. TET3 Function is Flowering Response Related .................................................................................
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