A COMPARATIVE PHYSIOLOGICAL, PROTEOMIC AND BIOCHEMICAL ANALYSIS OF SORGHUM SEEDLINGS UNDER SALT STRESS Sellwane Jeanette Moloi 2007105915 A dissertation submitted in fulfilment of the requirements in respect of the Masters Degree qualification in the Department of Plant Sciences in the Faculty of Natural and Agricultural Sciences at the University of the Free State, QwaQwa Campus. Supervisor: Dr. Rudo Ngara Co-supervisor: Dr. Nemera Shargie November 2018 DECLARATION I, Sellwane Jeanette Moloi, declare that the Masters Degree research dissertation that I herewith submit for the Masters Degree qualification in Botany at the University of the Free State is my independent work and that I have not previously submitted it for a qualification at another institution of higher education. I, Sellwane Jeanette Moloi, hereby declare that I am aware that the copyright is vested in the University of the Free State. I, Sellwane Jeanette Moloi, hereby declare that all royalties as regards intellectual property that was developed during the course of and/ or in connection with the study at the University of the Free State will accrue to the University. Sellwane Jeanette Moloi ii DEDICATIONS I dedicate this work to the most special person in my life my daughter, Lindokuhle Mosa Moloi. You have been a blessing in my life and I will always try my best to make you proud and be the mother you deserve. iii ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my supervisor Dr. Rudo Ngara for allowing me the opportunity to pursue this degree and guidance throughout the course of this study. Dr. Ngara, I am forever grateful for always believing in me even when I did not believe that I could make it. I would also like to thank my co-supervisor Dr. Nemera Shargie for his input in my studies. My sincere gratitude also goes to Dr. Stephen Chivasa and his research group at Durham University, United Kingdom for the iTRAQ and osmolyte analyses of my samples. I am also grateful for the financial support I received from the National Research Foundation. To Tatenda Goche, the Plant Biotechnology Research group, colleagues and friends, thank you all for the support and everything we shared during the course of this study. To my family, more especially my mother and siblings, thank you all for the support and encouragement you have given me. The year 2017, was the hardest in my life and without your support I do not think I would have made it. Thank you. Everything I have accomplished in life is by the grace of God. iv TABLE OF CONTENTS DECLARATION ....................................................................................................................... ii DEDICATIONS ........................................................................................................................iii ACKNOWLEDGEMENTS ...................................................................................................... iv LIST OF TABLES .................................................................................................................... ix LIST OF FIGURES ................................................................................................................... x LIST OF ABBREVIATIONS .................................................................................................. xii ABSTRACT ............................................................................................................................xiii CHAPTER 1 .............................................................................................................................. 1 LITERATURE REVIEW .......................................................................................................... 1 1.1 Sorghum, Origin and Importance ........................................................................................ 1 1.2 Abiotic Stresses and their General Effects on Plants ........................................................... 2 1.3 Salt Stress and its Effects on Plants ..................................................................................... 4 1.3.1 Responses of Plants to the Effects of Salt Stress .......................................................... 6 1.3.1.1 Osmotic Stress ........................................................................................................ 6 1.3.1.2 Ionic Stress ............................................................................................................. 7 1.3.1.3 Nutritional Imbalance ............................................................................................. 8 1.3.1.4 Oxidative Stress ...................................................................................................... 8 1.4 Metabolomics and Proteomics Approaches in Studying Plant Stress Response Mechanisms ............................................................................................................................. 10 1.4.1 Metabolomics .............................................................................................................. 10 1.4.2 Proteomics ................................................................................................................... 12 1.4.2.1 iTRAQ Analysis and its Application in Plant Stress Response Studies ............... 13 1.5 Aim, Objectives and Significance of the Study ................................................................. 15 CHAPTER 2 ............................................................................................................................ 16 MATERIALS AND METHODS ............................................................................................. 16 2.1 Plant Material ..................................................................................................................... 16 2.2 Methods.............................................................................................................................. 17 2.2.1 Pot Planting ................................................................................................................. 17 2.2.2 Preliminary Salt Stress Treatment Experiment ........................................................... 17 2.2.3 Measurement of Growth and Physiological Parameters ............................................. 18 2.2.3.1 Growth Measurements .......................................................................................... 18 2.2.3.2 Relative Shoot Water Loss ................................................................................... 18 v 2.2.3.3 Chlorophyll Content ............................................................................................. 19 2.2.3.4 Stomatal Conductance .......................................................................................... 19 2.2.3.5 Leaf Relative Water Content ................................................................................ 19 2.2.3.6 Calculation of Physiological Indices .................................................................... 20 2.2.4 Determination of Osmotyle Concentrations ................................................................ 20 2.2.4.1 Chromatographic Separation of Root and Leaf Extracts for Proline Content Analysis ............................................................................................................................ 21 2.2.4.2 Chromatographic Separation of Leaf and Root Extracts for Glycine Betaine Content Analysis............................................................................................................... 22 2.2.5 Salt Stress Treatments Using 100 mM NaCl .............................................................. 22 2.2.6 Protein Extraction from Sorghum Leaf and Root Tissue ............................................ 23 2.2.7 Protein Quantification ................................................................................................. 23 2.2.8 One Dimensional (1D) Polyacrylamide Gel Electrophoresis (PAGE) ....................... 24 2.2.9 Acetone Precipitation of Protein Samples ................................................................... 25 2.2.10. iTRAQ Analysis ....................................................................................................... 25 2.2.10.1 Sample Preparation for iTRAQ Analysis ........................................................... 25 2.2.10.2 Sample Labelling ................................................................................................ 26 2.2.10.3. iTRAQ Sample Clean-up................................................................................... 27 2.2.10.4 LC-MS Analysis ................................................................................................. 27 2.2.10.5 Mass Spectra Data Analysis ............................................................................... 28 2.2.10.6 Bioinformatic Analysis ....................................................................................... 29 2.2.11 Statistical Analysis .................................................................................................... 29 CHAPTER 3 ............................................................................................................................ 30 PHYSIOLOGICAL AND BIOCHEMICAL ANALYSIS OF SORGHUM PLANTS UNDER SALT STRESS ........................................................................................................................ 30 3.1 Introduction .......................................................................................................................