Physiological, biochemical and molecular responses of different barley varieties to drought and salinity Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Muhammad Tauhid Iqbal aus Vehari, Pakistan Bonn, 2018 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn 1. Gutachter: Prof. Dr. Dorothea Bartels 2. Gutachter: PD Dr. Ali Ahmad Naz Tag der Promotion: 21.02.2018 Erscheinungsjahr: 2018 DECLARATION I hereby declare that the whole PhD thesis is my own work, except where explicitly stated otherwise in the text or in the bibliography. Bonn, 2018 ------------------------------------ Muhammad Tauhid Iqbal III Contents ABBREVIATIONS ................................................................................................................. VII FIGURES AND TABLES ........................................................................................................ X SUMMARY ............................................................................................................................... 1 1. INTRODUCTION .................................................................................................................. 3 1.1 Barley- Hordeum vulgare as experimental plant .............................................................. 3 1.2 Stress ................................................................................................................................. 4 1.2.1 Relationship between global warming and abiotic stress .......................................... 4 1.2.2 Drought Stress ............................................................................................................ 5 1.2.3 Salt stress ................................................................................................................... 5 1.2.4 Effect of Stress on Morphology of Plant ................................................................... 7 1.3 Stress Tolerance ................................................................................................................ 8 1.3.1 Regulatory Mechanism .............................................................................................. 8 1.4 Objectives of the studies ................................................................................................. 16 1.5 Varieties of Barley .......................................................................................................... 17 2. Materials and Methods ......................................................................................................... 19 2.1 Materials ......................................................................................................................... 19 2.1.1 Plant material ........................................................................................................... 19 2.1.2 Chemicals ................................................................................................................. 19 2.1.3 Kits ........................................................................................................................... 20 2.1.4 Enzymes and DNA-marker ...................................................................................... 20 2.1.5 Microorganisms ....................................................................................................... 20 2.1.6 Vector ....................................................................................................................... 20 2.1.7 Machines and other devices ..................................................................................... 21 2.1.8 Buffers and Solutions ............................................................................................... 22 2.1.9 Primers ..................................................................................................................... 24 2.2 Methods .......................................................................................................................... 26 2.2.1 Growth Conditions ................................................................................................... 26 2.2.2 Morphological Analysis ........................................................................................... 26 2.2.3 Plant Material Storage .............................................................................................. 26 2.2.4. Water loss rate ......................................................................................................... 26 2.2.5 Leaf relative water contents of leaves ...................................................................... 27 2.2.6 Extraction of Nucleic Acids ..................................................................................... 28 2.2.7 First strand cDNA synthesis .................................................................................... 30 IV 2.2.8 Polymerase chain reaction (PCR) ............................................................................ 30 2.2.9 Semi quantitative gene expression level determination ........................................... 31 2.2.10 DNA extraction from an agarose gel/PCR product purification ............................ 31 2.2.11 Subcellular localization of Protein ......................................................................... 32 2.2.12 Protein analysis ...................................................................................................... 34 2.2.13 Physiological and Biochemical Assays ................................................................. 36 3. RESULTS ............................................................................................................................. 41 3.1 Growth of the plant ......................................................................................................... 42 3.1.1 Number of leaves ..................................................................................................... 42 3.1.2 Shoot length ............................................................................................................. 43 3.1.3 Root length ............................................................................................................... 44 3.2 Water Loss Rate (WLR) ................................................................................................. 44 3.3 Leaf Relative water content (RWC) ............................................................................... 45 3.4 Total chlorophyll content ................................................................................................ 46 3.5 Proline Determination Assay .......................................................................................... 47 3.6 Lipid peroxidation assay ................................................................................................. 49 3.7 Hydrogen Peroxide (H2O2) Measurement ...................................................................... 50 3.8 Anti-oxidative enzymes activities in different barley varieties after drought and salt stress ..................................................................................................................................... 52 3.8.1 Super Oxide Dismutase (SOD) Activity .................................................................. 52 3.8.2 Catalase Activity ...................................................................................................... 53 3.8.3 Glutathione Reductase Activity ............................................................................... 54 3.8.4 Peroxidase Acitvity .................................................................................................. 55 3.9 Dehydrins ........................................................................................................................ 57 3.9.1 Physico-chemical Analysis of different barley dehydrins ....................................... 57 3.9.2 Barley dehydrin transcript analysis .......................................................................... 59 3.9.3 Immuno Blots Analysis ............................................................................................ 70 3.9.4 Sub-cellular Localization of dehydrin proteins ........................................................ 73 4. DISCUSSION ...................................................................................................................... 76 4.1 Growth parameters ......................................................................................................... 76 4.2 Water retaining capability .............................................................................................. 77 4.3 Total chlorophyll contents .............................................................................................. 77 4.4 Proline contents .............................................................................................................. 78 4.5 MDA level and Hydrogen peroxide ............................................................................... 78 V 4.6 Activity of Antioxidative enzymes ................................................................................ 79 4.8 Dehydrins in barley ........................................................................................................ 81 4.8.1 Sub cellular localization of Dehydrin in barley