Faculty of Science and Technology MASTER’S THESIS Study program/ Specialization: Spring Semester, 2017 Master´s degree in Biological Chemistry Open/Restricted Writer: Ope Samuel Olabode ………………………………………………….. (Writer’s signature) Faculty Supervisor: Prof. Cathrine Lillo External Supervisor(s): Title of Thesis: The effect of plant growth-promoting bacteria on wild type, protein phosphatase 2A catalytic subunit mutants of Arabidopsis thaliana and Solanaceae lycopersicum (Gemini tomato) Credits (ECTS): 60 Key words: Arabidopsis Pages:54 thaliana, PP2A, RT-PCR, VOCs, ISR, + Enclosures:77 Stavanger, June 15, 2017 Date/Year Table of contents Table of contents .....................................................................................................................................i-iii Acknowledgement .................................................................................................................................... iv Abstracts ..................................................................................................................................................... v Abbreviation ............................................................................................................................................. vi List of Figures ................................................................................................................................... vii-viii List of Tables ............................................................................................................................................. ix 1. INTRODUCTION ......................................................................................................................... 1-16 1.1 History of Tomato ............................................................................................................................ 1 1.2 Solanum pennellii ............................................................................................................................ 1 1.3 Economic Importance of Tomato .................................................................................................... 2 1.4 Hybrid Seeds of Tomato Plants ....................................................................................................... 2 1.5 Plant Growth-promoting Bacteria (PGPB) ...................................................................................... 2 1.6 Plant Growth Promoting Effects ...................................................................................................... 3 1.6.1 Rhizosphere ............................................................................................................................... 3 1.6.2 Endosphere Bacteria ................................................................................................................. 4 1.6.3 Gram Positive Bacteria ............................................................................................................. 4 1.6.4 Gram Negative Bacteria ............................................................................................................ 5 1.6.5 Sphingobium lamneticum .......................................................................................................... 5 1.6.6 Acidovorax delafieldii ............................................................................................................... 6 1.6.7 PGPR Pseudomonas simiae WCS417r ..................................................................................... 6 1.7 Mechanism of Plant Growth Promotion .......................................................................................... 7 1.8 Biological Nitrogen Fixation ........................................................................................................... 8 1.9 Production of indolic compounds .................................................................................................... 8 1.10 Siderophore productions ................................................................................................................ 9 1.11 ACC deaminase activity .............................................................................................................. 10 1.12 Phosphate Solubilisation .............................................................................................................. 11 1.13 Production of Volatile Organic Compounds ............................................................................... 12 1.14 Induced Systemic Resistance ....................................................................................................... 13 1.15 Protein phosphatases .................................................................................................................... 14 1.15.1 Protein phosphorylation and dephosphorylation ................................................................... 14 i 1.15.2 The PPP Family of Protein Phosphatases ............................................................................. 15 1.15.3 Protein Phosphatase 2A (PP2A) ........................................................................................... 15 1.15.4 PP2A Catalytic subunits in physiological processes in Arabidopsis .................................... 16 2. MATERIAL AND METHODS .................................................................................................. 17-26 2.1 Materials ........................................................................................................................................ 17 2.1.1 Plant Materials ........................................................................................................................ 17 2.1.2 Hoagland Plant Nutrient Solution ........................................................................................... 17 2.1.3 Preparation of Gammborg Medium for Sowing Seeds ........................................................... 18 2.1.4 Preparation of MS Medium for Sowing Seeds ....................................................................... 18 2.2 Methods ......................................................................................................................................... 19 2.2.1 Soil Seed Sowing .................................................................................................................... 19 2.2.2 Sterilizing of Seeds ................................................................................................................. 19 2.2.3 Plant Growth Conditions ......................................................................................................... 19 2.2.4 Isolation of Bacteria from Rhizosphere .................................................................................. 20 2.2.5 Preparation of Enzymatic Lysis Buffer (Stock) ...................................................................... 20 2.2.6 Pre-treatment for Gram-Positive Bacteria ............................................................................... 20 2.2.7 Pre-treatment for Gram-Negative Bacteria ............................................................................. 21 2.2.8 Concentration Measurement ................................................................................................... 21 2.2.9 Polymerase Chain Reaction (PCR) ......................................................................................... 21 2.2.10 Primer used for Genotyping .................................................................................................. 22 2.2.11 PCR Mix and PCR program used when Genotyping ............................................................ 22 2.2.12 Agarose Gel Electrophoresis ................................................................................................. 23 2.2.13 DNA Bands Visualization ..................................................................................................... 23 2.2.14 Scaling up for DNA extraction ............................................................................................. 24 2.2.15 DNA Extraction from Agarose Gels ..................................................................................... 25 2.2.16 Sequencing of G-positive and G-negative Bacterial ............................................................. 25 2.2.17 Procedure for Rhizosphere and Endospheric Bacteria .......................................................... 25 2.2.18 Procedure for Pseudomonas simiae WCS417r Bacterial Inoculation .................................. 25 2.2.19 Procedure for Sphingobium limneticum and Acidovorax delafieldii strains ......................... 26 2.2.20 Preparation for Tomato plants (Gemini original) ................................................................. 26 2.2.21 Growth Media ....................................................................................................................... 26 3. RESULTS .................................................................................................................................... 27-47 3.1 Phenotype of Arabidopsis thaliana and mutants ............................................................................ 27 ii 3.2 DNA Bands Visualization by using PCR, Gel Electrophoresis, and DNA Extraction ................. 27 3.3 Effect of Plant Growth Promoting Bacteria ................................................................................... 29 3.4 Observation made for tomato plants (Gemini original) ................................................................