The biochemical consequences of ascorbate deficiency in Arabidopsis thaliana A thesis submitted by Nighat Sultana for the degree of Doctor of Philosophy at the University of Exeter, College of Life and Environmental Sciences, School of Biosciences. December, 2011 This thesis is available for Library use on the understanding that it is a copyright material and the no quotation from the thesis may be published without the proper acknowledgement. I certify that all material in this thesis which is not my own work has been identified and that no material is included for which a degree has been previously approved from this or any other University Signature.................................................. 1 © Copyright 2011 Nighat Sultana All rights reserved. 2 Table of Contents Acknowledgments .............................................................................................................. 8 List of Figures .................................................................................................................... 10 List of Tables ...................................................................................................................... 12 List of abbreviations .......................................................................................................... 14 Abstract ............................................................................................................................... 15 Chapter 1: General Introduction ..................................................................................... 19 1.1 Introduction ................................................................................................................. 19 1. 2 Reactive oxygen species and oxidative stresses ..................................................... 20 1. 3 ROS and their generation .......................................................................................... 21 1. 4 Antioxidant system .................................................................................................... 24 1. 5 AsA: an antioxidant ................................................................................................... 26 1. 6 2-Oxoglutrate dependent dioxygenases (2-ODD) ................................................. 32 1. 7 AsA biosynthesis: the D-mannose/L-galactose (D-Man/L-Gal) pathway .... 33 1. 8 AsA-deficient mutants (vtc) of Arabidopsis thaliana ............................................... 36 1. 9 Arabidopsis thaliana: a model system ........................................................................ 37 1. 10 Aims of thesis ............................................................................................................ 39 Chapter 2: Experimental Method .................................................................................... 41 2. 1 Plant Material .............................................................................................................. 41 2. 2 Growth and maintenance of seeds........................................................................... 41 2.2. 1 Surface sterilisation and plant growth ............................................................. 41 2.2. 2 Sowing seeds on soil and growth room conditions ........................................ 42 2.2.3 Harvesting and storage of seeds ......................................................................... 42 2.2.3.1 Tissue harvesting ........................................................................................... 42 2.2.3.1 Seed collection ................................................................................................ 43 2. 3 Metabolite extractions and assays ........................................................................... 43 2.3. 1 Inactivation of the metabolic pathways ........................................................... 43 2.3. 2 Ascorbate assay .................................................................................................... 43 2.3. 3 Metabolite extraction and analysis from whole leaf ....................................... 45 2.3. 4 Glucose-6-P assay (an extracellular marker) ................................................... 46 2.3. 5 Extraction of hormones and flavonoids ........................................................... 47 3 2. 4 Mass Spectrometry method development .............................................................. 47 2.4. 1 Development and optimisation of method for hormone and flavonoid analysis ............................................................................................................................ 47 2. 5 Plants stress treatments ............................................................................................. 49 2.5. 1 Optimisation of growth chamber conditions for high light stress experiments .................................................................................................................... 49 2.5.2 Mechanical wounding .......................................................................................... 50 2.5.3 Salt stress treatment .............................................................................................. 50 2. 6 Proteomic analysis...................................................................................................... 51 2.6.1 Ionically-bound cell wall protein extraction ..................................................... 51 2.6. 2 Total protein assay ............................................................................................... 52 2.6.3 Peroxidase assay ................................................................................................... 52 2.6. 4 Lectin affinity chromatography ......................................................................... 53 2.6. 5 Tryptic Digestion ................................................................................................. 53 2.6. 6 Proteins analysis by HPLC-Chip ....................................................................... 54 2. 7 Statistical Analyses ................................................................................................. 55 Chapter 3: Metabolite Profiling of Arabidopsis thaliana vtc mutants ........................... 57 3. 1 Introduction ................................................................................................................ 57 3. 2 Analytical techniques................................................................................................. 60 3.2. 1 High Performance Liquid Chromatography-Mass Spectrometry (LC-MS) 60 3.2. 2 Electrospray Ionisation ....................................................................................... 62 3.2. 3 Analyser ................................................................................................................ 64 3.2. 4 Detector ................................................................................................................. 65 3. 3 Untargeted Metabolites Analysis by LC-MS .......................................................... 68 3. 4 Material and Methods................................................................................................ 69 3.4. 1 Compound deconvolution and alignment ...................................................... 70 3.4. 2 Noise filtering and Analysis of Variance ......................................................... 74 3. 5 Metabolite Identification ........................................................................................... 76 3.5. 1 Visualisation of the raw data ............................................................................. 76 3.5. 2 Generation of Molecular Formulae and Database Searches .......................... 77 3. 6 Results .......................................................................................................................... 82 3.6. 1 Ascorbate concentration in the apoplastic fluid .............................................. 82 4 3.6. 2 Metabolite profiling ............................................................................................. 84 3.6.2. 1 Total ion Chromatogram (TIC) ................................................................... 84 3.6.2. 2 Deconvolution and Alignment ................................................................... 88 3.6.2. 3 Hierarchical Clustering Analysis (HCA) ................................................... 91 3.6.2. 4 Profile plot .................................................................................................... 100 3.6. 3 Metabolite Identification ...................................................................................... 103 3.6.3. 1 Identification of selected metabolites differentially expressed between WT and vtc mutants ................................................................................................. 103 3.6.3.2 Other metabolites affected by ascorbate deficiency ................................ 114 3. 7 Discussion .................................................................................................................. 125 3.7. 1 Identification of compounds differentially expressed between WT and vtc mutants .......................................................................................................................... 127 3. 8 Conclusions ............................................................................................................... 132 Chapter 4: Response of flavonoids and hormones to abiotic stress