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Leeds Thesis Template Determining the Link Between Genome Integrity and Seed Quality Robbie Michael Gillett Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds Faculty of Biological Sciences School of Biology January, 2017 - ii - The candidate confirms that the work submitted is his own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. The right of Robbie M. Gillett to be identified as Author of this work has been asserted by him in accordance with the Copyright, Designs and Patents Act 1988. © 2016 The University of Leeds and Robbie Michael Gillett - iii - Acknowledgements I would like to thank my supervisor Dr Christopher West and co-supervisor Dr. Wanda Waterworth for all of their support, expertise and incredible patience and for always being available to help. I would like to thank Aaron Barrett, James Cooper, Valérie Tennant, and all my other friends at university for their help throughout my PhD. Thanks to Vince Agboh and Grace Hoysteed for their combined disjointedness and to Ashley Hines, Daniel Johnston and Darryl Ransom for being a source of entertainment for many years. I would like to thank my international Sona friends, Lindsay Hoffman and Jan Maarten ten Katen. Finally unreserved thanks to my loving parents who supported me throughout the tough times and to my Grandma and Granddad, Jean and Dennis McCarthy, who were always very vocal with their love, support and pride. - iv - Abstract Seed quality is of paramount importance to agriculture, food security and plant conservation programs. However, our understanding of the molecular aspects determining seed quality is far from complete, and the influence of environmental conditions during seed development and post-harvest storage are poorly characterised. There is accumulating evidence that DNA damage, response and repair mechanisms are major factors that control germination performance. Here, levels of DNA damage were analysed to determine the effects of unfavourable environments during seed development and deterioration in storage on genome integrity. In response to DNA damage, plants display a highly specific transcriptional response to double strand breaks (DSBs). This response was found to be highly sensitive to seed ageing. A slight reduction in seed vigour accompanied a reduced ability to respond effectively to DNA damage upon imbibition, indicating that an impaired DNA damage response is an early symptom of seed deterioration. Analysis identified the specific forms of DNA damage associated with seed ageing. Levels of single strand DNA breaks (SSBs) increased with loss of seed viability. Similarly DNA base damage, in the form of 8oxoG residues, increased in the dry seed following accelerated ageing. Evidence of repair of this base damage was identified within nine hours imbibition. Suboptimal temperature in the maternal environment did not significantly influence levels of these lesions, consistent with pathways mitigating DNA damage active during seed development. A requirement for antioxidant activity in genome protection was also studied using mutants with reduced levels of the antioxidant vitamin C. Understanding the molecular differences seen between high quality seeds and those that have undergone different degradation conditions provides insight into the process by which seeds lose the capacity to germination. Future analysis in different species will determine the utility of DNA damage related biomarkers for seed quality and identify potential genetic targets to improve seed performance. - v - Table of Contents Acknowledgements ................................................................................ iii Abstract ................................................................................................... iv Table of Contents..................................................................................... v List of Tables .......................................................................................... xi List of Figures ........................................................................................ xii Abbreviations ........................................................................................ xiv 1. Introduction ...................................................................................... 1 1.1 DNA damage in plants .............................................................. 2 1.1.1 Ultraviolet damage ............................................................. 2 1.1.2 Alkylation and hydrolysis .................................................... 3 1.1.3 Oxidative damage .............................................................. 3 1.1.4 DNA Strand Breaks............................................................ 6 1.2 DNA repair in plants.................................................................. 7 1.2.1 DNA repair synthesis ......................................................... 8 1.2.2 Single strand DNA damage and base lesions ..................... 9 1.2.2.1 Photoreactivation and direct reversal ................... 10 1.2.2.2 Base excision repair ............................................ 10 1.2.2.3 Nucleotide excision repair.................................... 11 1.2.3 Repair of double stranded DNA breaks............................. 12 1.2.3.1 Homologous recombination ................................ 13 1.2.3.2 Non-homologous end joining ............................... 15 1.3 Responses to DNA damage in plants ...................................... 16 1.3.1 ATM and ATR: the signalling kinases ............................... 16 1.3.2 Transcriptional changes upon detection of DNA damage 17 1.3.3 Cell cycle control............................................................. 18 1.4 Seed Biology .......................................................................... 19 1.4.1 Seed development and maturation ................................... 20 1.4.2 Seed Storage................................................................... 21 1.4.3 Germination ..................................................................... 22 1.4.4 Seed vigour ..................................................................... 24 1.4.5 Oxidative damage, repair and seed vigour ........................ 25 1.4.6 Important roles for genome maintenance pathways in seeds ............................................................................... 26 - vi - 1.4.7 Antioxidant protection....................................................... 27 1.5 Summary and Aims ............................................................... 28 2. Materials and Methods ...................................................................... 31 2.1 Suppliers ............................................................................... 32 2.2 Equipment ............................................................................. 32 2.3 Plant material and mutants ..................................................... 32 2.3.1 Isolation of double T-DNA insertion knock-out mutants ..... 34 2.4 Plant Growth Conditions ........................................................ 35 2.4.1 Seed Sterilisation and Imbibition ...................................... 35 2.4.2 Chlorine Gas Seed Surface Sterilisation ........................... 36 2.4.3 Growth of Arabidopsis ...................................................... 36 2.4.5 Plant growth on soil.......................................................... 36 2.5 Seed production and treatments ............................................. 37 2.5.1 Seed production and after-ripening ................................... 37 2.5.2 Germination assays ......................................................... 37 2.5.3 Accelerated ageing of seeds ............................................ 38 2.5.4 X-ray treatment of seeds .................................................. 38 2.5.4 EcoSeed growth and CDT conditions ............................... 38 2.6 DNA and RNA Extraction from Seed and Leaf Tissue ................ 39 2.6.1 DNA Extraction from Leaf and Seedling Tissue................. 39 2.6.2 DNA Extraction from Seed Tissue .................................... 40 2.6.3 RNA Extraction ................................................................ 41 2.6.4 cDNA Synthesis ............................................................... 42 2.7 DNA Amplification via PCR reaction ........................................ 43 2.7.1 DNA amplification ............................................................ 43 2.7.2 PCR programmes ............................................................ 44 2.7.3 RT-PCR programmes ...................................................... 44 2.8 Enzymatic Digestion of DNA ................................................... 44 2.8.1 Uracil DNA glycosylase treatment of DNA ........................ 44 2.8.2 FPG treatment of DNA ..................................................... 45 2.9 Gel Electrophoresis ................................................................ 45 2.9.1 Agarose Gel Electrophoresis ............................................ 45 2.9.2 Alkaline Agarose Gel Electrophoresis ............................... 46 2.9.3 Urea Poly-Acrylamide Gel Electrophoresis (Urea PAGE) 47 2.9.4 DNA purification using Agarose Gels ................................ 49 - vii - 3. Characterisation of EcoSeed
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