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Leeds Thesis Template Roles and mechanisms of DNA repair factors and pathways in Maintaining Seed Quality Aaron James Barrett Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds Faculty of Biological Sciences School of Biology October, 2015 - 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 Aaron James Barrett to be identified as Author of this work has been asserted by him in accordance with the Copyright, Designs and Patents Act 1988. © 2015 The University of Leeds and Aaron James Barrett - iii - Acknowledgements I would like to thank my supervisor Dr Christopher West for all his advice, support and incredible patience throughout my PhD. I would also like to express my gratitude to Dr Wanda Waterworth for all of her encouragement and guidance with my work. Thank-you to all of my colleagues at the University, in particular, Robbie Gillett, Thomas Lanyon-Hogg, Thomas Torode, Valerie Tennant, Vincent Agboh, Grace Hoystead, Jack Goode, Debs Roebuck. I would also like to thank Sue Marcus and all other members of the Knox, Foyer and Baker groups for their support. I owe a great debt to my friends outside the University for their support, including Brown, Broadley and all my friends from home. A big thank-you must also go to Maria and Dave Duxbury (particularly for driving me to the viva) and all associates of The Crown Inn for listening to my ramblings and to all of my incredibly supportive friends. Lastly, I would like to thank Kerry Rostron for her love and support through- out the tough times, my mother Julia, father Joe, sisters, Emma and Isobel, my Grandparents Mavis and Max, and all members of my family, all of whom I appreciate very much. - iv - Abstract Successful germination is a major determinant of crop yields and survival of plants in the natural environment. Our knowledge of molecular factors important to seed quality is far from complete, yet such understanding is vital in the endeavour of mitigating the detrimental effects of extended storage on seed vigour and viability. Genome integrity is crucial for cellular survival and transmission of genetic information. A number of pre-genomic era studies identified strong correlations between DNA damage accumulated in the quiescent seed and seed ageing. If unrepaired, DNA damage results in delayed growth, mutagenesis and cell death. Damage products incurred by DNA are remarkably heterologous and plants have evolved multiple pathways to facilitate the repair of specific damage products. Through the isolation and analysis of knockout Arabidopsis mutants, this work identifies and characterises DNA repair genes whose action is required during seed imbibition. These studies examined the importance of four major DNA repair pathways in germination and seed quality. Mutants deficient in non- homologues end joining (KU70 and KU80), homologues recombination (XRCC2) and base excision repair (ARP) DNA repair pathways were all found to be hypersensitive to accelerated ageing treatment but those deficient in nucleotide excision repair (ERCC1) were not. Therefore this work establishes roles for multiple DNA repair pathways in seed longevity. Comparative analysis also defined non-homologues end joining as the most important DNA repair pathway to seed quality. LIG6 encodes a unique plant specific DNA ligase with roles in seed longevity and implicated in repair of DSBs but remains largely uncharacterised to date. Here studies using extra-chromosomal recombination assays demonstrate LIG6 functions in the promotion of recombination activities in Arabidopsis protoplasts. Further studies demonstrate the involvement of LIG6 in the maintenance of root meristem genome stability. Collectively, this work provides an increased understanding of the early events central to the germination process and seed longevity. - v - Table of Contents Acknowledgements ....................................................................................................... iii Abstract .............................................................................................................................. iv Table of Contents ............................................................................................................ iv List of Tables ....................................................................................................................xii List of Figures ................................................................................................................ xiii Abbreviations .................................................................................................................. xv 1. Introduction .................................................................................................................. 1 1.1 Seed Biology ........................................................................................................... 2 1.1.1 Classification of seeds ................................................................................. 2 1.1.2 Orthodox seeds and Arabidopsis ............................................................ 3 1.1.2.1 Seed maturation, desiccation, storage and potential damage ......................................................................... 5 1.1.2.2 Seed dormancy and hormonal control of germination ................................................................................... 7 1.1.3 Physical, morphological and biochemical changes associated with germination and radicle emergence ..................... 8 1.1.3.1 Phase I: germination and resumption of metabolic activities ..................................................................11 1.1.3.2 Phase II: pre-germination and radicle emergence .......12 1.1.3.3 Germination and cell cycle ....................................................13 1.1.4 Seed quality, vigour and longevity ......................................................14 1.1.4.1 Mean germination time correlates with seed lot quality ............................................................................................16 1.1.4.2 Seed ageing and the repair hypothesis .............................16 1.1.4.3 Improving seed vigour ............................................................17 1.2 DNA structure and the Arabidopsis genome ...............................................19 1.3 Sources and types of DNA damage to plants ...............................................21 1.3.1 UV damage ....................................................................................................21 1.3.2 Alkylation and hydrolysis .......................................................................23 1.3.3 Reactive oxygen species and oxidative damage ............................23 1.3.4 DNA strand breaks ....................................................................................26 1.4 Repair of single strand damage of the DNA duplex ..................................28 1.4.1 Direct reversal and photoreactivation ..............................................28 1.4.2 Mismatch repair .........................................................................................29 - vi - 1.4.3 Base excision repair (BER) .....................................................................29 1.4.4 Nucleotide excision repair (NER) ........................................................30 1.5 Repair of DNA double strand breaks ..........................................................32 1.5.1 DNA double strand break detection and signalling ......................33 1.5.2 Homologous Recombination .................................................................34 1.5.3 Non-homologous end joining ................................................................39 1.5.3.1 The canonical non-homologous end joining pathway ........................................................................................39 1.5.3.2 Back-up non-homologous end joining pathways .........40 1.6 Responses to DNA damage in plants ...........................................................42 1.6.1 The role of ATM and ATR ........................................................................42 1.6.2 Transcriptional changes in response to DNA damage ................45 1.6.3 DNA damage checkpoints and cell cycle ...........................................45 1.6.4 Deoxyribonucleotide synthesis for DNA repair and cell cycle .................................................................................................................46 1.7 DNA damage and repair in seeds .................................................................49 1.7.2 Single strand break repair ......................................................................52 1.7.2.1 Base excision repair .................................................................53 1.7.2.2 Nucleotide excision repair .....................................................53 1.8 Reverse genetics in the study of plant DNA repair ...................................54 1.9 Summary and aims................................................................................................55 2. Materials and Methods ........................................................................................... 56 2.1 Suppliers ...................................................................................................................57
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  • Leeds Thesis Template
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