INVESTIGATING THE TARGETS AND MECHANISMS REGULATING SELF INCOMPATIBILITY IN PAPAVER RHOEAS POLLEN by TAMANNA HAQUE A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Biosciences The University of Birmingham September 2015 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT Many higher plants use self-incompatibility (SI) mechanism to prevent inbreeding and thus encouraging out-crossing. Upon a self-challenge in Papaver rhoeas, a Ca2+-dependent-signalling-cascade is initiated resulting in the destruction of the self-pollen by Programmed Cell Death (PCD). Upstream of PCD, several SI-specific events are triggered in incompatible pollen, including phosphorylation of soluble inorganic pyrophosphatases (sPPases); alterations to actin; increases in Reactive Oxygen Species (ROS) and Nitric Oxide (NO). In Papaver pollen, sPPases play an important role, as they provide the driving force for biosynthesis; data suggested that Ca2+ and phosphorylation inhibits the sPPases activities, contributing to pollen tube inhibition. Work presented in this thesis characterized Pr-p26.1 sPPases and the role of phosphomimic mutants in the SI signalling cascade. These studies 2+ provide good evidence that, together with Ca , phosphorylation, H2O2 and pH dramatically affect sPPase activity. As previous studies showed that increases in ROS and NO are triggered by SI in incompatible pollen, to provide insights into SI-mediated events, this project investigated protein-targets in pollen modified by oxidation and S- nitrosylations after SI, including actin and actin-associated proteins. Using a mass spectrometry approach we identified several proteins that were modified by oxidation and S-nitrosylation. This has provided us with several potential new mechanisms involved in SI. Acknowledgement Firstly, I would like to thank my supervisor, Professor Noni Franklin-Tong, for all her support and encouragement over the previous almost four years and for giving me the opportunity to work in a project that I have enjoyed a lot. I am also very thankful to Professor Chris Franklin for his critical advices and discussions, especially about actin cloning. I would also like to express my gratefulness to the other members, staff and PIs on the second floor, past and present, who encouraged and helped me on many different levels, gave me the confidence, and provided an enjoyable working environment. In particular, I would like to thank Deborah J. Eaves for her valuable suggestions, support and comments during the experimental work and also on my thesis. Special thanks to Carlos Flores and Zongcheng Lin for being so helpful, for their valuable support and answering my questions all the time. I would like to thank Andrew Beacham, Javier Andrés Juárez Díaz, Katie Wilkins, Lijun Chai and Nianjun Teng for being wonderful lab mates. I also thank Kim Osman, Ruth Perry, Lisa Burke, Stefan Heckmann, Allan West for their valuable suggestions and help. I would also like to acknowledge Nick Cotton and Dr. White for providing me some chemicals, giving me scientific and technical advices on the Pr-p26.1 project. My thanks go to the Proteomics Department, especially Laine Wallace and Cleidi Zampronio, who helped me with my samples for mass spectrometry and took the time to discuss the results with me. I would like to thank the Commonwealth Scholarship Commission (CSC) for funding the PhD project. Finally, but most importantly I would like to thank my friends and family members, specially my brother, sister and in-laws for all their moral support and encouragement. I would like to give a massive thank to my husband Zia and my daughter Zeba, who have been great support throughout the PhD, especially during the writing-up stage. I would like to thank my mum, without her moral support and unconditional love I would never have had the courage to pursue my PhD. Table of contents CHAPTER 1………………………………………………………………........….......1 Introduction………………………………………………………........……..............1 1.1. Sexual Plant Reproduction...…………………………………………………………... ..2 1.1.1. Self-Incompatibility (SI)……...……………………………………………………......2 1.1.1.1. Different mechanisms of self-incompatibility…………………...………..........3 1.1.2. Pollen tubes……...……………………………………………………………….........5 1.1.2.1. The role of calcium in pollen tube growth…...…………………………............8 1.1.2.2. Protons (H+) in pollen tube growth…………....…………………………….......9 1.1.2.3. Reactive oxygen species (ROS) and nitric oxide (NO)................................11 1.1.2.3.1 ROS and NO in pollen tube growth........................................................12 1.2. The cytoskeleton……………………………………………………………………........13 1.2.1. The actin cytoskeleton………………………………………………………...........13 1.2.1.1. The actin cytoskeleton in pollen tubes……………………………………......14 1.2.1.2. Actin binding proteins (ABPs)……………………………………………….....15 1.3. Soluble inorganic pyrophosphatases (sPPases)…………………………………......16 1.3.1. Family I PPases…………………………………….....……………………….........17 1.3.2. Plant soluble inorganic pyrophosphatases………………………………….........20 1.4. Programmed cell death (PCD)………………………………………...………….........21 1.4.1. Apoptosis in mammalian cells……………………………………………………...22 1.4.2. Plant PCD……………………………………………………………………….........22 1.4.2.1. Vacuolar cell death (VCD)…………………………...…………......................23 1.4.2.2. Necrotic PCD……………………………………………………………….........24 1.4.2.2.1. Plant caspase-like activities………………………………………….........24 1.4.2.3. The hypersensitive response (HR) and induction of PCD………….............25 1.4.2.4. The role of ROS and NO in plant PCD……………………………...…..........27 1.5. Oxidation and Nitrosylation of Proteins…………………………………………..........29 1.5.1. Oxidation of proteins………………………………...…………………………........30 1.5.2. S-nitrosylation…………………………………………………………………….......33 1.6. Molecular mechanisms involved in the three main SI systems…………..…...........34 1.6.1. Mechanisms involved in SI in Brassicaceae………………………………….......35 1.6.2. Mechanisms involved in S-RNase based gametophytic SI………………..........37 1.6.2.1. Degradation Model…………………….……………………………….............39 1.6.2.2. The Compartmentalization Model……………………………………...……...39 1.6.3. Gametophytic Self-incompatibility in Papavaraceae…………………………....41 1.6.3.1. Mechanisms involved in Self-Incompatibility in Papaver…………………...42 1.6.3.1.1. Role of Ca2+ in the SI response…………………………………..…..…..43 1.6.3.1.2. PCD and caspase-like activity in Papaver………………………..……...44 1.6.3.1.3. SI-induced pollen tube acidification………...…………………………….45 1.6.3.1.4. Role of actin in poppy SI…...………………………………………………46 1.6.3.1.5. Recruitment of signalling for SI events in other species………....……..46 1.6.3.1.6. Role of ROS and NO in Papaver SI……...……………………………….48 1.6.3.1.7. Role of soluble in organic pyrophosphatases in poppy SI……....……...49 1.7. Aims of this project………………………………………………………………….…....50 CHAPTER 2………………………………………………………........…................52 Materials and methods……….…………………………………....…..................52 2.1. Pollen tube growth in vitro......................................................................................53 2.2. Treatments to pollen tubes.....................................................................................53 2.2.1. In vitro induction of SI in pollen........................................................................54 2.2.2. H2O2 treatment.................................................................................................54 2.2.3. NO donor GSNO..............................................................................................54 2.3. Visualisation of F-actin in Papaver pollen tube......................................................55 2.4. Fluorescence microscopy.......................................................................................56 2.4.1. Epifluorescence imaging for F-actin analysis...................................................56 2.5. Extraction of Pollen Proteins..................................................................................56 2.5.1. Protein extraction for ROS/NO experiments.....................................................56 2.5.2. Pollen protein extraction for F-actin isolation....................................................57 2.6. SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)..........................................57 2.7. Western blot............................................................................................................58 2.7.1. Protein transfer.................................................................................................58 2.7.2. Antibody probing...............................................................................................58 2.7.3. Enhanced chemiluminescence detection (ECL)...............................................59 2.8. F-actin enrichment using ultracentrifugation...........................................................59 2.9. F-actin pull-down assay..........................................................................................60