Evolutionary development of the plant spore and pollen wall A thesis submitted by Simon Wallace for the degree of Doctor of Philosophy Department of Animal and Plant Sciences August 2013 Acknowledgements I am very grateful to my supervisors, Prof. Andrew Fleming, Prof. David Beerling and Prof. Charles Wellman, for their support and encouragement throughout this project. I would like to express my gratitude to Bob Keen, Heather Walker and Marion Bauch for their invaluable assistance and advice in D59 lab; my postgraduate colleagues, Dr Hoe Han Goh, Dr Xiaojia Yin, Dr Adam Hayes, Dr Chloé Steels, Supatthra Narawatthana, Rachel George, Amin Adik, Dr Jen Sloan, Bobby Caine, Thomas Harcourt, Julia Van Campen, Sam Amsbury, Jenn Dick, Dr Phakpoom Phraprasert, Ross Carter, Dr Kate Allinson, Dr Janine Pendleton, Dr Brian Pedder, Faisal Abuhmida, Doreen Mkuu, Sam Slater, Dr Joe Quirk, Dr Lyla Taylor and Dr Claire Humphreys for their support and banter during my time in Sheffield; and to all my other friends in the Department of Animal and Plant Sciences. Many thanks also go to Dr Steve Rolfe for offering his expertise and time with regards to microarray analysis; James Mason for his generous help with Raman microspectroscopy; Prof. Mike Burrell for his general laboratory advice and guidance; Dr Steve Ellin for his assistance in preparing palynological samples; Chris Hill and Svet Tzokov for sharing their electron microscopy expertise; and Dr Katie Field and Dr Timmy O’Donoghue and other members of the Beerling lab for their jollity and support. Special thanks go to Dr Caspar Chater for his unstinting and generous support, advice and friendship. Enormous thanks also go to various collaborators and correspondents; Dr Andrew Cuming, Dr Yasuko Kamisugi, Scott Schuette, Prof. Roy Brown and Prof. Ralf Reski. I gratefully acknowledge the Natural Environment Research Council (NERC) for funding my PhD studentship. I also would like to acknowledge my undergraduate tutors, Dr Richard Lindsay and Dr John Rostron whose love of the natural world assured me that quitting a career in insurance to enrol at university was a good decision. i Finally, I would like to dedicate this thesis to my parents who I am very lucky to have in my life. ii Abstract The origin of land plants required aquatic green algal ancestors to evolve a number of key adaptations that enabled them to overcome physiological difficulties associated with survival in the harsh subaerial environment. One of these key adaptations was the development of a durable spore wall structure, containing a sporopolleninous exine layer, to withstand attrition, desiccation and exposure to UV-B radiation. All land plants (embryophytes) possess such walled spores (or their homologue pollen). However, the spore/pollen wall became more complicated over time, via a series of additional developmental steps, as it began to serve an increasing number of functions. A significant amount of study has been conducted with regards to the molecular genetics of pollen wall development in the angiosperm, Arabidopsis thaliana (L.), particularly with respect to the exine layer and sporopollenin biosynthesis. However, research into the molecular genetics of spore wall development in basal plants has thus far been extremely limited. In this thesis, the results of a fully replicated microarray analysis at early and mid stages of sporogenesis in the free-sporing model moss Physcomitrella patens, have allowed up and down regulated genes to be compared with those known to be involved in pollen wall development, therefore facilitating the identification of candidate genes likely to be involved in the development of the spore wall. Additionally, by way of a gene knock-out experiment with P. patens, this study demonstrates that the MALE STERILITY 2 (MS2) gene, which is involved in wall development in the pollen of A. thaliana, is highly conserved and has a similar function in P. patens. However, the moss homologue of MS2 is shown to be unable to recover functionality in A. thaliana indicating that the MS2 gene, although conserved, has evolved in angiosperms as their pollen walls have increased in complexity. Portions of this thesis have been published in peer-reviewed journals. The references for these publications are as follows: Wallace S, Fleming A, Wellman CH, Beerling DJ. 2011. Evolutionary development of the plant spore and pollen wall. AoB Plants, plr027 doi:10.1093/aobpla/plr027. O’Donoghue M, Chater C, Wallace S, Gray JE, Beerling DJ, Fleming AJ. 2013. Genome-wide transcriptomic analysis of the sporophyte of the moss Physcomitrella patens. Journal of Experimental Botany, 64(12): 3567-3581. iii Contents Page Acknowledgements i Abstract iii Contents iv List of figures viii List of tables x List of appendices xi List of abbreviations/acronyms xii CHAPTER 1 General Introduction 1-25 1.1 Introduction 1 1.2 Spore/pollen wall structure and development 2 1.2.1 Sporogenesis 3 1.2.2 Modes of sporopollenin deposition in spore and pollen walls 3 1.2.3 Spore wall development in bryophytes 5 1.2.4 Spore wall development in pteridophytes 6 1.2.5 Pollen wall development in gymnosperms 9 1.2.6 Pollen wall development in angiosperms 9 1.3 Molecular genetics of pollen wall development 11 1.3.1 Arabidopsis thaliana genes implicated in sporopollenin biosynthesis and exine formation 11 1.3.2 Arabidopsis thaliana transcription factors involved in sporopollenin and exine formation 16 1.3.3 Arabidopsis thaliana genes associated with probaculae formation 18 1.3.4 Arabidopsis thaliana genes connected to intine formation 19 1.3.5 Arabidopsis thaliana genes implicated in callose wall formation 20 1.3.6 Arabidopsis thaliana genes involved in tetrad separation 21 1.4 The utility of Physcomitrella patens 22 1.5 Research outline 23 1.5.1 Aims 24 1.5.2 Objectives 24 1.5.3 Hypotheses 25 1.6 Thesis outline 25 CHAPTER 2 Materials and Methods 26-58 2.1 Materials 26 2.1.1 General laboratory materials 26 2.1.2 Plasmids 27 2.1.3 Bacterial strains 27 2.1.4 Plant Materials 28 2.2 Plant growth conditions and tissue culture 28 2.2.1 Arabidopsis thaliana 28 iv 2.2.2 Physcomitrella patens 28 2.2.3 Protonemal culture and maintenance 28 2.2.4 Gametangia and sporophyte induction 29 2.2.5 Spore germination 29 2.3 Bioinformatics 30 2.3.1 Genome searches 30 2.3.2 Transcriptomic microarray preparation, design and analysis 30 2.3.3 Sequence alignment and phylogenetic analyses 32 2.4 Nucleic acid techniques 33 2.4.1 Plant genomic DNA extraction 33 2.4.2 Plant total RNA extraction 35 2.4.3 DNA and RNA quantification 36 2.4.4 Reverse transcription/cDNA synthesis 36 2.4.5 Polymerase chain reaction (PCR) 37 2.4.6 Semi-quantitative reverse transcription PCR (sq-RT-PCR) 39 2.4.7 Agarose gel electrophoresis 39 2.4.8 DNA gel extraction 40 2.4.9 Ethanol precipitation of DNA 41 2.4.10 Southern blot 41 2.5 Cloning techniques 45 2.5.1 Generation of ATMS2pro::PPMS2-1 and ATUBQ14pro::PPMS2-1 constructs 45 2.5.2 Generation of Physcomitrella patens MS2-1 knock-out construct 46 2.5.3 Generation of PPMS2-1pro::GUS construct 47 2.5.4 DNA ligation 49 2.5.5 pENTRTM/D-TOPO® topoisomerase reaction 50 2.5.6 LR clonase reaction 51 2.5.7 Transformation of competent Escherichia coli 51 2.5.8 Plasmid DNA preparation 52 2.6 Transformation of Arabidopsis thaliana with Agrobacterium tumefaciens 53 2.6.1 Transformation of Agrobacterium tumefaciens with plasmid DNA 53 2.6.2 Preparation of Agrobacterium tumefaciens for floral dipping 53 2.6.3 Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana by floral dipping 54 2.6.4 Polyethylene glycol (PEG)-mediated transformation of Physcomitrella patens 54 2.7 Phenotypic analyses 56 2.7.1 Scanning electron microscopy (SEM) 56 2.7.2 Transmission electron microscopy (TEM) 57 2.7.3 Spore germination test 57 2.7.4 Acetolysis 57 2.7.5 Raman microspectroscopy 58 v CHAPTER 3 The development of the Physcomitrella patens sporophyte 59-74 3.1 Introduction 59 3.2 Results 63 3.2.1 The life cycle of Physcomitrella patens 63 3.2.2 Sporogenesis and spore wall development in Physcomitrella patens 65 3.3 Discussion 68 3.3.1 Life cycle variation among Physcomitrella patens ecotypes 68 3.3.2 Revision of developmental staging of sporogenesis 68 3.3.3 Development of the Physcomitrella patens spore wall 70 3.4 Summary 73 CHAPTER 4 Identification of candidate spore wall genes in Physcomitrella patens 75-101 4.1 Introduction 75 4.2 Results 79 4.2.1 Physcomitrella patens and Selaginella moellendorffii genome search results 79 4.2.2 Microarray analysis: expression profiles of spore wall candidate genes during sporophyte development 79 4.2.3 Phylogenetic analyses 86 4.2.4 Electron microscope analysis of Arabidopsis thaliana ms2 and rpg1 pollen wall phenotypes 86 4.2.5 Sequence alignment and transcript analysis of MS2 homologues 92 4.3 Discussion 93 4.3.1 Flowering plant homologue genes for spore wall development are present in Physcomitrella patens and Selaginella moellendorffii 93 4.3.2 Identification of lead genes for further investigation 93 4.3.2.1 Putative Physcomitrella patens tetrad separation genes 95 4.3.2.2 Putative Physcomitrella patens sporopollenin biosynthesis/exine genes 96 4.4 Summary 101 CHAPTER 5 Functional analysis of the MS2-like gene in spore wall morphogenesis in Physcomitrella patens 102-120 5.1 Introduction 102 5.2 Results 104 5.2.1 Generation and molecular analysis of Physcomitrella patens PPMS2-1 knock-out lines and Arabidopsis thaliana PPMS2-1 gain-of-function lines 104 5.2.2 Phenotypic analysis of ppms2-1 spores 106 vi 5.2.3 Phenotypic