FLOWER HEAD DEVELOPMENT IN THE ASTERACEAE FAMILY Nicholas Andrew Zoulias Faculty of Life Sciences 2014 A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences Table of Contents ABSTRACT OF THESIS: ......................................................................................................................... 7 Declaration .......................................................................................................................................... 8 Copyright Statement ........................................................................................................................... 8 Research Contributions ....................................................................................................................... 9 List of Abbreviations ......................................................................................................................... 10 Acknowledgments ............................................................................................................................. 12 Chapter 1 ........................................................................................................................................... 13 Introduction ...................................................................................................................................... 13 1.1 The Asteraceae family ............................................................................................................. 14 1.1.1 Structure of the flower head............................................................................................ 15 1.2 Organ patterning ..................................................................................................................... 18 1.3 Auxin ....................................................................................................................................... 20 1.4 Polarised auxin transport ........................................................................................................ 21 1.4.1 PIN-FORMED auxin efflux transporters ........................................................................... 21 1.4.2 Long PIN-FORMED transporters....................................................................................... 24 1.4.3 PIN-FORMED cycling ........................................................................................................ 26 1.4.4 ATP-binding cassette auxin transporters ......................................................................... 27 1.4.5 Auxin influx transporters ................................................................................................. 29 1.4.6 Control of auxin distribution and auxin sensing .............................................................. 30 1.4.7 Auxin biosynthesis and flower initiation .......................................................................... 33 1.4.8 Floral organogenesis and pattern formation ................................................................... 33 1.4.9 Auxin maxima and minima ............................................................................................... 34 1.4.10 Auxin a morphogen? ...................................................................................................... 36 1.5 Flower development ............................................................................................................... 38 1.5.1 CYCLOIDEA ....................................................................................................................... 38 1.5.2 CYCLOIDEA-Like genes in model species .......................................................................... 40 1.5.3 Pseudanthia and CYCLOIDEA-Like .................................................................................... 41 1.5.4 RADIALIS ........................................................................................................................... 42 1.5.5 DIVARICATA ...................................................................................................................... 43 1.5.6 TCP genes and the flower head ....................................................................................... 45 1.6 LEAFY ....................................................................................................................................... 48 1.6.1 LEAFY and the ABC genes ................................................................................................. 50 1.6.2 LEAFY and auxin ............................................................................................................... 52 2 1.7 MADS-box genes and the flower head ................................................................................... 53 1.8 TERMINAL FLOWER1 ............................................................................................................... 53 1.9 Aims......................................................................................................................................... 55 Chapter 2 ........................................................................................................................................... 56 General Methods .............................................................................................................................. 56 2.1 Plant material and plant transformation ................................................................................ 57 2.1.1 Plant growth conditions ................................................................................................... 57 2.1.2 Exogenous auxin application............................................................................................ 57 2.1.3 Floral dipping method ...................................................................................................... 58 2.1.4 Virus induced gene silencing (VIGS) ................................................................................. 59 2.1.5 Plant transformation techniques-tissue culture method ................................................ 60 2.2 Bacterial growth conditions and transformation.................................................................... 61 2.2.1 E. coli transformation ....................................................................................................... 61 2.2.2 A. tumefaciens transformation ........................................................................................ 62 2.3 RNA, DNA and Cloning ............................................................................................................ 63 2.3.1 RNA and DNA extractions and cDNA synthesis ................................................................ 63 2.3.2 Degenerate Cloning of PIN1, LFY and PDS ....................................................................... 63 2.3.3 DR5::GUS Construct ......................................................................................................... 65 2.3.4 Colony PCR ....................................................................................................................... 65 2.3.5 DNA sequencing ............................................................................................................... 66 2.4 Expressional analysis ............................................................................................................... 66 2.4.1 Quantitiative RT-PCR ........................................................................................................ 66 2.4.2 Immunolocalisation ......................................................................................................... 67 2.4.3 In situ Hybridisation ......................................................................................................... 68 2.5 Histology and Scanning Electron Microscopy ......................................................................... 70 2.5.1 Beta-glucuronidase staining ............................................................................................. 70 2.5.2 Scanning Electron Microscopy ......................................................................................... 71 2.6 Software .................................................................................................................................. 71 Chapter 3 ........................................................................................................................................... 73 A Novel Morphogen-like Role of Auxin in Determining Capitulum Pattern Formation in Asteraceae .......................................................................................................................................................... 73 3.1 Abstract ................................................................................................................................... 74 3.2 Nature Letter ........................................................................................................................... 75 3.3 Method Summary ................................................................................................................... 84 3.4 Supplementary Data and Methods ......................................................................................... 86 3 Supplementary Figures ............................................................................................................. 86 Supplementary Tables .............................................................................................................