PhD thesis Sabrina Stanimirovic Elucidating the roles of MAP kinases in the moss Physcomitrella patens Supervisor: John Mundy Handed in: 17/05/2017 2 Table of contents Preface ................................................................................................................................................. 7 Acknowledgements............................................................................................................................. 8 Abstract ............................................................................................................................................... 9 Resume .............................................................................................................................................. 10 List of abbreviations ........................................................................................................................ 11 Introduction ...................................................................................................................................... 13 Physcomitrella patens .................................................................................................................... 13 Evolution .................................................................................................................................... 13 The life cycle and morphology ................................................................................................. 14 Homologous recombination ..................................................................................................... 16 Plant innate immunity.................................................................................................................. 17 2 layered defenses – PAMP and effector triggered immunity (PTI & ETI) ....................... 17 MAMPs/PAMPs and LRRs ..................................................................................................... 18 ROS and cytoplasmic calcium ................................................................................................. 19 Mitogen activated protein kinase signaling pathways ........................................................... 20 Immunity in Physcomitrella patens.......................................................................................... 24 Abiotic stress ................................................................................................................................. 26 Salinity and osmotic stress ....................................................................................................... 26 Phytohormones ......................................................................................................................... 28 Abscisic acid........................................................................................................................... 28 Auxin & cytokinin ................................................................................................................. 32 Strigolactone .......................................................................................................................... 36 Light ........................................................................................................................................... 38 Results ............................................................................................................................................... 40 Identification of Physcomitrella patens homologs of MPKs in Arabidopsis thaliana .............. 40 3 Generation of MPK knockout mutants ...................................................................................... 42 Phenotypic analysis of generated mutants ................................................................................. 44 Sporophyte induction ............................................................................................................... 44 Growth of protonemata ............................................................................................................ 53 3 weeks phenotypic analysis ................................................................................................. 55 6 weeks phenotypic analysis ................................................................................................. 58 Gravitropism ............................................................................................................................. 63 Finding the needle in the haystack – what activates the MPKs? ......................................... 66 Biotic stress ............................................................................................................................ 66 Abiotic stress – Drugs ........................................................................................................... 67 Calyculin A – protein Serine/Threonine phosphatase inhibitor ................................... 67 Zeomycin – DNA damage ................................................................................................. 69 Abiotic stress – Salt and osmotic stress ............................................................................... 71 Vertical growth .................................................................................................................. 72 Stress survival .................................................................................................................... 74 Abiotic stress – Hormones .................................................................................................... 78 Auxin................................................................................................................................... 78 Abscisic acid ....................................................................................................................... 80 Cytokinin ............................................................................................................................ 82 Strigolactone ...................................................................................................................... 83 Abiotic stress – Light ............................................................................................................ 86 Dark .................................................................................................................................... 86 Red and Blue light ............................................................................................................. 88 Expression of RAK1, RAK2, MPK3, MPK5 and NATH ...................................................... 94 Discussion ........................................................................................................................................ 101 Auto-phosphorylation ................................................................................................................ 101 4 Rosettas – RAK1/RAK2 and their single-domain NATH homolog ....................................... 102 Characterizations of mutant KO lines...................................................................................... 103 Protonemata and secondary growth ......................................................................................... 104 MPK phosphorylation and activation ...................................................................................... 105 MPK3 and MPK5 – involvement in ion homeostasis upon salt treatments? ........................ 106 MPK regulation by phytohormones? ....................................................................................... 108 Light responses ........................................................................................................................... 110 Concluding remarks ................................................................................................................... 111 Material and Methods ................................................................................................................... 111 Plant growth conditions ............................................................................................................. 111 Phenotypic analysis .................................................................................................................... 112 Generation of Physcomitrella patens mutants .......................................................................... 113 Microscopy and statistical analysis ........................................................................................... 113 Protein extraction and immunoblotting ................................................................................... 114 RNA extraction and quantitative RT-PCR .............................................................................. 114 Supplemental .................................................................................................................................. 115 References ....................................................................................................................................... 152 Manuscript 1: Remarkable Regulatory Rosettas in the bryophyte Physcomitrella patens ..... 162 Abstract ........................................................................................................................................... 162 Introduction ...................................................................................................................................