UVR8: a plant UV-B photoreceptor Inaugural-Dissertation zur Erlangung der Doktorwürde der Fakultät für Biologie der Albert-Ludwigs-Universität Freiburg im Breisgau Vorgelegt von Luca Rizzini Freiburg, Dezember 2010 Dekan: Prof. Dr. Gunther Neuhaus Promotionsvorsitzender: Prof. Dr. Samuel Rossel Prof. Dr. Stefan Rotter Prof. Dr. Karl-Friedrich Fischbach Betreuer der Arbeit: Prof. Roman Ulm Referent: Prof. Roman Ulm Koreferent: PD Gerhard Leubner Tag der Verkündigung des Prüfungsergebnisses: 21-02-2011 TABLE OF CONTENTS SUMMARY ................................................................................................................. 5 LIST OF ABBREVIATIONS .................................................................................................. 7 1 INTRODUCTION ............................................................................................... 11 1.1 The Photoreceptors ............................................................................................... 12 1.1.1 Phytochromes ....................................................................................................... 13 1.1.2 Cryptochromes ..................................................................................................... 17 1.1.3 Phototropins ......................................................................................................... 22 1.1.4 LOV Domains and Zeitlupe Photoreceptors ........................................................ 24 1.1.5 Chimeric Photoreceptors ...................................................................................... 26 1.2 Photoreceptor Systems not Present in Plants ....................................................... 28 1.2.1 BLUF .................................................................................................................... 28 1.2.2 Rhodopsins ........................................................................................................... 28 1.2.3 The Aryl Hydrocarbon Receptor .......................................................................... 29 1.3 UV-B Radiation .................................................................................................... 29 1.3.1 UV-B Damage ...................................................................................................... 30 1.3.2 UV-B Damage Signaling ..................................................................................... 31 1.3.3 UV-B non-Damage Response .............................................................................. 32 1.3.4 UV-B Perception .................................................................................................. 35 1.4 Components of the Low-Fluence Rate UV-B Pathway ....................................... 36 1.4.1 HY5 ...................................................................................................................... 39 1.4.2 COP1 .................................................................................................................... 40 1.4.3 UVR8 ................................................................................................................... 42 1.5 Aim of This Work ................................................................................................ 45 2 MATERIALS AND METHODS .......................................................................... 46 2.1 Materials ............................................................................................................... 46 2.1.1 Plant Material and Media ..................................................................................... 46 2.1.2 Bacterial Strains and Media ................................................................................. 46 2.1.3 Yeast Strains and Media ....................................................................................... 47 2.1.4 Plasmids, Oligonucleotides and Antibodies ......................................................... 47 2.1.5 Enzymes and Reagents ......................................................................................... 51 2.2 Methods ................................................................................................................ 52 2.2.1 Plant Growth ........................................................................................................ 52 2.2.2 Plant Protein Extraction ....................................................................................... 52 2.2.3 Cell-Free Degradation Assay ............................................................................... 53 2.2.4 Yeast Growth and Transformation ....................................................................... 53 2.2.5 Yeast Protein Extraction ....................................................................................... 54 2.2.6 HEK293T Cells Growth and Transformation ...................................................... 54 2.2.7 Protein Extraction from Transfected HEK293T Cells ......................................... 55 2.2.8 UV-B Treatments ................................................................................................. 55 2.2.9 Agrobacterium Mediated Plant Transformation .................................................. 57 2.2.10 DNA Isolation ...................................................................................................... 57 2.2.11 PCR ...................................................................................................................... 58 2.2.12 Agarose Gel Electrophoresis ................................................................................ 58 2.2.13 Site-Directed Mutagenesis ................................................................................... 58 2.2.14 CPRG Assay ......................................................................................................... 59 2.2.15 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) ..................................... 60 2.2.16 Immunoblot Analysis ........................................................................................... 60 2.2.17 BiFC ..................................................................................................................... 61 2.2.18 Luciferase Measurement ...................................................................................... 61 2.2.19 Bioinformatic Analysis ........................................................................................ 62 3 RESULTS .......................................................................................................... 63 3.1 COP1-UVR8 Interaction in Yeast ........................................................................ 63 3.2 UVR8 Homodimer ............................................................................................... 69 3.3 UV-B-Dependent Monomerization of UVR8 ...................................................... 71 3.3.1 UV-B-Dependent Monomerization of UVR8 in HEK293T Cells ....................... 71 3.3.2 UV-B-Dependent Monomerization of UVR8 in Yeast ........................................ 75 3.3.3 UV-B Dependent Monomerization of UVR8 in Planta ....................................... 78 3.3.4 UV-B-Dependent UVR8 Degradation ................................................................. 81 3.4 Evolutionary and Structural Considerations......................................................... 83 3.5 Site-Directed Mutagenesis ................................................................................... 88 3.6 Mixing-Extracts Experiment ................................................................................ 92 3.7 Physcomitrella UVR8 Ortholog ........................................................................... 93 3.8 UVR8 and HY5 Compete for COP1 Interaction .................................................. 95 3.9 RUPs ..................................................................................................................... 96 3.9.1 RUPs UV-B-Dependent Interaction with UVR8 ................................................. 96 3.9.2 RUPs Mechanism ................................................................................................. 96 4 DISCUSSION .................................................................................................... 98 4.1 UVR8 and COP1 Interaction in Heterologous System ........................................ 98 4.2 UVR8 Self-Interaction and UV-B Dependent Monomerization .......................... 99 4.3 UVR8 Protein Putative Conformational Change ............................................... 101 4.4 Phylogenetic and Structural Considerations....................................................... 103 4.5 UV-B Perception by UVR8 ................................................................................ 104 4.6 UVR8 Mechanism .............................................................................................. 106 4.7 RUPs Mechanism ............................................................................................... 109 4.8 Conclusions and Outlook ................................................................................... 110 ACKNOWLEDGEMENTS ...................................................................................... 112 5 REFERENCES ................................................................................................ 113 Summary Ultraviolet-B radiation is part of the sunlight spectrum reaching the Earth. The high energy per photon of this wavelength range can cause ROS production,