O'Hara, Andrew (2012) The importance of specific tryptophans to UVR8 function: an intrinsic chromophore for a UV-B photoreceptor. PhD thesis. http://theses.gla.ac.uk/4012/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] THE IMPORTANCE OF SPECIFIC TRYPTOPHANS TO UVR8 STRUCTURE AND FUNCTION: AN INTRINSIC CHROMOPHORE FOR A UV-B PHOTORECEPTOR By Andrew O’Hara Thesis submitted for the degree of Doctor of Philosophy College of Medical, Veterinary & Life Sciences Institute of Molecular, Cell and Systems Biology University of Glasgow August, 2012 © Andrew O’Hara, 2012 I “This new power for the driving of the world’s machinery will be derived from the energy which operates the universe, the cosmic energy, whose central source for the earth is the sun and which is everywhere present in unlimited quantities” Nikola Tesla “What is a weed? A plant whose virtues have not yet been discovered” Ralph Waldo Emerson II To my Mum and Dad, Elizabeth and Thomas O’Hara and my son Ben III CONTENTS Title……………………………………………………………………………………… i Dedication……………………………………………………………………………….iii Contents…………………………………………………………………………………iv Figures………………………………………………………………………..…………xi Tables……………………………………………………………………………………xiv Acknowledgments………………………………………………………………………xv Publications……………………………………………………………………………..xvi Author’s Declaration…………………………………………………………………..xvii Abbreviations…………………………………………………………………………..xviii Summary………………………………………………………………………………..xxi Chapter 1 UV-B Perception and Signal Transduction in Plants 1.1 Introduction: UV-B Perception and Signal Transduction in Plants …………. 1 1.1.1 What was known when I first started this project? …………………………… 4 1.1.2 Recent advances………………………………………………………………… 8 1.1.3 Aims of the chapter……………………………………………………………… 8 1.2 Photoreception and photoreceptors…………………………………………… 9 1.2.1 Phytochromes………………………………………………………………….… 9 1.2.2 Cryptochromes…………………………………………………………………..12 1.2.3 Phototropins and other LOV containing photoreceptors and proteins …………………………………………………………………………………………….14 1.3 Potential harmful effects of UV-B………………….………………………….17 1.4 UV-B specific and non-specific responses…………………………………..18 1.5 UV-B perception and signalling………………………………………………..20 1.6 UVR8 and downstream components………………………………………….24 1.7 The multi functional E3 ubiquitin ligase COP1……………………………….26 1.8 The brakes of the UVR8 signalling pathway, RUP1 and RUP2 control the negative feedback loop…………………………………………………………………29 1.9 Possible importance of tryptophan to UVR8 function and UV-B perception and signalling……………………………………………………………………………30 1.10 Aims and probable and possible experimental approaches………………..32 IV Chapter 2 Materials and Methods 2.1 Materials…………………………………………………………………………35 2.1.1 Chemicals……………………………………………………………………….35 2.1.2 Primers…………………………………………………………………………...35 2.1.3 Antibiotics………………………………………………………………………..35 2.1.4 Enzymes…………………………………………………………………………36 2.1.5 Reagents for Protein Quantification, Electrophoresis and Immunoblot…..36 2.1.6 Yeast and Bacterial Strains……………………………………………………36 2.1.7 Plasmid Vectors…………………………………………………………………36 2.1.8 Antibodies………………………………………………………………………..37 2.2 Preparation of Solutions and Media…………………………………………38 2.2.1 pH Measurements……………………………………………………………..38 2.2.2 Autoclave Sterilisation…………………………………………………………38 2.2.3 Filter Sterilisation……………………………………………………………….38 2.3 Plant Material……………………………………………………………………38 2.3.1 Seed Stocks……………………………………………………………………..38 2.3.2 Growth of Arabidopsis Plants on Soil…………………………………………38 2.3.3 Surface Sterilisation of Arabidopsis Seeds…………………………………..39 2.3.4 Growth of Arabidopsis Plants on Agar Plates………………………………..39 2.4 Plant, E. coli and Yeast Treatments............................................................39 2.4.1 Light Sources……………………………………………………………………39 2.4.2 Light Fluence Rate Measurements……………………………………………40 2.4.3 UV-B Sensitivity and Hypocotyl Length Assay……………………………….40 2.5 Nucleic Acid Isolation and Manipulations …………………………………….41 2.5.1 RNA Isolation from Arabidopsis Leaf Tissue…………………………………41 2.5.2 Removal of Genomic DNA from Purified RNA by DNase Treatment..........41 2.5.3 Transformation of E. coli Cells…………………………………………………42 2.5.4 Isolation of Genomic DNA from Arabidopsis Plants…………………………42 2.5.5 Isolation of Plasmid DNA……………………………………………………….42 2.5.6 Restriction Enzyme Endonuclease Digestion………………………………43 2.5.7 DNA Ligation ……………………………………………………………………43 2.5.8 DNA Sequencing………………………………………………………………43 2.5.9 Quantification of DNA and RNA………………………………………………43 2.5.10 Amplification of DNA by Polymerase Chain Reaction……………………..44 V 2.5.11 Agarose Gel Electrophoresis of DNA…………………………………………44 2.6 Quantitative and Semi-quantitative Reverse-Transcriptase Polymerase Chain Reaction…………………………………………………………………………45 2.6.1 cDNA synthesis…………………………………………………………………45 2.6.2 qPCR- Quantitative PCR………………………………………………………45 2.6.3 Semi-quantitative RT-PCR…………………………………………………….47 2.6.4 Site Directed Mutagenesis of plasmid DNA…………………………………47 2.7 Generation of stable transgenic Arabidopsis lines…………………………48 2.7.1 Generation of UVR8 trp mutant Fusion Constructs for Stable Expression Studies in Arabidopsis…………………………………………………………………48 2.7.2 Preparation of Competent Agrobacterium Cells for Electroporation……...48 2.7.3 Transformation of Competent Agrobacterium Cells by Electroporation ……………………………………………………………………………............49 2.7.4 Agrobacterium-mediated Transformation of Arabidopsis by Floral Dip ……………………………………………………………………………...49 2.7.5 Transient Expression of Gene Constructs in N. benthamiana by A. tumefaciens Infiltration…………………………………………………………..50 2.8 Protein Methods…………………………………………………………..51 2.8.1 Protein Isolation from Arabidopsis and Nicotiana benthamiana plants ……………………………………………………………………………...51 2.8.2 Quantification of Protein Concentration……………………………….51 2.8.3 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Native PAGE…………………………………………………………………52 2.8.4 Western Blot Transfer……………………………………………………52 2.8.5 Stripping of Immunolabelled Protein Membrane ………………………53 2.8.6 Co-immunoprecipitation of GFP Tagged UVR8 from Plant Extracts using uMAC TM Beads…………………………………………………………………53 2.8.7 Monomer/dimer Kinetics………………………………………………….54 2.8.8 Immunodetection………………………………………………………….54 2.9 Chromatin Immunoprecipitation (ChIP) Assay………………………….54 2.9.1 ChIP of Arabidopsis Plant Tissue…………………………………………54 2.9.2 Conditions of PCR on ChIP……………………………………………….56 2.10 Confocal Microscopy……………………………………………………….56 VI 2.10.1 Confocal Microscopy of Nicotiana benthamiana for Transient Expression of GFP-UVR8 and GFP-UVR8 Trp mutants and stable expression of GFP-UVR8 Trp mutants in Arabidopsis Plants……………………………………………………..56 2.11 Yeast-Two-Hybrid Methods…………………………………………………57 2.11.1 Yeast Transformation with Plasmid DNA………………………………….57 2.11.2 Isolation and Determination of Protein Expression from Cells Used for Yeast-2-hybrid…………………………………………………………………………57 2.11.3 Yeast Expression of UVR8 and Extraction…………………………58 2.12 Dose Response Curves and Action Spectrum of UVR8 Monomer/dimer Kinetics………………………………………………………………………………...59 Chapter 3 Identification of Trp mutant candidates of UVR8, by site directed mutagenesis, transient expression in Nicotiana benthamiana to check for stability, and Y2H to test for homodimerization and interaction with COP1, RUP1 and RUP2. 3.1 Introduction……………………………………………………………………64 3.2 Primary and Predicted Secondary structure of UVR8…………………….64 3.3 Transient expression of Trp UVR8 mutants in Nicotiana benthamiana…66 3.4 Yeast-2-hybrid, using UVR8 Trp mutant variants as bait, for the detection of homodimerization and interaction with COP1………………………………………67 3.5 UVR8 forms homodimers that dissociate at low fluence rates of narrowband UV-B and interacts with COP1 only after UV-B irradiation…………………………68 3.6 W39A, W144A and W352A cause loss of interaction with COP1 and loss of homodimerization whereas W39F or Y, W144F or Y and W352F or Y restores COP1 interaction but also causes loss of homodimerization………………………69 3.7 Mutation of W233A, W285A or W337A singly or collectively causes constitutive interaction with COP1……………………………………………………70 3.8 Mutation of W233, W285 and W337 to Y or F cause loss of COP1 interaction and constitutive homodimerization………………………………………71 3.9 Mutation of W196/198A, W92/94A, W300/302A, W250A or W400A does not affect COP1 interaction or homodimerization………………………………………71 3.10 Mutation of glycines 197 or 199, within the 5 amino acid deletion WGWGR of uvr8-1, to alanine causes loss of interaction with COP1 and loss of homodimerization………………………………………………………………………72 VII 3.11 The effect of the Trp mutants of UVR8 on RUP1 and RUP2 interaction, negative regulators of the UVR8 pathway……………………………………………72 3.12 Mutation of W39, W144 and W352 to Y or F do not affect RUP1 and RUP2 interaction but W39A, W144A and W352A cause a loss of RUP1 and RUP2 interaction in both dark and UV-B conditions……………………………………….73 3.13 Mutation of Trps 233, 285 and 337 to Ala, Tyr or Phe do not affect interaction with RUP1 or RUP2……………………………………………………….73 3.14 Mutation