Evolution, function and manipulation of methyl halide production in plants Evelyn Koerner A thesis submitted to the University of East Anglia for the degree of Doctor of Philosophy John Innes Centre Norwich, UK September 2012 © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. Acknowledgements Firstly, I would like to thank my supervisor Lars Østergaard for giving me the opportunity to work on this exciting project in his lab and for his guidance and support. I would also like to thank current and past members of the Østergaard lab for their help, especially Pauline Stephenson, Thomas Girin, Sara Fuentes and Alice Baillie. A special thanks goes to Bill Sturges, Hannah Newton and Stephen Humphrey for analysing the methyl halide samples. I would also like to thank Anna Jordan, Gavin Hatt and Ian Bedford for their help with the insect experiments, and Andrzej Tkacz and Phil Poole for hosting me in their lab to do the rhizosphere work. Moreover, I would like to thank Rene Dreos and Sam Mugford for their help with the microarray analysis. I am grateful to Nick Brewin, Mike Merrick, Rita Galhano, Ane Sesma, Georgina Fabro and Jonathan Jones for making the Rotation Year such a fantastic experience and another thank you to Jonathan for his support as my secondary supervisor. I would also like to thank Chie Hattori, Anastasia Gardiner, Cintia Kawashima and Kate Bailey for cheering me up during lunch or tea breaks when things were not going so well. Finally, I would like to thank my parents, my sister and my partner Moritz for their love and support during the last couple of years. The work presented here was funded by the John Innes Foundation and the Earth and Life Systems Alliance (ELSA). i Abstract Methyl halides (CH3Cl, CH3Br and CH3I) are a group of volatile organic compounds which contribute to natural ozone degradation in the atmosphere. Plants are important emitters of these compounds due to the activity of halide/thiol methyltransferases (HTMTs), however, the function of HTMTs or methyl halide production is not known. In Arabidopsis thaliana, one HTMT is primarily responsible for the production of methyl halides and encoded by the HARMLESS TO OZONE LAYER (HOL) gene. In this study, an A. thaliana hol mutant and 35S::HOL lines were used to investigate the function of HOL. No support was found for the hypothesis that HOL contributes to salt stress tolerance via the disposal of excessive amounts of halides. On the contrary, increased HOL activity made 35S::HOL plants more susceptible towards salt stress. Despite the toxicity of methyl halides, differences in HOL activity in hol mutant and 35S::HOL plants did not affect the performance of insect herbivores, nor did it alter the microbial diversity of the rhizosphere in these lines. Microarray analysis of WT and hol mutant plants pointed to a function of HOL in starch/carbon metabolism and stress response pathways in A. thaliana. Brassica crops are significant emitters of methyl halides. A HOL-homologous gene (BraA.HOL.a) was identified in Brassica rapa. It was confirmed that this gene contributes to methyl halide production in B. rapa since braA.hol.a mutants had significantly reduced emission levels compared to WT. HOL-homologous genes were also found in various plant species throughout the plant kingdom including the moss Physcomitrella patens, which was shown to produce CH3Br. These data show that methyl halide production is an ancient mechanism in land plants. Moreover, phylogenetic analysis revealed a clear separation between HTMTs from glucosinolate (GL)-containing plants and HTMTs from eudicots without GLs supporting the hypothesis of a novel function of HTMTs in the order Brassicales. ii Table of Contents Table of Contents Acknowledgements .................................................................................................................... i Abstract ..................................................................................................................................... ii Table of Figures ...................................................................................................................... vii Table of Tables ......................................................................................................................... x List of Abbreviations ............................................................................................................... xi Chapter 1 – General Introduction ............................................................................................. 1 1.1 Methyl halides and ozone degradation ................................................................................ 1 1.2 Sources and sinks of methyl halides ................................................................................... 3 1.3 Abiotic methyl halide production from senescent and dead leaves .................................... 7 1.4 Production of methyl halides by methyltransferases .......................................................... 9 1.5 Function of methyl halide production and HTMTs .......................................................... 12 1.6 Objectives of this thesis .................................................................................................... 16 Chapter 2 – Materials and Methods ........................................................................................ 17 2.1 Plant material and growth conditions ................................................................................ 17 2.1.1 Arabidopsis thaliana .................................................................................................. 17 2.1.2 Brassica rapa ............................................................................................................. 17 2.1.3 Oryza sativa ............................................................................................................... 17 2.1.4 Physcomitrella patens ................................................................................................ 18 2.2 DNA extraction ................................................................................................................. 18 2.3 RNA extraction and cDNA synthesis ............................................................................... 19 2.4 Creation of transgenic A. thaliana lines ............................................................................ 19 2.4.1 Plasmid construction .................................................................................................. 19 2.4.1.1 Generation of 35S::HOL, 35S::PpHOL, 35S::OsHOL1, 35S::OsHOL2 and 35S::PtHTMT constructs................................................................................................. 19 2.4.1.2 Generation of HOL::GUS construct.................................................................... 20 2.4.1.3 Generation of HOL::HOL:GFP and 35S::GFP constructs................................. 20 2.4.2 Sequencing ................................................................................................................. 20 2.4.3 Transformation into A. thaliana ................................................................................. 21 2.5 Creation of DR5::GFP x hol and DR5::GFP x 35S::HOL crosses .................................. 21 2.6 Generation of Physcomitrella Pphol knockout lines ........................................................ 21 2.7 Genotyping of braA.hol.a mutants .................................................................................... 22 2.8 RT-PCR and Quantitative Real-Time PCR ...................................................................... 22 2.9 Microarray analysis ........................................................................................................... 23 2.10 Methyl halide measurements .......................................................................................... 24 2.11 Salt stress and KSCN experiments .................................................................................. 25 iii Table of Contents 2.12 Sucrose experiment ......................................................................................................... 25 2.13 Auxin experiments .......................................................................................................... 25 2.14 Chlorophyll measurements ............................................................................................. 26 2.15 Glucosinolate analysis .................................................................................................... 26 2.16 GUS activity.................................................................................................................... 26 2.17 Iodine staining for visualisation of starch ....................................................................... 27 2.18 Microscopy ..................................................................................................................... 27 2.19 Herbivore experiments .................................................................................................... 28 2.20 Rhizosphere community profiling .................................................................................. 28 2.20.1 Experimental setup ..................................................................................................