Comparison of the chloroplast peroxidase system and characterization of the Physcomitrella patens thylakoid-bound APx Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) Submitted to the Department of Biology, Chemistry and Pharmacy of the Freie Universität Berlin by Nicola Tatjana Pitsch from Stuttgart (Germany) Berlin, July 2013 The investigations described in the following thesis were started under supervision of Prof. Dr. Margarete Baier at the Institute of Plant Sciences of the Heinrich-Heine University Düsseldorf (12/2007 - 9/2010) and continued after moving of the group at the Institute of Biology, Section Plant Physiology of the Freie Universität Berlin (10/2010 – 7/2013). 1st Reviewer: Prof. Dr. Margarete Baier (FU Berlin) 2nd Reviewer: Prof. Dr. Tina Romeis (FU Berlin) Date of defense: 17. September 2013 This thesis is dedicated to my mother… R. I. P. Acknowledgements I would like to thank Prof. Dr. Margarete Baier for the opportunity to prepare my PhD thesis in her labs and research group. Thank you for your supervision and long discussions about science. Many thanks to Prof. Dr. Tina Romeis for being the second reviewer of my thesis. I thank the research groups in Düsseldorf and Berlin for their support and nice discussions about diverse topics - especially, Dr. Hans-Peter Haschke and Dr. Christiane Hedtmann, who spend time for reading and discussing my PhD thesis. I am thanking my mom who was always there for me. She supported me in good and bad times. You will always stay in my heart. R. I. P. A big thank you to Joachim Meeßen and Marc Wenczek! You are the best friends in the world, helping me through all times! Thank you so much! I would like to thank Ilona Juszczak for being my friend, for nearly endless shopping tours, and deep discussions about science and other things. Many thanks to Manfred Loell who became a friend for many grocery shopping tours and long discussions in nice evenings. A big thank you to my remaining family in Stuttgart. Thank you for your help and support! For many relaxed evenings with nice food and discussions on sunny and rainy days. Furthermore I would like to thank Benjamin Witsch, who helped in the lab work and who brought so many gummibears to survive long computer and lab days. Finally, I would like to thank all my friends and the people I got to know in Düsseldorf and Berlin for their support and having an open ear for PhD problems. Content i List of abbreviations ................................................................................................................. 2 1 Introduction ..................................................................................................................... 6 1.1 Plant evolution .................................................................................................................. 6 1.2 Chlamydomonas reinhardtii.............................................................................................. 8 1.3 Physcomitrella patens ....................................................................................................... 9 1.4 Selaginella moellendorffii ............................................................................................... 10 1.5 Arabidopsis thaliana ....................................................................................................... 11 1.6 Reactive oxygen species ................................................................................................. 12 1.7 Antioxidative defense systems ........................................................................................ 13 1.7.1 Non-enzymatic antioxidative defense systems: Low molecular weight antioxidants .......................................................................................................... 13 1.7.1.1 Ascorbate ............................................................................................................. 13 1.7.1.2 Glutathione .......................................................................................................... 15 1.7.1.3 Other low molecular weight antioxidants ............................................................ 16 1.7.2 Enzymatic antioxidative defense systems ........................................................... 16 1.7.2.1 Ascorbate peroxidases ......................................................................................... 18 1.7.2.2 Peroxiredoxins ..................................................................................................... 20 1.7.2.3 Glutathione peroxidases ....................................................................................... 21 1.8 Aim of the study .............................................................................................................. 22 2 Material and Methods .................................................................................................. 24 2.1 Plant material and growth conditions .............................................................................. 24 2.1.1 Cultivation of Arabidopsis thaliana .................................................................... 24 2.1.1 Cultivation of Selaginella moellendorffii ............................................................ 24 2.1.2 Cultivation of Physcomitrella patens .................................................................. 24 2.1.3 Cultivation of Chlamydomonas reinhardtii ......................................................... 25 2.1.4 Cultivation of Nicotiana benthamiana ................................................................ 25 2.2 Stress treatments.............................................................................................................. 26 2.2.1 Exposure to varying light intensities and chilling temperatures .......................... 26 2.2.2 Application of ascorbate, dehydroascorbate, reduced and oxidized glutathione . 26 2.2.3 Application of abscisic acid ................................................................................. 26 2.2.4 Treatment with methylviologen ........................................................................... 27 2.3 Isolation of DNA from plant material ............................................................................. 27 2.3.1 Isolation of DNA from Arabidopsis thaliana ...................................................... 27 2.3.2 Isolation of DNA from Selaginella moellendorffii and Physcomitrella patens .. 28 2.3.3 Isolation of DNA from Chlamydomonas reinhardtii .......................................... 28 2.4 Polymerase chain reactions (PCRs) ................................................................................ 28 2.4.1 Primer design ....................................................................................................... 28 2.4.2 Standard PCRs ..................................................................................................... 31 2.4.3 Electrophoretic separation of DNA ..................................................................... 31 2.4.4 Gel extraction of PCR products ........................................................................... 32 2.5 Escherichia coli modifications ........................................................................................ 32 2.5.1 Generation of competent Escherichia coli .......................................................... 32 2.5.2 Transformation of Escherichia coli ..................................................................... 33 2.5.3 Escherichia coli colony PCRs ............................................................................. 34 2.5.4 Plasmid isolation from Escherichia coli .............................................................. 34 Content ii 2.5.5 Preparation of Escherichia coli glycerol stocks .................................................. 35 2.6 Plasmid modifications ..................................................................................................... 35 2.6.1 Ligation of PCR products into pCR8/GW/TOPO ............................................... 35 2.6.2 Ligation of PCR products into pJET1.2/blunt ..................................................... 36 2.6.3 Gateway cloning (LR reactions) .......................................................................... 36 2.7 Cleavage of double-stranded DNA with restriction enzymes ......................................... 37 2.8 Sequencing ...................................................................................................................... 37 2.9 Agrobacteria tumefaciens modifications ........................................................................ 38 2.9.1 Generation of chemically competent Agrobacteria tumefaciens ......................... 38 2.9.2 Transformation of Agrobacteria tumefaciens ...................................................... 38 2.9.3 Preparation of Agrobacteria tumefaciens glycerol stocks ................................... 39 2.10 Isolation and transfection of Arabidopsis thaliana mesophyll protoplasts ..................... 39 2.11 Transfection of Arabidopsis thaliana, Selaginella moellendorffii, Physcomitrella patens, and Nicotiana benthamiana ............................................................................................ 40 2.12 Detection of reporter gene activity .................................................................................. 41 2.12.1 Detection of luciferase activity ...........................................................................