Molecular and physiological characterisation of the two Arabidopsis thaliana mutants atpd and petc I n a u g u r a l – D i s s e r t a t i o n zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Daniela Sabine Maiwald aus Hilden Kopier Center Süd, Düsseldorf 2003 Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf. Refernt: Prof. Dr. P. Westhoff 1. Koreferent: Prof. Dr. W. Martin 2. Koreferent: Prof. Dr. F. Salamini Tag der mündlichen Prüfung: 02.07.2003 CONTENTS I CONTENTS ABBREVIATIONS............................................................................................................IV 1 INTRODUCTION ....................................................................................................... 1 1.1 The chloroplast and photosynthesis........................................................................... 1 1.2 The photosynthetic electron transport chain.............................................................. 2 1.3 The cytochrome b6/f complex (cyt b6/f)..................................................................... 4 1.4 Mutational analysis of the cyt b6/f complex .............................................................. 5 1.5 The chloroplast ATP synthase................................................................................... 8 1.6 The d-subunit of the ATP synthase ......................................................................... 11 1.7 Mutational analysis of the ATP synthase ................................................................ 12 1.8 Chlorophyll fluorescence, photochemical and non-photochemical quenching and the role of the xanthophyll cycle....................................................................... 14 1.9 Aim of the thesis...................................................................................................... 16 2 MATERIAL & METHODS ..................................................................................... 17 2.1 Chemicals and enzymes........................................................................................... 17 2.2 Strains ...................................................................................................................... 17 2.3 Media and cultivation .............................................................................................. 17 2.3.1 E. coli: Luria Broth (LB) Medium .................................................................. 17 2.3.2 A. tumefaciens: YEB medium ......................................................................... 18 2.3.3 A. thaliana axenic culture: Murashige & Skoog (MS) medium...................... 18 2.4 Plant propagation..................................................................................................... 18 2.4.1 On soil ............................................................................................................. 18 2.4.2 Sterile culture................................................................................................... 18 2.5 Oligonucleotide sequences ...................................................................................... 19 2.6 Gel electrophoresis .................................................................................................. 19 2.6.1 Agarose gel electrophoresis............................................................................. 19 2.6.2 Polyacrylamide gel electrophoresis................................................................. 20 2.7 Enzymatic reactions................................................................................................. 20 2.7.1 PCR.................................................................................................................. 20 2.7.2 Ligation of DNA fragments............................................................................. 20 2.7.3 Restriction analysis.......................................................................................... 20 2.7.4 Radioactive labelling of DNA ......................................................................... 20 CONTENTS II 2.7.5 cDNA single strand synthesis.......................................................................... 21 2.8 Sequence analyses ................................................................................................... 21 2.9 Plasmids................................................................................................................... 21 2.9.1 pGEM-Teasy ................................................................................................... 21 2.9.2 pPVC702 ......................................................................................................... 21 2.9.3 pPVC702-AtpD................................................................................................ 21 2.10 Nucleic acid preparation.......................................................................................... 22 2.10.1 DNA preparation ............................................................................................. 22 2.10.2 RNA preparation ............................................................................................. 22 2.11 Northern analysis..................................................................................................... 22 2.12 Transformation of E. coli......................................................................................... 23 2.13 Complementation analysis of Arabidopsis plants via A. tumefaciens ....................... transformation.......................................................................................................... 23 2.13.1 Transformation of Arabidopsis using A. tumefaciens ..................................... 23 2.13.2 Selection of transformed plants ....................................................................... 23 2.14 Thylakoid preparation from Arabidopsis leaves ..................................................... 23 2.14.1 For 2-D gel electrophoresis and Western analysis .......................................... 23 2.14.2 For electron transport measurements............................................................... 24 2.15 Western blot analysis............................................................................................... 24 2.16 2-D gel electrophoresis............................................................................................ 24 2.17 Pigment analysis...................................................................................................... 25 2.18 Chlorophyll fluorescence measurements and redox state of P700.......................... 25 2.19 Membrane potential measurements......................................................................... 26 2.20 Electron transport measurements ............................................................................ 26 2.21 Expression profiling ................................................................................................ 27 2.21.1 Probe synthesis ................................................................................................ 27 2.21.2 Hybridisation procedure .................................................................................. 27 2.21.3 Data analysis.................................................................................................... 28 3 RESULTS................................................................................................................... 29 3.1 Characterisation of the petc-2 mutant...................................................................... 29 3.1.1 Identification of the Rieske protein mutant lines............................................. 29 3.1.2 Phenotype of the petc mutant lines.................................................................. 30 3.1.3 Expression analysis of the PetC gene.............................................................. 31 3.1.4 The pigment composition in the petc-2 mutants ............................................. 32 3.1.5 The composition of the photosynthetic apparatus in the petc-2 plants............ 33 CONTENTS III 3.1.6 Photosystems are functional in petc-2, but there is no linear electron ............... transport........................................................................................................... 35 3.2 Characterisation of the atpd-1 mutant ..................................................................... 39 3.2.1 Phenotype of the atpd-1 mutant line ............................................................... 39 3.2.2 Expression analysis of the AtpD gene and complementation analysis of the atpd-1 mutation ............................................................................................... 40 3.2.3 ATP synthase activity in atpd-1 mutant plants................................................ 41 3.2.4 Leaf pigment composition in the atpd-1 mutants............................................ 42 3.2.5 The composition of the photosynthetic apparatus in the atpd-1 plants ........... 43 3.2.6 Photosystems are functional in atpd-1 mutants, but linear electron transport .... is impaired ....................................................................................................... 45 3.3 mRNA expression analysis for nucleus-encoded chloroplast proteins in the mutants petc-2 and atpd-1 ....................................................................................................