Assembly and function of photosystem I in higher plants [Elektronische Ressource] / vorgelegt von Katrin Amann

Katrin Amann

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 22
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Assembly and Function of Photosystem I
in Higher Plants





Dissertation
zur Erlangung des Doktorgrades der Fakultät für Biologie
der Ludwig-Maximilians-Universität München
Department Biologie I, Botanik




vorgelegt von
Katrin Amann
aus Schliersee
1





Erstgutachter: Prof. R.G. Herrmann
Zweitgutachter: Prof. H.-U. Koop

Datum der mündlichen Prüfung: 20.01.2006












2

Content

Abbreviations 7-9

I. Introduction 10-18
1. Oxygenic photosynthesis 10-11
2. Structure and function of photosystem I 11-12
3. Photosystem II, cytochrome b f-complex, ATP-synthase complexes 12-13 6
4. Biogenesis of thylakoid membrane protein complexes 13-14
5. PSI-LHCI-biogenesis 14-15
6. Nuclear control of plastid gene expression 15-16
7. Arabidopsis thaliana as a model organism 16
8. Functional genomics 17
9. Function of PSI-J in higher plants 17-18

II. Materials and Methods 19-41
1. Materials 19-27
1.1 Working materials 19
1.2 Plant material 19
1.3 Bacterial strains and vectors 19-20
1.4 Hybridization probes for Northern analysis 21
1.5 Oligonucleotides 22-23
1.6 cDNA library 23
1.7 Media, solutions and buffers 23-25
1.8 Antibodies 26-27

2. Methods 28-41
2.1 Cultivation of bacteria 28
2.2 Seed sterilization, medium and culture conditions 28
2.3 Molecular biological methods 28-29
2.4 Vector construction, chloroplast transformation and plant material 29
2.5 DNA analysis 29
2.5.1 Polymerase chain reaction (PCR) 29-30
3Content

2.5.2 DNA isolation 30
2.5.3 Inverse PCR (iPCR) 30
2.5.4 GFP fusion 30- 31
2.5.5 Radioactive labelling of DNA 31
2.5.6 Hybridization of nucleic acids 31
2.6 RNA analysis 31-34
2.6.1 Isolation of total RNA 31
2.6.2 Northern analysis 32
2.6.3 RNA gel blot analysis 32
2.6.4 Primer extension 32
2.6.5 Reverse transcription (RT)-PCR 33
2.6.6 Quantitative real-time RT-PCR 33
2.6.7 Polysome analysis 33-34
2.7 Translation inhibition experiment 34
2.8 Protein and pigment analysis 34-37
2.8.1 Measurement of protein and chlorophyll concentration in Arabidopsis 34
2.8.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 34
2.8.3 Immunological analysis 34-35
2.8.4 Radioactive labelling of chlorolast proteins 35
2.8.5 Separation of thylakoid membrane complexes 35
2.8.6 Protein import into chloroplasts 35-36
2.8.7 Isolation of thylakoid membranes and PSI assemblies from tobacco 36
2.8.8 Chlorophyll content per leaf area 36
2.8.9 Immunoblotting 36-37
2.9 Spectroscopic and fluorimetric methods 37-40
2.9.1 Chlorophyll a fluorescence analysis 37
2.9.2 Light-induced change of the P700 redox state 37
2.9.3 Photochemical and non-photochemical chlorophyll a fluorescence quenching 37
2.9.4 Low temperature chlorophyll fluorescence spectra 38
2.9.5 Fluorescence measurements in tobacco 38
+
2.9.6 NADP photoreduction measurements and Chl/P700 38
2.9.7 Antenna size of PSI 38-39
2.9.8 Kinetic measurements 39
4Content

2.9.9 P700 oxidation state in WT and ΔPSI-J leaves 39-40
2.10 Genetic methods 40
2.10.1 Mapping of the apo1 mutation 40
2.10.2 Complementation of the apo1 mutant 40
2.11 Sequence analysis 40-41
2.12 Chloroplast ultrastructure 41
2.13 GenBank accession numbers 41

III. Results 42-69
Part A: Characterisation of the apo1 mutant 42-58
1. Selection, phenotype and growth of apo1 42
2. Fluorometric and spectroscopic characteristics of the photosynthet