Identification of novel nuclear factors required for chloroplast gene expression and photosystem I assembly [Elektronische Ressource] / vorgelegt von Lina Lezhneva

Lezhneva Lina

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 9 Mo

Extrait

Identification of novel nuclear factors
required for chloroplast gene expression and
photosystem I assembly
Dissertation
zur Erlangung des Doktorgrades der Fakultät für Biologie
der Ludwig-Maximilians-Universität München
vorgelegt von
Lina Lezhneva
aus Minsk, Weißrußland
20051. Gutachter: Prof. Dr. Reinhold G. Herrmann
2. Gutachter: Prof. Dr. H. Scheer
Tag der mündlichen Prüfung: 14.07.2005Abbreviations
µE microeinstein (1 E = 1 mol of photons)
Å Ångstrom
A PSI primary electron acceptor composed of chlorophyll a0
A PSI secondary electron acceptor composed of phylloquinone1
ATP adenosine 5′-triphosphate
bp base pairs
BSA bovine serum albumin
cDNA complementary DNA
CES control by epistasy of synthesis
Ci Curie
cpm counts per minute
DNA deoxyribonucleic acid
dNTPs deoxynucleoside triphosphates
EDTA ethylenediaminetetraacetic acid
EGTA ethylene glycol-bis(2-aminoethylether)-N,N,N_,N_-tetraacetic acid
EMS ethyl methanesulfonate
ESTs expressed sequence tags
F [4Fe-4S] cluster on PsaC subunitA
FB
FTR ferredoxin-thioredoxin reductase
F interpolypeptide [4Fe-4S] cluster between PsaA and PsaB subunitsx
g gravity force, gramme
GTP guanosine 5′-triphosphate
hcf high chlorophyll fluorescence
HEPES N-[2-Hydroxyethyl]piperazine-N’-[2-ethanesulfonic acid]
K Kelvin
kb kilobases
kDa kilodalton
LHCI chlorophyll-binding PSI light-harvesting complex
Mb megabases
MES 2-Morpholinoethanesulfonic acid
MOPS 3-[N-Morpholino]propanesulfonic acid
2mRNA messenger RNA
+NADP nicotinic adenine dinucleotide phosphate
NPQ non-photochemical chlorophyll a fluorescence quenching
P700 PSI primary electron donor chlorophyll a
PAM pulse amplitude–modulated fluorometer
PCR polymerase chain reaction
PEG polyethylene glycol
PSI photosystem I
PSII photosystem II
PVDF polyvinylidene difluoride
qP photochemical chlorophyll a fluorescence quenching
RF release factor
RNA ribonucleic acid
rpm revolutions per minute
rRNA ribosomal RNA
RT-PCR reverse transcription PCR
S svedberg unit
SD standard deviation
SDS sodium dodecyl sulfate
SDS-PAGE SDS-polyacrylamide gel electrophoresis
SSLP simple sequence length polymorphism
Suc sucrose
T-DNA transferred DNA
Tricine N-Tris-(hydroxymethyl)-methylglycine
Tris Tris-(hydroxymethyl)-aminomethane
tRNA transfer RNA
Tween polyoxyethylenesorbitan monolaurate
U unit, enzyme activity
UTR untranslated region
v/v volume per volume
w/v weight per volume
3Contents
1 Introduction 9
1.1 Evolution, development and function of chloroplasts............................................. 9
1.1.1 Evolution of chloroplasts ................................................................................ 9
1.1.2 Plastid development...................................................................................... 10
1.1.3 Function of plastids 10
1.2 Biogenesis and structure of photosynthetic complexes of thylakoid membranes 11
1.2.1 Photosystem I ............................................................................................... 11
1.2.2 Photosystem II, cytochrome b f and chloroplast ATP synthase complexes .... 136
1.3 Role of nuclear-encoded factors in the expression of chloroplast genes .............. 13
1.4 Chloroplast translation.......................................................................................... 15
1.5 Arabidopsis thaliana is a model organism for the study of chloroplast
biogenesis ................................................................................................................ 16
1.6 Goals of the project................................................................................................ 18
2 Materials and methods 19
2.1 Materials 19
2.1.1 Chemicals, enzymes, radioactive substances, working materials and devices 19
2.1.2 Plant material................................................................................................ 19
2.1.3 Bacterial strains and vectors.......................................................................... 20
2.1.4 Hybridisation probes..................................................................................... 20
2.1.5 Oligonucleotides........................................................................................... 21
2.1.6 cDNA library 24
2.1.7 Media, solutions and buffers......................................................................... 24
2.1.8 Antibodies .................................................................................................... 26
42.2 Methods.................................................................................................................. 28
2.2.1 Cultivation of bacteria .................................................................................. 28
2.2.2 Seed sterilization, plant growth and mutant selection .................................... 28
2.2.3 General molecular biological methods .......................................................... 29
2.2.4 Hybridisation of nucleic acids....................................................................... 29
2.2.5 DNA analyses............................................................................................... 30
2.2.5.1 Polymerase chain reaction (PCR)................................................ 30
2.2.5.2 Rapid DNA isolation for PCR ...................................................... 30
2.2.5.3 Dot-blot transfer of DNA onto nylon membrane........................... 30
2.2.5.4 Radioactive labelling of DNA 31
2.2.6 RNA analyses 31
2.2.6.1 Isolation of total RNA .................................................................. 31
2.2.6.2 Northern analyses........................................................................ 31
2.2.6.3 Reverse transcription (RT)-PCR .................................................. 31
2.2.6.4 Quantitative real-time RT-PCR.................................................... 32
2.2.6.5 Isolation of transcriptionally active chloroplasts from
Arabidopsis ................................................................................. 33
2.2.6.6 Run-on transcription analysis...................................................... 34
2.2.7 Translation inhibition experiment ................................................................. 35
2.2.8 Protein and pigment analyses........................................................................ 35
2.2.8.1 Measurement of protein and chlorophyll concentration ............... 35
2.2.8.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE)................................................................................ 35
2.2.8.3 Western analyses ......................................................................... 35
2.2.8.4 In vivo labelling of chloroplast proteins ...................................... 36
2.2.8.5 Polysome analysis........................................................................ 36
2.2.8.6 Separation of thylakoid membrane complexes.............................. 36
2.2.8.7 Protein import ............................................................................. 37
2.2.9 Spectroscopic and fluorimetric methods........................................................ 37
2.2.9.1 Chlorophyll a fluorescence analyses ............................................ 37
2.2.9.2 Light-induced change of the P700 redox state.............................. 37
2.2.9.3 Photochemical and non-photochemical chlorophyll a
fluorescence quenching................................................................ 38
2.2.9.4 Low temperature chlorophyll fluorescence spectra ...................... 38
2.2.10 Genetic methods ........................................................................................... 38
52.2.10.1 Mapping of the hcf145 mutation .................................................. 38
2.2.10.2 Complementation of the hcf101 mutant........................................ 39
2.2.10.3hcf109 39
2.2.11 Electron microscopy ..................................................................................... 39
2.2.12 Sequence analyses ........................................................................................ 40
3 Results 41
3.1 High chlorophyll fluorescence (hcf) phenotypes of the hcf145, hcf109 and
hcf101 mutants....................................................................................................... 41
3.2 Characterisation of the hcf145 mutant ................................................................. 41
3.2.1 Activity of PSI complexes is primarily abolished in hcf145 .......................... 41
3.2.2 Accumulation of PSI subunits is dramatically affected whereas LHCI
complexes are still present in hcf145 ............................................................. 43
3.2.3 hcf145 has reduced rates of the psaA and psaB mRNA translation ................ 44
3.2.4 Amounts of the plastid psaA-psaB-rps14 transcript unit are specifically
decreased in hcf145....................................................................................... 45
3.2.5 The hcf145 mutation primarily affects the psaA-psaB-rps14 mRNA stability 45
3.2.5.1 Comparison of the transcription rates of hcf145 and wild-type .... 45
3.2.5.2 Translational events