La lecture en ligne est gratuite
Le téléchargement nécessite un accès à la bibliothèque YouScribe
Tout savoir sur nos offres
Télécharger Lire

Molecular and physiological characterisation of the two Arabidopsis thaliana mutants atpd and petc [Elektronische Ressource] / vorgelegt von Daniela Sabine Maiwald

De
155 pages
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 b /f complex (cyt b /f)..................................................................... 4 6 61.4 Mutational analysis of the cyt b /f complex.............................. 5 61.5 The chloroplast ATP synthase................................................................................... 8 1.6 The d-subunit of the ATP synthase......... 11 1.
Voir plus Voir moins





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 b /f complex (cyt b /f)..................................................................... 4 6 6
1.4 Mutational analysis of the cyt b /f complex.............................. 5 6
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 -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..... 51
4 DISCUSSION............................................................................................................. 54
4.1 The petc-2 mutant.... 54
4.1.1 The absence of the Rieske protein blocks the linear electron transport .......... 54
4.1.2 The Rieske protein is involved in the stability of the cytochrome b /f ...............6
complex and has an effect on the thylakoid composition................................ 55
4.1.3 The disruption of the cyt b /f complex causes a relative increase of the 6
chloroplast ATP synthase................................................................................ 57
4.2 The atpd-1 mutant ................................... 58
4.2.1 The d-subunit is important for the stability of the chloroplast ATP synthase. 58
4.2.2 Absence of a functional ATP synthase changes the thylakoid composition ......
and favours a limited cyclic electron transport................................................ 59
4.2.3 The low luminal pH in the atpd-1 mutant induces non-photochemical
quenching even at low light intensities............................ 60
4.3 Effects on plastid signalling in the petc-2 and atpd-1 mutants................................ 61
4.4 Outlook .................................................................................................................... 62
5 SUMMARY / ZUSAMMENFASSUNG .................................................................. 63
6 REFERENCES .......................................... 65
7 APPENDIX................................................................................ 73
7.1 Detailed image of hierarchical clustering of the expression profiles ...................... 73
7.2 Direct comparison of the differential expression profiles of petc-2 and atpd-1 plants ...... 78
7.3 Complete list of significantly differentially expressed genes.................................. 84

ABBREVIATIONS IV
ABBREVIATIONS
_as anti-sense
_s sense
°C degree Celsius
1-D one-dimensional
2-D two-
A. th. Arabidopsis thaliana
aa amino acid
Amp ampicillin
ATP adenosine triphosphate
bp base pairs
Bq Becquerel
BSA bovine serum albumin
Carb carbenicillin
Chl chlorophyll
Col-0 Arabidopsis thaliana, ecotype Colmbia 0
cpTP chloroplast transit peptide
cyt cytochrome
cyt b /f cytochrome b /f complex 6 6
DCMU 3-(3-4-di-chlorophenyl)-1,1-dimethylurea
DEPS de-epoxidation state = (violaxanthin + 0.5 antheraxanthin)/
(violaxanthin + antheraxanthin + zeaxanthin)
DNA deoxyribonucleic acid
E. coli Escherichia coli
EDTA ethylenediaminetetraacetic acid
F minimal fluorescence 0
F initial fluorescence in the steady-state 0S
Fd soluble ferredoxin
F maximal fluorescence M
F ’ maximal fluorescence in light adapted leaves M
F stationary (quenched) level of maximum fluorescence MS
+FNR ferredoxin-NADP oxidoreductase
F transient fluorescence T
F /F maximal fluorescence yield of PSII V M
g gram
GST gene sequence tags
hcf high-chlorophyll-fluorescence
HPLC high pressure liquid chromatography
Kan kanamycin
l litre
LDS lithium dodecyl sulphate
Ler Arabidopsis thaliana, ecotype Landsberg erecta
LHC light harvesting complex
M molar
mol mole
mRNA messenger RNA
mV milliVolt
+NADP oxidized nicotinamide adenine dinucleotide phosphate
NPQ non-photochemical quenching
ABBREVIATIONS V
o/n over night
OD optical density
OEC oxygen evolving complex
effective quantum yield of PSII F II
P680 reaction centre of PSII
P700 reaction centre of PSI
PA polyacrylamide
PAGE polyacrylamide gel electrophoresis
PAM pulse amplitude modulation
PBQ 1-4 benzochinone
Pc plastocyanin
PCR polymerase chain reaction
Pet photosynthetic electron transport
PFD photon flux density
Phe pheophytin
PQ plastoquinone
PQH plastoquinol 2
PSI photosystem I
PSII photosystem II
Q bound plastoquinone A
Q plastoquinone able to exchange with plastoquinone (PQ) pool B
qE energy-dependent quenching
qI photoinhibition
Q plastoquinol binding pocket o
qP photochemical quenching
RNA ribonucleic acid
rpm revolutions per minute
RT room temperature
rv relative values
s second
SD standard deviation
SDS sodium dodecyl sulphate
T-DNA transfer DNA
TMPD 2,3,5,6-tetramethyl-p-phenylen-diamine
Tris Tris(hydroxymethane)aminomethane
U unit of enzyme activity given by supplier
v/v volume per volume
VAZ sum of xanthophyll cycle pigments (violaxanthin + antheraxanthin +
zeaxanthin)
w/v weight per volume
WT wild-type
.
1 INTRODUCTION 1
1 INTRODUCTION
1.1 The chloroplast and photosynthesis
By the process of photosynthesis solar light energy is converted into chemical energy,
thereby enabling most life on earth. The photosynthetic reactions are performed in a
specialized organelle, the chloroplast, which is enclosed by three systems of membranes
(Figure 1.1). It is believed that chloroplasts derive from an ancestral cyanobacterial
endosymbiont (Douglas, 1998). Chloroplasts have their own genome (plastome) and a
transcription and translation apparatus which closely resembles that of the prokaryotes.
The chloroplast is not only involved in photosynthesis, but also in the synthesis of
compounds such as amino acids, nucleotides, fatty acids and lipids, vitamins, plant
hormones and secondary metabolites and it also plays a central role in the assimilation of
sulphur and nitrogen.

Figure 1.1 Structure of the chloroplast shown as a diagrammatical representation
and an electron microscope micrograph.

Photosynthesis in higher plants consists of a series of light-driven reactions which in turn
lead to the generation of ATP and NADPH used for the fixation of atmospheric CO into 2
organic compounds. The protein complexes involved in photosynthesis are photosystem II
(PSII), the cytochrome (cyt) b /f complex, photosystem I (PSI) and the ATP synthase, all 6
1 INTRODUCTION 2
located in the thylakoid membranes of the chloroplast (Figure 1.2). Photons are absorbed
mainly by the light-harvesting complexes (LHCs) of PSI and PSII.

Figure 1.2 Schematic view of the photosynthetic apparatus.
Membrane-located multi-protein complexes: LHCII, light harvesting complex of PSII;
PSII, photosystem II; Cyt b /f complex, cytochrome b /f complex; PSI, photosystem I; 6 6
LHCI, light harvesting complex of PSI. The soluble electron carriers: PC,
plastocyanin; Fd, ferredoxin; Cyt c , cytochrome c and the membrane-bound electron 6 6
carrier FNR, ferredoxin-NADPH-reductase. Nuclear-encoded subunits are indicated in
grey, or black if they are plant specific. Chloroplast encoded subunits are drawn in
white.
1.2 The photosynthetic electron transport chain
The first complex in the photosynthetic electron transport chain, accepting electrons from
H O, is PSII which acts as a water-plastoquinone oxireductase (Renger and Govindjee, 2
1985). When the reaction centre of PSII (P680) becomes photoexcited it reduces the
quinone Q via a rapid oxidoreduction of a pheophytin molecule (Phe) (Figure 1.3). Q A A
then reduces the two-electron carrier plastoquinone (Q ). When Q is fully reduced, it B B
binds two protons and is thereby converted to plastoquinol (PQH ) which is not any longer 2
bound to PSII. PQH moves freely in the thylakoid membrane and connects PSII and the 2
cyt b /f complex to which it transfers the electrons. The cyt b /f complex acts as a 6 6
plastoquinol-plastocyanin oxidoreductase. It catalyses the oxidation of the lipophilic two-
electron carrier donor PQH and the reduction of the hydrophilic one-electron acceptor 2
protein plastocyanin (Pc).
1 INTRODUCTION 3

Figure 1.3 A scheme of the electron transport in oxygen evolving photosynthetic
organisms (Hill and Bendall, 1960).
M, components of the oxygen evolving complex; Z, primary electron donor to P680;
P680, the reaction centre of PSII; Phe, pheophytin; Q , bound plastoquinone; Q , A B
plastoquinone able to exchange with the plastoquinone (PQ) pool; ReFeS, Rieske iron-
sulphur centre; cyt, cytochrome; Pc, plastocyanin; P700, reaction centre of PSI; A , A , 0 1
A , primary and secondary electron acceptors of PSI; FeS , bound iron-sulphur 2 A,B
+centres A and B; Fd, soluble ferredoxin; FNR, ferredoxin-NADP oxidoreductase;
+NADP , oxidized nicotinamide adenine dinucleotide phosphate.

Three subunits of the cyt b /f complex bear prosthetic groups necessary for the electron 6
transport: cyt b bearing two b-type heme moieties (E = -84 mV and – 158 mV) (Pierre et 6 m
al., 1995); cyt f bearing a covalently bound c-type heme moiety (E = + 330 mV), (Pierre m
et al., 1995); and the Rieske protein bearing a [Fe S ] cluster (E = + 290 mV), (Nitschke 2 2 m
et al., 1992). According to the Q-cycle model the transfer of the two electrons is branched
in the complex between the low-potential chain (composed of the two b type hemes, b 6 l
and b ) and the high-potential chain (formed by the Rieske protein and cyt f) (Mitchell, h
1975; Crofts et al., 1983). This mechanism postulates both an oxidation and a reduction of
plastoquinol at two distinct sites of the protein, the Q and the Q sites, on opposite sites of o i
the membrane. The oxidation of plastoquinol at the Q site is associated with the reduction o
of cyt f and cyt b (Hope, 1993; Hauska et al., 1996) and the release of two protons into the 1
lumen. Two in-series turnovers of the complex are required to reduce both b - and b -l h
hemes and trigger the generation of a PQH molecule at the Q site. The cyt f then finally 2 i
reduces the plastocyanin which can (re-) reduce PSI. PSI acts as a plastocyanin-ferredoxin