Calibration of NPQ as a function of the external pH

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Table of contents

1 GENERAL INTRODUCTION
1.1 LIGHT AS THE FUEL OF LIFE
1.2 BIODIVERSITY OF PHOTOSYNTHETIC EUKARYOTES
1.2.1 Acquisition of photosynthesis by multiple endosymbiosis events
1.2.2 Diatoms are key players in modern oceans
1.3 PHOTOSYNTHESIS AND CHLOROPLAST
1.3.1 Chloroplast architecture in diatoms and plants
1.3.2 Photosynthetic reactions
1.3.3 The proton motive force as transient energy storage
1.4 LIGHT PROTECTION
1.4.1 Need for light protection
1.4.1.1 Light and oxidative stress
1.4.1.2 Timescales of photoprotection
1.4.2 Principle of non-photochemical quenching measurements
1.4.3 Effectors of energy quenching (qE)
1.4.3.1 Components of NPQ
1.4.3.2 Xanthophyll cycle
1.4.3.3 Specialized proteins
1.4.4 Regulation of NPQ effectors by the proton motive force
1.5 ION CHANNELS AND TRANSPORTERS IN PHOTOSYNTHESIS REGULATION
1.5.1 Chloroplastic ion channels and transporters
1.5.2 The KEA and Kef families
1.6 AIM OF THE THESIS: ION TRANSPORTERS AS A TOOL TO INVESTIGATE PHOTOPROTECTION MECHANISMS IN DIATOMS
2 NPQ IS STRICTLY CONTROLLED BY LUMEN PH IN DIATOMS
2.1 NPQ INDUCTION IN THE DARK
2.1.1 Induction of NPQ in the dark by an acid shift
2.1.2 NPQ depends on the external pH in permeabilized cells
2.1.2.1 Calibration of NPQ as a function of the external pH
2.1.2.2 NPQ dependency to pH in a natural LHCX1 knock-down
2.1.2.3 pH equilibration in the acid-quenching setup
2.2 CALIBRATION OF THE PH-DEPENDENCY OF NPQ IN P. TRICORNUTUM
2.2.1 Principle
2.2.2 Calibration of the pH-dependency of NPQ using the b6f turnover
2.3 ESTIMATION OF THE LUMEN PH BASED ON NPQ
2.4 CONCLUSION
3 IDENTIFICATION OF A HOMOLOGUE OF KEA3 IN P. TRICORNUTUM
3.1 THE KEA FAMILY IN P. TRICORNUTUM
3.1.1 The KEA family in photosynthetic eukaryotes
3.1.2 Functional protein domains in the KEA family in P. tricornutum
3.1.3 Putative subcellular targeting
3.2 EXPRESSION PATTERN OF THE KEA FAMILY IN P. TRICORNUTUM
3.3 IDENTIFICATION OF KEA3 IN P. TRICORNUTUM
3.4 MOLECULAR CHARACTERIZATION OF THE MUTANT LINES
3.4.1 Presentation of mutants
3.4.2 Detection of KEA3 by immunolabelling
3.4.2.1 Production of anti-peptide antibodies
3.4.2.2 Characterization of anti-peptide antibodies on total protein extracts
3.4.2.3 Characterization of anti-peptide antibodies on membrane-enriched protein extracts
3.4.2.4 Production an antibody directed against the soluble part of KEA3
3.4.3 Expression pattern of KEA3 and in the WT and complemented strain
3.5 CELL GROWTH OF MUTANT STRAINS
3.6 LOCALISATION OF KEA3
3.7 CONCLUSION
4 REGULATION OF PHOTOPROTECTION BY THE TRANSPORTER KEA3 IN P. TRICORNUTUM
4.1 USE OF NIGERICIN TO CHARACTERIZE KEA3 MUTANTS
4.2 EFFECTS OF KEA3 AT DIFFERENT LIGHT INTENSITIES ON NPQ KINETICS
4.2.1 KEA3 does not affect photoprotective capacities
4.2.1.1 KEA3 leaves the maximum NPQ capacity unchanged
4.2.1.2 Photosynthetic complexes are unaffected by the presence of KEA3
4.2.2 KEA3 slows down NPQ induction in high light
4.2.3 KEA3 affects the extent of photoprotection in moderate high light
4.2.4 KEA3 affects NPQ relaxation kinetics in low light
4.3 KEA3 MODULATES LIGHT UTILIZATION IN P. TRICORNUTUM
4.4 KEA3 IS INACTIVE DURING NPQ RELAXATION IN THE DARK
4.5 KEA3 MODULATES THE PROTON MOTIVE FORCE
4.6 REGULATION OF KEA3 BY CALCIUM
4.6.1 KEA3 binds calcium in vitro
4.6.2 Construction of an EF-hand mutant
4.6.3 NPQ phenotype of EF-hand deprived mutants (preliminary results)
4.7 CONCLUSION
5 MODELLING NPQ DEPENDENCY TO PH
5.1 KINETIC MODELLING OF NPQ
5.1.1 First order kinetic model and theoretical frame
5.1.2 Verification of hypothesis
5.1.2.1 Absence of photoinhibition
5.1.2.2 Proportionality between DES and NPQ
5.1.3 Experimental results
5.1.4 Possibility of a pH-dependency of DEP
5.2 MODELLING OF THE XANTHOPHYLL CYCLE WITH MICHAELIS-MENTEN KINETICS
5.2.1 Simple Michaelis-Menten kinetics
5.2.2 pH dependency of Michaelis-Menten parameters
6 DISCUSSION AND PERSPECTIVES
6.1 NPQ IS A UNIVOCAL FUNCTION OF LUMEN PH IN DIATOMS
6.1.1 Mimicking NPQ in the dark
6.1.2 Clarifying pH control of NPQ
6.1.3 A model for NPQ dynamics in P. tricornutum
6.1.4 Refining NPQ model in diatoms
6.1.4.1 Diatoxanthin formation is kinetically limiting in NPQ induction
6.1.4.2 Perspective: Modelling of NPQ, the xanthophyll cycle and pH
6.2 MODULATION OF NPQ BY AN ION TRANSPORTER
6.2.1 KEA3 controls NPQ dynamics in light transitions
6.2.2 Role of KEA3 in a transition from light to dark
6.2.2.1 The proton gradient controls NPQ decay in the dark
6.2.2.2 Perspective: Activation and deactivation of ATP synthase in diatoms
6.2.3 Regulation of KEA3 activity in P. tricornutum
6.2.3.1 Perspective: Regulation of KEA3 through the RCK domain
6.2.3.2 Perspective: Diatoms, calcium fluxes and photosynthesis
6.2.4 Mode of action of KEA3 in P. tricornutum
6.3 ENERGETICS OF THE PROTON MOTIVE FORCE
6.3.1 Energy conversion by KEA3
6.3.2 Perspective: Regulation of the proton motive force by a network of ion channels
7 CONCLUSION: AN INTEGRATIVE MODEL OF NPQ REGULATION BY PH AND KEA3 IN P. TRICORNUTUM
8 MATERIALS AND METHODS
8.1 ANALYSIS OF PROTEIN SEQUENCES
8.1.1 Phylogenetic tree construction
8.1.2 Analysis of protein sequences
8.2 ANALYSIS OF TRANSCRIPTOMIC DATASETS
8.2.1 Microarray datasets
8.2.2 RNA-sequencing dataset
8.3 DIATOM CULTURE CONDITIONS
8.3.1 Diatom culture in liquid cultures
8.3.2 Diatom culture on agar plates
8.3.3 Diatom storage
8.4 MOLECULAR CLONING
8.4.1 DNA cloning
8.4.2 Contents of used plasmids
8.4.3 Plasmid transformation in E. coli
8.4.4 DNA sequencing
8.4.5 Recombinant production of the soluble part of KEA3
8.5 DIATOM GENETIC ENGINEERING
8.5.1 Biolistic transformation
8.5.1.1 Principle of the biolistic transformation
8.5.1.2 Generation of KO-mutants
8.5.1.3 Mutant screening
8.5.2 Transformation by bacterial conjugation
8.5.2.1 Principle of bacterial conjugation
8.5.2.2 Heterogeneity in gene expression on conjugation transformed cells
8.5.3 Comparison between the two transformation strategies available in the laboratory
8.6 PROTEIN ANALYSIS
8.6.1 Preparation of protein extracts
8.6.1.1 Preparation of total protein extracts
8.6.1.2 Preparation of membrane-enriched protein extracts
8.6.2 Protein quantification
8.6.3 Separation of proteins by electrophoresis (SDS-PAGE)
8.6.4 Transfer
8.6.5 Immunodetection of proteins
8.7 PIGMENT ANALYSIS
8.7.1 Pigment extraction
8.7.2 HPLC analysis
8.8 ANALYSIS OF PHOTOSYNTHETIC ACTIVITY IN VIVO
8.8.1 Fluorescence measurements
8.8.1.1 Principle of the measurement and main parameters
8.8.1.2 Fluorescence measurements at the Pulse Amplification Modulator (PAM) fluorometer
8.8.1.3 Fluorescence measurements in multi-well plates with a Speed Zen imaging fluorometer
8.8.1.4 Comparison between the two devices
8.8.1.1 NPQ induction by acid shift in the dark
8.8.2 Electrochromic shift measurements
8.8.2.1 Principle
8.8.2.2 ECS spectrum in P. tricornutum
8.8.2.3 Experimental setup
8.9 STATISTICAL ANALYSIS
9 ANNEXES
9.1 HOMOLOGUES OF KEA3
9.2 LIST OF MUTANTS CREATED
9.3 POTENTIAL REGULATORS OF THE P.M.F. IN P. TRICORNUTUM