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NPQ IS STRICTLY CONTROLLED BY LUMEN PH IN DIATOMS
The dependency of NPQ to thylakoid lumen acidification is well established in the green lineage, as the proteins PSBS, LHCSR and the enzyme VDE are all activated by low pH. In diatoms, there are indications that NPQ also depends on lumen pH acidification.
These studies have all been performed either as enzymatic assays on partially purified thylakoids (Jakob, Goss and Wilhelm, 2001; Grouneva et al., 2006) or in vivo using ammonium chloride (NH4Cl) as an uncoupler of the proton gradient (Mewes and Richter, 2002; Ruban et al., 2004; Goss et al., 2006). Existing protocols for diatom chloroplast identification do not leave the organelles as functionally preserved as for plants (Schober et al., 2018).
FIGURE 2.1. MECHANISM OF PENETRATION OF AMMONIUM CHLORIDE INSIDE BIOLOGICAL MEMBRANES.
Ammonium ions freely diffuse inside compartments. Upon reaching a membrane, they may lose a proton and form ammoniac gas that freely diffuses across the membrane. If the other side of the membrane (compartment 2) is acidic, ammoniac is protonated into ammonium and diffuses into the next compartment. If the second compartment is less acidic than the second, no proton equilibration takes place.
Besides, the use of ammonium chloride to study photosynthesis may be criticized. Ammonium chloride allows for protons to cross a membrane only if the pH on the other side of the membrane would be low enough (Crofts, 1967). Since in diatoms chloroplasts are surrounded by four different membranes, NH4Cl would penetrate from the external medium to the thylakoid lumen only if all the compartments were crossed in descending pH (Figure 2.1). Penetration could also be forced by adding NH4Cl in excess to the external medium. Ammonium is also the nitrogen source in some diatom culture media (Ryther and Guillard, 1962), i.e. ammonium can be transported and metabolized by a diatom. For these reasons, ammonium chloride deeply perturbs algal physiology. It may therefore not be the most suitable option to study photosynthesis.
High doses of ammonium chloride abolished NPQ if added before or during illumination (Ruban et al., 2004). It was also suggested (Goss et al., 2006) that the proton gradient was only required for diatoxanthin synthesis and was no longer required once NPQ was activated. In order to solve this apparent contradiction, we induced NPQ in the diatom P. tricornutum solely by modifying the pH in all cellular compartments.
NPQ INDUCTION IN THE DARK
Induction of NPQ in the dark by an acid shift
We monitored the external pH of P. tricornutum cells and measured the fluorescence parameters using a Pulse Amplitude Modulation (PAM) Fluorometer. All experimental details of these experiments are described in the Methods Section (see Section 8.8.1).
Nigericin is an ionophore catalyzing K+/H+ exchange across biological membranes (Figure 2.2). This chemical occurs naturally as a bacterial toxin (Graven, Estrada-O and Lardy, 1966). At low concentrations, nigericin decreases ΔpH and compensates part of the proton motive force with ΔΨ (Ahmed and Booth, 1983; Pu, Wang and Chen, 1995). It has been widely used to investigate functional response to pH in vivo (Ahmed and Booth, 1983; Johnson and Ruban, 2014; Armbruster et al., 2016) in several photosynthetic and non-photosynthetic organisms.
FIGURE 2.2. CARTOON SHOWING THE CHEMICAL STRUCTURE OF NIGERICIN AND THE MECHANISM OF NIGERICIN-DRIVEN K+/H+ EXCHANGE ACROSS A BIOLOGICAL MEMBRANE IN RESPONSE TO A PROTON GRADIENT.
In the experiment presented below, WT P. tricornutum cells were first exposed to high light to reach their maximum NPQ level (NPQmax). NPQ was then relaxed in low light (Figure 2.3). The light was switched off and nigericin was added (black arrow). At this stage, the value of the external pH was 7.
The nigericin concentration used (20 µM) was sufficient to drive diffusion of protons added from the external medium to the inner compartments of the cell. Indeed, addition of acetic acid (red arrow) to the cell suspension successfully decreased the external pH to 4.5, while promoting a sharp decrease in PSII maximum fluorescence Fmax. Adding potassium hydroxide (blue arrow, Figure 2.3) increased the medium pH to its original value and unquenched PSII. This indicates that acid-induced quenching of PSII is reversible.
P. tricornutum cells (15.106 cells/ml) were exposed to high light (1200 µmol.photons.m-2, white box) followed by low light (30 µmol.photons.m-2, grey box) and dark (black box). Arrows indicate the successive addition of 20 µM nigericin (black arrow), 4 µM acetic acid (red arrow) and 2 µM potassium hydroxide (blue arrow). Addition of acetic acid caused a shift in the external pH from7.8 (blue box) to 4.5 (red box), while potassium hydroxide addition restored the pH to its original value. The raw fluorescence trace and NPQ calculation are shown for the same experiment.
Adding acetic acid without nigericin did not trigger any detectable change in fluorescence (not shown). Therefore, in the experiment reported here (Figure 2.3), nigericin acted as a proton shuttle across the membranes of P. tricornutum. We can thus assume that without nigericin, penetration of acidic acid is too slow or not sufficient to acidify the various cell compartments, especially since they are buffered to maintain cytoplasmic homeostasis.
Both the high light induced quenching and acid-induced quenching were reversible and did not affect PSII stability: indeed Fv/Fm (see Section 1.4.2) reaches the same value as the initial one at the end of the neutral shift (Figure 2.3). The acid-induced quenching also reached the same level as the high light induced quenching (~1.8 in our conditions).
The maximum quenching in high light is characteristic of a specific cell culture at a given time (Bailleul et al., 2010) and will be called NPQmax in the rest of this manuscript. We exposed cells to acid quenching in the same conditions as previously described and the pigment content of these cells was analyzed by HPLC (see Section 8.7 for experimental procedures). Addition of acetic acid in the dark in the presence of 20 µM nigericin did trigger the conversion of diadinoxanthin into diatoxanthin since a de-epoxidation state of ~0.4 was reached for ~0.7 NPQ/NPQmax (2 technical replicates, data not shown).
Both the acid-induced quenching and the high light induced quenching are able to induce pigment de-epoxidation and therefore proceed from the same mechanism. NPQ induction and relaxation in P. tricornutum are therefore dependent on pH-related changes.
NPQ depends on the external pH in permeabilized cells
Calibration of NPQ as a function of the external pH
FIGURE 2.4 CALIBRATION OF NPQ AS A FUNCTION OF THE EXTERNAL PH.
pH changes were performed using various doses of acetic acid (acid shift, full squares) or potassium hydroxide (neutral shift, empty squares) in the presence of 20 µM of nigericin, in the same conditions as Figure 2.3. NPQ was normalized to the maximum NPQ value reached in high light (NPQmax). Data was fitted using a Hill function (R² = 0.90).
We performed the same experiment as described in the previous paragraph using various doses of acid acetic. Different NPQ levels were reached in the dark (Figure 2.4). NPQ induction was always reversible when neutralizing the external pH using potassium hydroxide.
Interestingly, the level of NPQ reached quantitatively depends on external pH (Figure 2.4). In a pH range between ~4 to 5.5, the extent of quenching is linearly correlated to the external pH. Overall, the shape of this dependency is sigmoidal, which is a characteristic shape for phenomena involving pH shifts around a pKa value.
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
