Photosynthetic physiology of the coccolithophore Emiliania huxleyi Major environmental determinants and signature of viral infection in the field

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Electron-proton coupling and ATP/NADPH ratios

As presented above, photosynthetic reactions can be separated into two steps: the photosynthetic chain that produces cell usable energy (ATP) and reducing power (NADPH) and the synthesis pathways, being mainly the CBB cycle which uses ATP and NADPH to produce organic matter. It has long been debated whether ATP/NADPH ratio produced by the linear electron flow (LEF) is equivalent to ATP/NADPH ratio required for CBB cycle and other metabolic process (e.g. Allen, 2003).
 ATP/ NADPH ratio produced by the LEF ATP and NADPH production by the LEF are coupled and, based on the molecular understanding of the mechanisms involved in electron transfer and ATP synthesis, it is possible to calculate the ratio ATP/NADPH produced by LEF. We presented earlier the sites of proton liberation in the lumen: 1H+ is transferred per electron at the OEC level and thanks to the Q-cycle, the cytochrome b6f couples the transfer of one electron to the translocation of two protons. The coupling between electron transfer and H+ translocation results thus in 6 protons translocation to the lumen every 2 electrons transferred from water to NADP+. The number of protons needed for 3 ATP production depends on the number of c-subunits of the ATP synthase CF0 domain. The group of Kühlbrandt recently obtained structures of the ATP synthase (CF1-F0) from spinach and confirmed that it contains 14 c subunits (Hahn et al., 2018). Hence, 14 H+ are needed to form 3 ATP. The 6 H+ translocated by the LEF would thus result in the synthesis of 1.29 ATP.
 ATP/NADPH ratio required for CO2 fixation and associated metabolic pathways As presented above, it is well admitted that the CBB cycle uses a ratio of 3 ATP per 2 NADPH under atmospheric conditions. But, more generally, the required ATP/NADPH ratio depends on CO2 partial pressure in the vicinity of the Rubisco (Kramer & Evans, 2011); it can vary from 1,5 to 1,65. As presented previously, microalgae also use carbon concentrating mechanisms (CCM) involving active transport of inorganic carbon inside the chloroplast. CCM thus need ATP and are more active when CO2 concentration is low. The carbon concentrating mechanisms have an extra cost in terms of ATP, and could in some conditions increase the required ATP/NAPH ratio for carbon concentration and assimilation to 2 ATP per NADPH. All in all, the overall CO2 reduction pathways need at least 1,5 ATP per NADPH (CBB cycle under atmospheric conditions) but this ratio may increase depending on environmental conditions such as inorganic carbon and nutrient availability.
 Coupling between CO2 reduction and LEF leads to an ATP shortfall The comparison of the ATP/NADPH ratio produced by the linear electron flow (1.29) and the one required for CO2 reduction (>1.5) reveals that LEF alone cannot fuel the CBB cycle with enough ATP (Allen, 2003). We have mentioned before that the plastid is not just the site of carbon assimilation, but also the site of nitrate and sulfate reduction and of amino acids and fatty acids synthesis (Jensen and Leister, 2014). All those metabolic pathways require ATP and/or NADPH with varying stoichiometry (Noctor and Foyer, 1998), making the simple comparison of the two above ratios quite naïve. Nevertheless, some level of ATP/NADPH adjustment of the “light phase” of photosynthesis is necessary to respond to varying demands of ATP and NADPH for downstream PSI metabolism (Noctor and Foyer, 1998).

Light absorption and photochemistry

The photosynthetic process of energy conversion begins when a pigment is excited by a quantum of light, a photon, and an electron is moved from one molecular orbital (ground state) to another of higher energy (excited state). Such an excited molecule is unstable (the lifetime of a chlorophyll in water is in the ns range) and tends to return to its original fundamental energy level with one of the following relaxation pathways (Figure 1-12): (1) by converting the energy into heat (non-radiative dissipation) or (2) by re-emiting a photon of lower energy, i.e. at a longer wavelength (fluorescence) (3) by transferring the energy – but not the electron – directly to a nearby pigment molecule by a process called Förster resonance energy transfer (FRET) (Förster, 1948), (4) by transferring the high-energy electron to another nearby molecule, an electron acceptor; this is the photochemical event or charge separation.
In the thylakoid membrane, the supercomplexes formed by the assembly of light harvesting complexes (LHC), involved in the capture of light energy, and of photosystem cores, where the electron transfers occur, enable an efficient photochemistry. The following paragraphs describe the structure of these supercomplexes and the reaction steps of photochemistry.

Light harvesting complexes and absorption cross-section of the PS

In the beginning of the 20th century, Willstätter determined the structure of the most abundant green pigment in photosynthetic eukaryotes, the chlorophyll (chl), and he was awarded the Nobel Prize in Chemistry in 1915 for this work. In 1932, Robert Emerson and William Arnold demonstrated that chloroplasts contain much more chlorophyll molecules than reaction centers and suggested that most of the chlorophylls serve as light harvesting pigments (Emerson and Arnold, 1932). Using spectrophotometric titration, they could calculate the concentration of chlorophyll contained in a Chlorella sample. They then measured the amount of O2 production of the sample which had been exposed to saturating brief flash of light. They calculated that each flash of saturating intensity produced only one molecule of O2 per ~ 2400 molecules of chlorophyll. In hindsight, since we now know that at least eight photons must be sequentially absorbed to liberate one O2 molecule, these results suggest that the photosynthetic unit contains about 300 chlorophyll molecules forming an antenna. In higher plants and green algae, the antenna system increases the fraction of light absorbed by more than two orders of magnitude funneling light energy to the photochemical converters (i.e. special pair of chlorophyll) and thereby enhance their absorption cross-section (Croce and van Amerongen, 2014).
We now know that all organisms that perform oxygenic photosynthesis have an antenna system that increases the optical section of photosystems. In plants and green algae, where most of the studies of antenna systems in eukaryotes have been performed, these antennae are made up of pigment-binding complexes integrated into the membrane and referred to as LHC (Light Harvesting Complex, reviewed in Caffarri et al., 2014). Pigment-protein complexes from PSII and PSI are called LHCb and LHCa, respectively.
In the case of plant PSII, the core generally forms a dimer surrounded by Lhcb proteins (also called CP24, CP26 and CP29) (Figure 1-13A). The external antenna system is composed of pigment-protein monomer complexes which are in direct contact with the PSII core and of the trimeric LHCII, which are generally less strongly associated with it (Dekker et al., 2005; Caffarri et al., 2009). The organization of the Lhc proteins around the photosystem cores is different between PSII and PSI. According to the crystallographic model and the analysis of single particles, in plant PSI, a single layer of Lhca proteins (also called LHCI) is linked on one side of the central complex from PsaG (Figure 1-13A) and the order of binding is: Lhca1, Lhca4, Lhca2, Lhca3 (Amunts et al., 2010).

Structure and function of photosystem II and photosystem I

The two photosystems are the sites of photochemistry. PSII and PSI differ in the reaction they catalyze: the PSII is the site of water oxidation at the lumenal side and of the reduction of the membrane soluble plastoquinones next to the stromal site, according to the reaction: 2H2O + 2PQ + 4H+ = O2 + 2PQH2 + 4H+ PSI catalyzes the oxidation of lumenal PC by stromal ferredoxins (Fd) according to the equation: PC + Fd = PC+ + Fd-.
What particular structural organizations of the PS enable their catalytic function? Are there structural and functional similarities between PSII and PSI? What are their differences? Hereafter, is presented a brief review of the state of the art on these issues.
 General structure of the PS The PSII core is a multisubunit complex composed of approximately 25-30 subunits (Guskov et al., 2009; Caffarri et al.,2009). Most of the chromophores involved in light harvesting, as well as the electron transfer reactions, are linked to four main subunits, called D1, D2, CP43 and CP47, which are all membrane proteins containing several transmembrane helices. The D1 and D2 subunits are homologous and form a heterodimer whose transmembrane alpha helices are organized in a handshake motif. The D1-D2 complex together with cyt. b559 (composed of the PsbE and PsbF subunits) is often called the PSII reaction center (RC) because it binds most of the cofactors involved in the photocatalytic activity of this photosystem. The CP43 and CP47 subunits bind a total of 29 chl a molecules (based on the cyanobacterial structural model) which function as an internal antenna and allow the transfer of excitation energy from the peripheral antenna system to the reaction center. The chl a composing the internal light collection system seem to be organized mainly in two layers parallel to the plane of the membrane and located near the lumenal and stromal sides of the membrane (Figure 1-13A).
The central core complex of PSI is made up of fewer proteins (~ 15 subunits) than PSII (Jensen et al., 2007). The large PsaA and PsaB subunits form a hetero-dimer which binds the vast majority of cofactors for light capture (~ 80 Chls a and ~ 20 – carotenes) as well as the cofactors involved in electron transfer reactions with the exception of the terminal electron acceptors (Fe-S FA and FB clusters that will be described below), which are linked by the PsaC subunit (Figure 1-13A and B). As in the case of PSII, the chl a molecules involved in light harvesting are organized in two parallel layers and located near the lumenal and stromal sides of the membrane, respectively, while the cofactors involved in stabilizing the charge separation form two parallel branches, perpendicular to the plane of the membrane. The other subunits are involved in different processes, such as docking of plastocyanin (donor site, PsaF), docking of ferredoxin (PsaC, PsaD, PsaE, acceptor side), stabilization of the LHCI antenna system (PsaK, PsaG) or the formation of the host site for LHCII binding to PSI.

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Measuring P700 redox state to investigate PSI activity

Photosynthesis consists in electron transfers that involve several redox cofactors. Some of these cofactors absorb in the visible light and do not display the same absorption spectrum depending on whether they are in their oxidized or reduced form. The measurement of absorption changes will specifically inform on the changes of the redox state of those electron carriers. For example, light-induced PSI photochemistry induces a main absorption band bleaching (in the Qy region) with a maximum at ∼695 nm. This absorption change comes from the oxidation of P700 (Ke 2001). The difference absorption spectrum of P700 (P700+ minus P700) is presented in Figure 2-2 and shows a bleaching region around 680-710 nm in the green alga Chlamydomonas reinhardtii (Webber et al. 1996).
PSI core complex proteins are highly conserved among cyanobacteria and photosynthetic eukaryotes (Mix et al. 2005). Moreover, in cyanobacteria, plants and green algae, P700 absorption change spectra is conserved (Drop et al. 2014; Hiyama and Ke 1972; Webber et al. 1996).

Table of contents :

Chapter 1: General introduction
1. 1. Marine ecosystems, phytoplankton and photosynthesis
1. 1. 1. Most of the genetic diversity in the Ocean is known
1. 1. 2. Phytoplankton plays a crucial role in fluxes of matter and energy in the ocean
1. 1. 3. Origin and taxonomic diversity of phytoplankton
1. 2. Reactions and mechanisms involved in eukaryotic photosynthesis
1. 2. 1. Different types of photosynthesis
1. 2. 2. The chloroplast: an organelle hosting photosynthesis
1. 2. 3. The Calvin-Benson-Bassham cycle synthesizes organic carbon from CO2
1. 2. 4. The photosynthetic electron transfer chain uses light energy to supply the CBB cycle
1. 2. 5. From light absorption to energy storage
1. 2. 6. Monitoring photosynthesis with chlorophyll fluorescence
1. 3. Regulation of the photosynthetic process and acclimation to changing environments
1. 3. 1. Light stress and photo-inhibition
1. 3. 2. Protecting photosystem I and II from light stress
1. 3. 3. Photosynthetic alternative pathways
1. 3. 4. CEF, a crucial regulative pathway remaining mysterious
1. 4. Thesis outline
1. 5. Bibliography
2 Exploring the diversity of cyclic electron flow around photosystem I in microalgae species
Chapter 2: Probing PSI activity
2.1. How to probe photosystem I with absorption spectroscopy?
2.1.1. Some reminders about photosystem I
2.1.2. Measuring P700 redox state to investigate PSI activity
2.1.3. Electrochromic Shift: an internal voltmeter
2.2. Article: Critical reappraisal of methods to measure photosystem I activity
2.3. Discussion
2.3.1. The P700 pulse method underestimates Y(I) because of reduction of PSI acceptors during the multiple turnover pulse
2.3.2. Technical considerations regarding P700 measurements
2.3.3. Generalization to the case of an active photosystem II
2.3.4. Revisiting the literature based on the P700 pulse method
2.3.5. Partial conclusions and transition
2.4. Bibliography
Chapter 3: Diversity of cyclic electron flow in microalgae
3.1 Introduction: CEF, a still mysterious alternative pathway
3.1.1. Role of CEF in ATP/NADPH adjustment and photo-protection
3.1.2. Regulation of CEF rate
3.1.3. Measuring CEF is a methodological challenge
3.1.4. Chapter outlines
3.2 Material and methods
3.2.1 Strains, growth and sampling
3.2.2 Chemicals
3.2.3 In vivo spectroscopy
3.2.4 ECS spectra and linearity with electric field
3.2.5 Absorption cross section assessment
3.3 Results (I): Exploring CEF diversity
3.3.1 DCMU titration reveals CEF behavior
3.3.2 Which observables?
3.3.3 CEF is not essential to photosynthesis in the dinoflagellate Amphidinium carterae
3.3.4 CEF is independent on LEF photosynthesis in the dinoflagellate Symbiodinium sp.
3.3.5 Chlamydomonas reinhardtii displays a CEF which is dependent on LEF
3.3.6 Partial conclusion on CEF diversity
3.4 Results (II): Evaluating CEF and LEF absolute rates as a function of light irradiance in the green alga Chlamydomonas reinhardtii
3.4.1 ECS-based estimations of the PSI and PSII absorption cross sections
3.4.2 Validation of the method for measurement of absorption cross sections
3.4.3 Evaluating CEF absolute rates from ETR(total) and ETR(II)
3.4.4 Calculations of the ATP/NADPH ratio produced by the photosynthetic chain
3.5 Discussion and future perspectives
3.5.1 A simple and robust protocol to investigate CEF diversity highlights three different behaviors
3.5.2 Limitations of the DCMU titration method
3.5.3 Despite a complex relationship, CEF and LEF remain proportional at all light intensities in Chlamydomonas reinhardtii
3.6 Bibliography
Chapter 4: Photosynthetic physiology of the coccolithophore Emiliania huxleyi Major environmental determinants and signature of viral infection in the field
4.1 Introduction
4.2 Material and methods
4.2.1 Mesocosm setup, treatments and sampling
4.2.2 Flow cytometry/ qPCR
4.2.3 Measurement of environmental (abiotic) parameters
4.2.4 Photophysiology by Fast Induction and Relaxation fluorometry (FIRe)
4.2.5 Statistical analyses
4 Exploring the diversity of cyclic electron flow around photosystem I in microalgae species
4.3 Results
4.3.1 A two phases phytoplankton bloom occured
4.3.2 Photosynthetic physiology
4.3.3 Fluorescence signals were mainly due to Emiliania huxleyi from day 9 to 24th of the experiment
4.3.4 Evolution of environmental parameters during the experiment
4.3.5 Environmental determinants of E. huxleyi photosynthesis
4.3.6 A photosynthetic signature of viral infection?
4.4 Conclusion and discussion
4.5 Bibliography
Chapter 5: General discussion
5. 1. 1. The importance of cross validations of methods
5. 1. 2. Using the flash-induced ECS to estimate the photochemical rate: a good choice?
5. 1. 3. Studying CEF and its abiotic and biotic determinants in the field: an accessible project?
5. 2. Roles of CEF and mechanisms of the regulation of the CEF and LEF
5. 2. 1. ATP:NADPH ratio equilibration
5. 2. 2. A role of CEF when PSII is inhibited?
5. 2. 3. CEF and LEF regulation
5. 3. Three years were too short for…
5. 3. 1. Revisiting literature using P700 method
5. 3. 2. Exploring cyclic electron flow using our methods
5. 3. 3. Harness all data collected in the field
5. 4. Bibliography


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